The relationship between hydrophobicity and interfacial tension of proteins. Charge-free hydrophobic gels of Hjerten et al. (Hjerten, S., Rosengren, J. and Pahlman, S. (1974) J. Chromatogr. 101, 281--288) were used for hydrophobic affinity chromatography. The effective hydrophobicity of proteins was expressed as their retention volumes from columns of butylepoxy- and hexylepoxy-Sepharose 4B. The effective hydrophobicity was also estimated by a partition method of Shanbhag and Axelsson ((1975) Eur. J. Biochem. 60, 17--22) from the partition coefficients of proteins between two phases, poly (ethylene glycol) and dextran. The former contained a hydrophobic ligand, palmitate. A close correlation was observed between the hydrophobicities determined by the two methods. However, no significant relationship was observed between these effective hydrophobicities and the average hydrophobicity of Bigelow ((1967) J. Theoret. Biol. 16, 187--211) that was calculated from the total amino acid composition of each protein. The interfacial tensions at the 0.2% protein/corn oil interface revealed negative correlations with the effective hydrophobicities determined by both methods indicating lower interfacial tensions with more hydrophobic proteins.


Preparation of dipeptidyl aminopeptidase IV for polypeptide sequencing. Dipeptidyl aminopeptidase IV is a dipeptidylpeptide hydrolase (EC 3.4.14) which hydrolyzes bond at the carboxyl group of proline releasing X-Pro dipeptides from the amino-terminus of polypeptides. The enzyme was purified 440-fold in 37% yield from swine kidney by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration and affinity chromatography with dipeptide-substituted Sepharose 4B. The enzyme released X-Pro from all X-Pro-beta-naphthylamides and polypeptides tested. The released dipeptides were not further degraded, and were readily identified in digests. The enzyme is suitable for use in the dipeptidyl aminopeptidase method for sequence analysis of polypeptides.


Renin in the mouse submaxillary gland has a molecular weight of 40,000. The availability of pure submaximillary renin, its antibody and pure specific immunoreactive Fab fragments of the antirenin molecule were used in an attempt to detect in which form renin is stored in the submaxillary gland. The proteolytic activity of serine-, metallo- and sulfhydryl enzymes during homogenisation was inhibited, but no inactive or high molecular weight form could be detected enzymatically or antigenically after gelfiltration. Nor were they demonstrable in crossed immuno-electrophoresis by using antibodies elicited against pure renin. Furthermore, pepstatin which additionally inhibits acid proteases, including a possible autoactivation of renin, and renin specific Fab fragments, were added, the latter in order to steric hinder proteolytic attack on a possible renin precursor. The renin-Fab complex was purified by precipitation with anti-Fab antibodies. No high molecular weight renin was demonstrable in SDS polyacrylamide gel electrophoresis. The only form of renin demonstrable in the submaxillary gland of mice was the fully active 40,000 dalton form. Its specific enzymatic activity was identical to that of pure submaxillary renin, being 0.4 . 10(-3) Goldblatt unit . ng-1.


Species-dependent immunological differences between various mammalian cardiac tropomyosins. Antisera were produced from guinea-pigs against purified pig or rat cardiac tropomyosins and antigen-antibody interactions were analyzed by the micro-complement fixation technique. Immunoadsorption with purified tropomyosins coupled with CN Br-activated Sepharose 4B enabled us to establish that these antisera were only specific to tropomyosin and not to other contractile proteins. Direct cross-reactions and competition experiments performed with both the above antisera indicated quantitative differences in the maximum amount of complement fixed by tropomyosins from various heterologous species (man, beef, pig, rabbit, rat and mouse). These data provide direct evidence that mammalian cardiac tropomyosin is species-specific.


Immunochemistry of serum albumin. VI. A dynamic approach to the immunochemical cross reactions of proteins using serum albumins from various species as models. Antisera against bovine serum albumin were raised in two rabbits. Serial bleedings were obtained at different times after the first immunization, and antisera from these serial bleedings were not mixed but were kept and studied separately. The immunochemical cross-reactions of these antisera with serum albumins from bovine, goat, sheep, porcine, horse, human and chicken were determined by immunoadsorbent studies. These were done by titration so that the values of maximum (plateau) binding by each albumin of radioiodinated antibodies were determined. In each rabbit, the immunochemical cross-reactivity was not static but increased progressievly with time after the first immunization. In the interval 7 days to 398 days the increases in cross-reaction were extremely large. pH dissociation studies revealed that, together with the increase in cross-reactivity of a given albumin with time after immunization, there was a restriction in the antibody heterogeneity towards populations possessing higher affinity. These results provide a rational explanation for the different values of cross-reactivities for a given albumin from different laboratories. The findings are analyzed in relation to the antigenic structure of albumin and their significance in evolutionary studies discussed.


Cow kappa-casein: structure of the carbohydrate portion. The detailed sugar sequences of the two main carbohydrate portions of cow kappa-casein were established by enzymic and chemical methods and by mass spectrometry. The sugar sequences correspond to widespread sugar parts occurring in many glycoproteins.


Isolation and partial characterization of low sulfated chondroitin 4-sulfate-proteoglycan in bovine blood. Bovine plasma low sulfated chondroitin sulfate-proteoglycan (34 microgram/ml plasma), accounting for the main component of acidic glycosaminoglycans in blood, has been purified by isoelectric precipitation, dissociation with 4 M guanidine chloride followed by DEAE-chromatography, Sephadex G-200 chromatography and by preparative polyacrylamide gel electrophoresis. The proteoglycan, having a molecular weight of approx. 44,000, is composed of about 77% protein and 23% glycosaminoglycan at a molar ratio of 1 : 1 which could be cleaved by alkaline treatment into each component. Amino acid analysis of the proteoglycan and its glycosylpeptide has shown that the material is derived from a different origin from other tissue proteoglycans, though the amino acid residues surrounding O-glycosidic linkage to serine residue are quite similar to that of cartilage proteoglycan. Characteristic features of plasma low sulfate chondroitin sulfate-proteoglycan are discussed, compared with tissue materials.


[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage. Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.


Fractionation of granule proteins of granulocytes by copper chelate chromatography. The tendency of lactoferrin to associate with other proteins at low salt concentrations defeated attempts to fractionate the acetate-extracted granule proteins of guinea pig granulocytes by ion-exchange chromatography. This problem was overcome by using copper-chelate chromatography at high salt concentrations to separate lactoferrin from the other granule proteins. Lysozyme and a 'cationic protein' were purified to apparent homogeneity in the same operation. The chromatographic profile of proteins extracted from purified secondary granules differed from that of proteins extracted from total granules chiefly in the absence of a substantial 'cationic protein' peak.


The interaction of dodecyl and tetradecyl sulfate with proteins during polyacrylamide gel electrophoresis. 1. The affinity of tetradecyl sulfate for many unfolded proteins is greater than that of dodecyl sulfate. 2. It is the presence of tetradecyl sulfate that results in the staining of proteins by pinacryptol yellow seen by Stoklosa and Latz (Stoklosa, J.T. and Latz, H.W. (1974) Biochem. Biophys. Res. Commun. 58, 74--79), as some tetradecyl sulfate remains associated with proteins during electrophoresis at room temperature (as opposed to dodecyl sulfate which, within the limit of detection, is completely removed). 3. Tetradecyl sulfate has a greater capacity to dissociate protein aggregates which consist of identical peptide chains, such as Glycophorin dimers and bovine serum albumin dimers, than does dodecyl sulfate.


X-ray diffraction study of bovine lens capsule collagen. The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.


Ring current effects in the conformation dependent NMR chemical shifts of aliphatic protons in the basic pancreatic trypsin inhibitor. Previously, a highly refined crystal structure and energy refined atomic coordinates were obtained for the basic pancreatic trypsin inhibitor, as well as numerous individual resonance assignments in the 1H NMR spectrum. These data were now used to investigate the contributions from the local ring current fields of the aromatic rings to the overall conformation dependent chemical shifts in this globular protein. A program was written which allowed the consideration of certain aspects of internal mobility of the protein, and the different commonly used ring current equa tions were compared. These studies indicate that ring current shifts are the dominant contribution to the observed conformation dependent chemical shifts of the peripheral aliphatic side chain protons. On the other hand, it appears that ring current shifts do not make dominant contributions to the conformation dependent shifts of the backbone alpha- and amide protons or the aromatic protons in the inhibitor. On the basis of the empirical calibration with the peripheral aliphatic side chain protons, the Johnson-Bovey ring current equation was selected for an analysis of the ring geometries of two prolines in the inhibitor.


Resonance Raman study of the dark-adapted form of the purple membrane protein. The resonance Raman spectrum of the dark-adapted form of the purple membrane protein (bacteriorhodopsin) has been obtained and is compared to the light-adapted pigment and model chromophore spectra. As in the light-adapted form, the chromophore-protein linkage is found to be a protonated Schiff base. Electron delocalization appears to play the dominant role in color regulation. The dark-adapted spectrum indicates a conformation closer to 13-cis than the light-adapted spectrum.


Protamines. II. Circular dichroism study of the three main components of clupeine. The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.


Fluorescence properties of o-phthaldialdehyde derivatives of amino acids. The fluorescence properties of the products formed by reaction of o-phthaldialdehyde with amino acids and their derivatives, in the presence of thiol compounds, have been studied. The emission spectra, quantum yields, and lifetimes depend on the primary amine and thiol compound used; the observations confirm the report (Simsons, S.S., Jr. and Johnson, D.F. (1978) J. Org. Chem 43, 2886--2891) that the product incorporates molecules of all three types of compounds. The fluorescence quantum yields of o-phthaldialdehyde derivatives of the naturally occuring amino acids ranged from 0.33 to 0.47, using 2-mercaptoethanol as the thiol compound. The fluorescence lifetimes were about 18--20 ns. Lower quantum yields were obtained when mercaptoethanol was replaced by dithiothreitol or ethnethiol. Derivatives of amino acid amides and peptides had quantum yeilds as low as 0.03, due to quenching by the carboxamide group. The intramolecular quenching was relieved by the detergent, sodium dodecyl sulfate, and by dimethylsulfoxide. Monosubstituted lysine exhibited a normal fluorescence, but the di-substituted product was largely quenched, presumably due to interaction between the two isoindole fluorophors. Fluorscence stopped-flow experiments showed that the alpha- and epsilon-amino groups reacted at different rates, with the epsilon-amion group reacting 10 times faster, with a t 1/2 of about 6 s under pseudo first order conditions at pH 9.0 with 10(-3) M o-phthaldialdehyde. The amount of instability shown by the o-phthaldialdehyde derivatives depended on the thiol compound used, the primary amine involved, and the solvent. Cysteine and o-phthaldialdehyde reacted to give an unstable, weakly fluorescent product; but cysteine could be assayed normally if its sulfhydryl was blocked. The o-phthaldialdehyde reagent was discussed in relation to fluorescamine, another reagent for primary amines.


Planorbis corneus haemoglobin. Circular dichroism and susceptibility to proteases. The purified haemoglobin of Planorbis corneus was subjected to protease digestion and the resulting products characterised by gel filtration and detergent-gel electrophoresis. Small functional subunits of molecular weights approximately 20,000 were obtained corresponding to a single haem group, but multiples of this unit were also always obtained even at high proteolytic enzyme: haemoglobin ratios. This suggested that the subunits of the native molecule (one-tenth containing perhaps ten O2-binding sites) were made up of single-binding site domains linked by regions of polypeptide chains having different susceptibilities to proteases. The far ultraviolet CD of the native haemoglobin indicated the presence of a high helix content (75--80%) in the protein. The near ultraviolet and visible CD spectra of oxy- deoxy-, and CO-haemoglobin were reported. Planorbis haemoglobin CD was more like that of vertebrate haemoglobins than that of haemoblobins. Nevertheless the Soret CD of Planorbis oxyhaemoglobin had only about half the rotational strength of that of human haemoglobin A, and was halved again upon removal of the ligand. Also in contrast to Lumbricus and human haemoglobins there was only a small decrease in rotational strength in the 260 nm band when Planorbis oxyhaemoglobin was deoxygenated.


Haemoglobin Avicenna (beta 47 (CD6) Asp replaced by Ala). A new abnormal haemoglobin. In a survey for abnormal haemoglobin variants in voluntary blood donors in Iran, a new variant was found in a young male who presented no clinical symptoms. It had the same electrophoretic mobility as haemoglobin D in alkaline buffers. Separation of the constituent polypeptide chains in acid urea buffer revealed it to be different from haemoglobin D previously found among Iranians. Analysis of its structure demonstrated a substitution to alanine (beta 47 Asp replaced by Ala) in the same residue as involved in haemoglobin G-Copenhagen (beta 47 Asp replaced by Asn).


Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome. Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.


Structure of human hemoglobin C: a disease with intraerythrocytic crystals. Crystals of human cyanomethemoglobin C (beta 6A3 glu leads to Lys) crystallized in the orthorhombic space group P212121, A = 158(1), B = 65.5(4), C = 54.9(5) A with Z =4. Single crystal electron micrographs show filaments parallel to the b direction. The molecules are unusually densely packed compared to other hemoglobin crystals, and this may be related to the ease of intraerythrocytic crystallization.


Renin binding proteins in plasma. Binding of renin to some of the plasma protease inhibitors, to lipoproteins, and to a non-trypsin-binding unidentified plasma protein. Renin is found in mouse plasma as high molecular weight forms, in addition to the fully active 40 000 dalton form. By using freshly 125 I-labelled 40 000 dalton pure submaxillary mouse renin, no binding to plasma proteins was demonstrable. However, unfolding and refolding of the labelled renin by guanidine facilitated binding to specific mouse and human plasma proteins. By using antibodies against individual human plasma proteins, the specific binding proteins were identified to be the plasma protease inhibitors: alpha2-macroglobulin, inter-alpha-trypsin inhibitor, alpha2-antithrombin. Binding was also demonstrated to alpha1- and beta1-lipoproteins, albumin and to a non trypsin binding unidentified plasma protein. No binding to 56 other tested proteins was demonstrable. It is concluded that the native 40 000 renin does not bind, but that a conformational change of the renin molecule most likely is necessary before binding occurs. It is discussed whether or not inactive or high molecular weight forms of renin in plasma are 40 000 renin bound to plasma protease inhibitors and lipoprotein.


Hydrophobicity of lectins. I. The hydrophobic character of concanavalin A. The hydrophobicity of Concanavalin A has been estimated by its tendency to adsorb to hydrophobic adsorbents. Experiments varying temperature, salt concentration and hydrophobicity of the absorbent were consistent with accepted criteria of hydrophobic interaction between biomolecules and hydrophobic ligands. The biological significance of the hydrophobic character of Concanavalin A is also discussed.


The collagenous matrix of bovine predentine. Predentine obtained from bovine teeth by microdissection was solubilized by cyanogen bromide cleavage. The electrophoretic mobility of the resultant peptides was established on polyacrylamide gel and the amino acid composition of several peptides was determined. The data clearly indicated that this collagen is entirely of the Type I genetic species. No differences were detected between the predentine and dentine collagens except that the mature tissue was more highly crosslinked. Nevertheless the amount of stable cross-link formed in the predentine was higher than expected for an immature tissue.


Charge separation of proteins complexed with sodium dodecyl sulfate by acid gel electrophoresis in the presence of cetyltrimethylammonium bromide. Globular proteins, casein, and membrane proteins which were reacted with sodium dodecyl sulfate were studied by acid urea gel electrophoresis. The sodium dodecyl sulfate bound tightly to the proteins, producing a more acidic charge which prevented migration into the gel. When cetyltrimethylammonium bromide was added to the sodium dodecyl sulfate-protein complexes, the sodium dodecyl sulfate apparently reacted with cetyltrimethylammonium bromide and dissociated so that the proteins migrated in acid gel in a normal manner as compared to the proteins without any added detergent. The sodium dodecyl sulfate-cetyltrimethylammonium bromide complex could be removed from the proteins by centrifugation. Thus, cetyltrimethylammonium bromide used in conjunction with acid gel electrophoresis allows direct comparison by charge of proteins fractionated in the presence of sodium dodecyl sulfate with the starting mixture of proteins not exposed to detergent. The reaction of cetyltrimethylammonium bromide with sodium dodecyl sulfate in acidic urea also provides a simple convenient method of removal of sodium dodecyl sulfate from proteins.


Resolution of the glycosyltransferase activities from two strains of Streptococcus mutans by polyacrylamide gel electrophoresis in the presence of Tween 80. The glycosyltransferase complex from Streptococcus mutans can be resolved by polyacrylamide gel electrophoresis following treatment with Tween 80. The enzyme complex was treated with Tween 80 and electrophoresis was performed in the presence of Tween 80. Discrete bands of glycosyltransferase activity were observed when the gels were incubated in buffered sucrose. The enzyme aggregation that occurs with the exocellular glycosyltransferase may involve lipophilic interactions that are disrupted by the Tween 80.


Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies. Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.


On the chromatographic heterogeneity of human fetal hemoglobin. Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.


Structure of 2-hydroxy-5-nitrobenzylated carboxypeptidase A. Bovine pancreatic carboxypeptidase A (EC 3.4.12.2) was treated with dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium chloride at pH 7.5, resulting in a preparation which consisted primarily of a monohydroxynitrobenzylated derivative of the enzyme. Samples of the hydroxynitrobenzylated enzyme were subjected to tryptic digestion and to cyanogen bromide cleavage, and resulting peptides were isolated chromatographically. One tryptic hydroxynitrobenzyl-containing peptide was isolated; its amino acid composition was that of the N-terminal tryptic segment of carboxypeptidase Agamma (residues 8--35). Likewise, CNBr cleavage of the hydroxynitrobenzylated enzyme revealed that the hydroxynitrobenzyl group resided in the N-terminal fragment, FN (residues 8--22). Neither of these hydroxynitrobenzylated peptides contains Trp, the amino acid residue which is characteristically the site of hydroxynitrobenzylation in proteins, and each was found to contain approximately one less Asx than the corresponding native peptide. Both dansylation and automated Edman degradation procedures revealed that the N-terminal Asn of carboxypeptidase Agamma had been modified by hydroxynitrobenzylation of the enzyme. Thus the sulfonium salt reacts with carboxypeptidase A in the same manner as that established earlier for 2-hydroxy-5-nitrobenzyl bromide (Radhakrishnan, T.M., Bradshaw, R.A., Deranleau, D.A. and Neurath, H. (1970) FEBS Lett. 7, 72--76). Such reactivity of the alpha-amino group presumably reflects its unique location with respect to Trp residues in the tertiary structure of the enzyme.


Improvement in the resolution of human sperm protamines by use of iodoacetamide as alkylating agent. By use of the neutral alkylating agent iodo [14C1] acetamide instead of ethylene imine or iodoacetate, the resolution of human protamines on gel electrophoresis and ion-exchange chromatography has been improved. Using 20-cm gels, human protamines may be fractionated into seven bands, including the two chromatographically distinct forms of HP1 and a hitherto undetected component HP4. On ion-exchange chromatography, HP2 and the two forms of HP1 may be isolated in sufficient purity for sequence analysis.


Fragmentation of actin by thrombin-like snake venom proteases. The effect of thrombin-like snake venom proteases (Ancrod of Agkistrodon rhodostoma and Batroxobins of Bothrops moojeni and Bothrops marajoensis) on skeletal muscle actin was studied and compared to the thrombic cleavage of this protein. Only EDTA-pretreated G- and F-actin were split by thrombin and Ancrod, while Batroxobins hydrolyzed native G-actin, too. The time course of digestion was followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A split product of 37 500 daltons appeared first which was cleaved further resulting in three lower molecular weight fragments. The sodium dodecyl sulfate gel pattern of thrombic fragmentation was well distinguishable from those caused by Ancrod and Batroxobins. The first split products of Batroxobin digestion--a smaller peptide and the 37 500 dalton fragment--were isolated and by estimating their N-, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actino-peptides (at Arg(28)-Ala(29) and Arg(39)-His(40) from the N-terminal end of the molecule only Arg(39)-His(40) was cleaved by Batroxobins.


Histones of Xenopus laevis erythrocytes. Purification and characterization of the lysine-rich fractions. Lysine-rich histones have been isolated from the terminally differentiated erythrocytes of Xenopus laevis. Three major proteins have been separated by ion-exchange chromatography. These proteins have been characterized by electrophoresis, amino acid analysis and immunochemical techniques. It is concluded that two 'typical' lysine-rich subfractions are present in Xenopus erythrocytes and, in addition, a serine-rich histone, that shares no common antigenic determinants with the other lysine-rich histones.


Purification and partial characterization of a mitogenic lectin from Vicia sativa. From the seeds of Vicia sativa a lectin has been purified by affinity chromatography on Sephadex G-100, followed by specific elution with D-glucose. The lectin is a glycoprotein with a molecular weight of 70 000. The aminoacid composition and the total sugar content have been determined. This lectin agglutinates horse, rabbit and human erythrocytes, with no specificity for human blood groups, but does not agglutinate calf and sheep erythrocytes. The agglutinating activity is inhibited by mono-, di-, and trisaccharides with a pyranosyl residue whose free hydroxyl group in position 4 has the configuration of glucose, and by fructose. The lectin has mitogenic activity on human peripheral blood lymphocytes.


The conformational properties of some fragments of the peptide hormone somatostatin. The conformational properties of some somatostatin fragments have been studied using high resolution NMR and semi-empirical calculations. The fragments are Thr10-Phe11, Phe11-Thr12, Thr10-Phe11-Thr12 and Thr10-Phe11-Thr12-Ser13. The results of high resolution 1H and 13C NMR using dimethylsulfoxide and 2H2O as solvents, combined with a new method for determining dihedral angles phi and psi from 13C and 1H spin lattice relaxation times are presented. A marked inequivalence of the two Thr10,12 residues is attributed to a shielding of the the Thr10 side chain by the Phe11 aromatic ring. The calculations show the existence of extended and folded low energy conformations in the tri and tetrapeptide. Only the folded structures give the observed shielding of Thr10. A temperature study of the tri and the tetrapeptide indicates that the folded structures are energetically the most favorable conformations at room temperature in dimethylsulfoxide. Increasing temperature reduces the nonequivalence of the Thr residues towards the differences that are observed in 2H2O. A detailed comparison of 3JalphaNH coupling constants and relaxation time measurements with the calculated conformations gives in general good agreement between both approaches. It is concluded that in these linear peptides, although several quite different low energy conformations exist, some of them are predominant. The continuity of both NMR parameters and calculated low energy conformations, when going from the smaller to the larger peptides, demonstrates the existence of structural properties far from the "random conformation".


Regulation of platelet adenylate cyclase by adenosine. The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5'-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.


Superinduction of phenylalanine ammonia-lyase in gherkin hypocotyls caused by the inhibitor, L-alpha-aminooxy-beta-phenylpropionic acid. The extractable activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) and the concentration of sugar esters of p-coumaric and ferulic acids in the hypocotyls of etiolated gherkin seedlings increase upon irradiation with white light. Treatment of intact seedlings with the phenylalanine ammonia-lyase inhibitors alpha-aminooxyacetic acid and L-alpha-aminooxy-beta-phenylpropionic acid during illumination causes enhanced formation of the lyase and reduces the accumulation of hydroxycinnamic acids. Enzyme activity in excised hypocotyl segments floating on buffer increases in the dark as well as in the light, while hydroxycinnamic acids accumulate only in the light. Phenylalanine ammonia-lyase formation in the segments is inhibited by cinnamic acid and, to a lesser extent, p-coumaric acid, while it is slightly enhanced by caffeic acid and is not affected by ferulic acid. Aminooxyphenylpropionate dramatically promotes phenylalanine ammonia-lyase formation in the segments in darkness and light prevents the accumulation of hydroxycinnamic acids in the light. Aminooxyphenylpropionate does not, however, affect the time course of apparent lyase formation and decay. Cinnamic acid, the product of the lyase reaction, antagonizes the effect of aminooxyphenylpropionate. It is proposed that the reaction product(s) are involved to some extent in the regulation of the pool of active lyase in the hypocotyl tissue.


Anti-microtubular agents as inhibitors of desensitization to catecholamine stimulation of adenylate cyclase in Ehrlich ascites tumor cells. Adenylate cyclase activity of the homogenate of Ehrlich ascites tumor cells pretreated with catecholamine at 37 degrees C was not stimulated by the addition of the same catecholamine, whereas that of the cells without the pretreatment was stimulated. Such a desensitization was induced hardly at all when the pretreatment was performed at low temperature. The desensitization of adenylate cyclase activity to catecholamine stimulation was prevented by pre-pretreatment of the cells with colchicine prior to the catecholamine pretreatment. The effect of colchicine was dependent on the period of the treatment and concentration of colchicine. Vinblastine had a similar effect, whereas cytochalasin B was without effect. Thus, involvement of microtubules was suggested in the desensitization of the membrane-associated enzyme to external stimulation.


The control by vasopressin of carbohydrate and lipid metabolism in the perfused rat liver. Vasopressin-induced glucose release from the perfused livers of fed rats is diminished in the presence of insulin or following adrenal ablation. The reduced rate of glucose release following vasopressin treatment in the perfused livers of adrenalectomized rats was restored towards the control value by cortisol treatment in vivo. Vasopressin did not influence the total rate of fatty acid synthesis in the livers of fed rats perfused with medium containing glucose and two concentrations of lactate. The contribution of these precursors to hepatic fatty acid synthesis and CO2 production was similarly uninfluenced by vasopressin. Vasopressin casued a transient increase in the release of K+ by the perfused liver which was observed within 2 min of hormone administration. These results are discussed in relation to the possible mode of action of vasopressin in the liver.


Clearance of circulating desialylated thyroglobulins in the rat. Canine and rat thyroglobulins were labeled with 125I either in vitro or in vivo, and were utilized for plasma clearance studies performed with rat. The half-life of physiologically radioiodinated asialothyroglobulins was about 6 min, while that of chemically radioiodinated asialothyroglobulin was about 12 min. No marked species difference was observed in this clearance. The label which had disappeared from the blood was recovered mainly in the liver, and this uptake was blocked by the simultaneous injection of desialylated orosomucoid but not by native orosomucoid. Radiolabeled monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine were detected in the liver 17 min after intravenous injection of asialothyroglobulin labeled with 125I in vivo, suggesting the possible production of thyroid hormones in extrathyroidal tissues.


Proteoglycan aggregates in adult human costal cartilage. 1. A sequential extraction procedure using isotonic KCl and 4.0 M guanidine hydrochloride was used to solubilise proteoglycans from adult human costal cartilage under conditions minimising autolysis. Up to 28% of the total tissue hexuronate was extracted. 2. Proteoglycan fractions were prepared from the extracts by CsCl equilibrium density gradient centrifugation. 3. Each fraction exhibited distinct electrophoretic heterogeneity. 4. Proteoglycans extracted by isotonic solutions are relatively small and may be in vivo degradation products of whole molecules. The major fraction (A1) from high ionic strength extracts has a composition similar to that of A1 proteoglycans from adult human articular cartilage. A2 fractions differ and are highly enriched in protein. 5. A1 and A2 fractions from high ionic strength extracts contain proteoglycan aggregates, but to a much lesser extent than found in other cartilages like bovine nasal or porcine epiglottal cartilage. 6. The aggregates can be dissociated and a 'subunit" proteoglycan isolated by CsCl density gradient centrifugation in 4.0 M guanidine hydrochloride. 'Subunits' can reaggregate partially, when mixed with fractions of lower density from the gradient. 7. Addition of hyaluronic acid to A1GuHCl promotes a large increase in the amount of aggregate present. 8. The hyaluronic acid content of costal cartilage is not deficient compared to that of other hyaline cartilages. 9. Polypeptides with molecular weights suggesting their identity as 'link proteins' are present in A1 and A2 fractions. 10. Additional polypeptides of higher molecular weight than link proteins are also present in A1 and A2 fractions. They may represent the hyaluronic acid binding  region of proteoglycans and compete with 'whole' proteoglycans for hyaluronate in the tissue.


The turnover catabolism and excretion of folate administered at physiological concentrations in the rat. This study examines the distribution of folate-derived compounds in rat urine on a daily basis after the administration of tracer doses of radioactive [3H]pteroylglutamic acid. The identification of 10-formyldihydropteroyl-glutamate in the rat urine, prior to equilibration of the tracer, is also reported for the first time.


Sensitivity variations in the visual system, contrast resolution and eye movements. Attention is drawn to the fact that under normal visual conditions the sensitivity of the receptor units of the visual system are subject to spatial and temporal variations, and that consequently in performing pattern recognition the visual cortex has to discriminate between external luminance structure and internal sensitivity structure. It is suggested that eye movements are the method by which this discrimination is performed. In a simplified model analysis it is shown that eye movements are a suitable mechanism for this discrimination. Implications of this model for detection threshold and stabilized retinal images are discussed. A new interpretation of the adaptation to sine wave grids is given.


Transformation and relational-structure schemes for visual pattern recognition. Two models tested experimentally with rotated random-dot patterns. Two models for visual pattern recognition are described; the one based on application of internal compensatory transformations to pattern representations, the other based on encoding of patterns in terms of local features and spatial relations between these local features. These transformations and relational-structure models are each endowed with the same experimentally observed invariance properties, which include independence to pattern translation and pattern jitter, and, depending on the particular versions of the models, independence to pattern reflection and inversion (180 degrees rotation). Each model is tested by comparing the predicted recognition performance with experimentally determined recognition performance using as stimuli random-dot patterns that were variously rotated in the plane. The level of visual recognition of such patterns is known to depend strongly on rotation angle. It is shown that the relational-structure model equipped with an invariance to pattern inversion gives responses which are in close agreement with the experimental data over all pattern rotation angles. In contrast, the transformation model equipped with the same invariances gives poor agreement to the experimental data. Some implications of these results are considered.


On a conjecture concerning population growth in random environment. Discrete stochastic models are constructed and their limit diffusion processes are derived to shed light on a controversial conjecture regarding the effects of environmental variance on the asymptotic behavior of a population subject to logistic growth in random environment.


Estimation of signal and noise spectra by special averaging techniques with application to a posteriori "Wiener" filtering. This paper deals with the problem of separating the spectra of signal and noise in ensembles where the signal can be considered as an invariant component and the noise as a stationary additive background. Several methods are discussed and compared on the basis of a statistical analysis of the first two moments of the estimators for signal and noise spectra. As a consequence a procedure is proposed which provides a flexible compromise between estimation accuracy and computational effort. The application of this procedure to a posteriori "Wiener" filtering is compared with a more common, but time consuming, technique.


A model for processing of movement in the visual system. Processing of spatio-temporal information in the human visual system has been investigated thoroughly during the past decade, but is still far from being properly understood. Moreover, the theory of separation of information by means of sustained and transient channels already at the retinal level is not satisfactory, as experimental results indicate that these two types of channels span a continuum of temporal characteristics. It is however obvious, that the process of pattern recognition and velocity perception calls for their separation at some level of the hierarchy. In this communication, we extend our model of three-dimensional spatio-temporal frequency expansion in the visual system (Gafni and Zeevi, 1977) to show how velocity-information extraction channels, sensitive to direction and velocity exclusively, can be formed by simple summation of signals from well-defined sets of channels representing points in the frequency space. Correspondence of these channels to characteristics of the cortical neurons is discussed.


Energy-barrier models for membrane transport. Energy-barrier models are analyzed to find hidden assumptions and establish ranges of validity. The analysis proceeds by comparison with integrated results for model continuum membranes. The main conclusions are that a simple energy-barrier model has a wide range of validity, is remarkably accurate even when its conditions of validity are not strictly met, and is almost always superior to the analogous equations of irreversible thermodynamics. Its major limitations are a possible nonphysical divergence at high electric fields or volume flows caused by breakdown of the transition-state approximation, and the inability to treat multicomponent mixtures except in a pseudobinary (Nernst-Planck) approximation.


Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements. Ultrasonic absorption and velocity dispersion curves have been measured in the temperature induced helix-coil transition range of poly-N5-(3-hydroxypropyl)-L-glutamine in a methanol/water mixture. The results clearly reflect an effect due to the kinetics of the conformational conversion. A practically single relaxation time is observed which passes through a maximum when plotted versus the degree of transition. This maximum occurs at definitely less than 50% helix as predicted for by the theory for the comparatively short chain length involved here. The results are discussed in relation to previous theoretical and experimental findings.


On the determination of association constants of proteins by electrophoresis measurements. During the electrophoresis of a reversibly associating substance the concentration profile is determined by diffusion, migration and reaction. The influence of the diffusion can be eliminated by extrapolating the concentration profiles, taken at different times and suitably transformed, to infinite time. This leads to a profile which reflects migration and reaction only (Gilbert profile). From this the association constant can be deduced. Preliminary experiments with beta-lactoglobulin A show the feasibility of the method.


Nanosecond spectroscopy of a dimeric enzyme: plasma amine oxidase. The fluorescence dye 1-anilino-naphthalene-8-sulphonic acid (ANS) was used as a probe of non-polar binding sites in the enzyme plasma amine oxidase. Steady fluorescence measurements indicate that ANS binds to a single binding site of the dimeric enzyme with a dissociation constant of 5 microns. This binding site is different from the catalytic binding site. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 105 ns) corresponds to the rotation of a rigid dimeric macromolecule. A rotational correlation time of 120 ns was obtained with the enzyme labelled with pyrenebutyric acid. It is concluded that the dimeric enzyme does not exhibit any modes of flexibility due to independent rotation of the subunits in the nanosecond range.


High electric field dielectric studies of aqueous myoglobin solutions. High field dielectric measurements of the Piekara factor deltaepsilon/E2 have been carried out for a range of concentrations of horse heart myoglobin in water at 293K. Using the literature value for the dipole moment of myoglobin and the established theory for the classical orientational dipolar non-linear effect predicts a value of deltaepsilon/E2 one order of magnitude greater than that for water. The measured effect, however, was found to be one order of magnitude less than for water. This difference is explained as being most probably due to the existence of antiparallel molecular dipole pairs in the myoglobin solution. The possibility of a positive deltaepsilon due to a field induced conformational change of the myoglobin cannot, however, be ruled out.


Nitellopsis obtusa internodal cell birefringence change during action potential. Birefringence change during excitation was studied by using Nitellopsis obtusa. The velocity change of cytoplasmic streaming during an action potential was measured simultaneously by fluctuation analysis of transmitted light intensity. The origin of the retardation change was discussed by comparing optical retardation change to the time course of the action potential, the cytoplasmic streaming velocity change and the cell contraction. By the time course analysis of retardation change, we concluded that the change of the birefringence might be the sum of the changes of cytoplasmic flow and that of the size of length and diameter of the cell. But it is still difficult to separate the change to its components.


Suction capillaries with U-shaped opening for holding moving cells during intracellular electrical recording. The production of suction capillaries with an U-shaped cross-section is described. The capillary may be used for holding contractile cells during intracellular recording to diminish movement artifacts. In addition microelectrode penetration is facilitated by stretching the surface of the cell.


Interaction of bovine rhodopsin with calcium ions. I: the metarhodopsin I--II reaction and the regeneration of rhodopsin. The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10(-9) M (using EGTA ro EDTA) to 15 x 10(-3) M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1). The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10(-7) M calcium (by adding EGTA), the other containing 10(-3) M calcium (Table 2).


Interaction of bovine rhodopsin with calcium ions. II: calcium release in bovine rod outer segments upon bleaching. The calcium content of bovine rod outer segment (ROS) suspensions was determined by flame spectrophotometry to be about 0.2 Ca2+ per molecule rhodopsin. After bleaching of rhodopsin, a release of 0.01--0.1 Ca2+ per molecule rhodopsin from ROS into the solution was observed. These figures agree with some data in the literature (Appendix). A measured absorption increase of the Ca2+-indicator phthalein purple (10 degrees C, 562 nm, pH 9.3) occurs apparently simultaneously with the formation of metarhodopsin ii in ROS. This indicates that a light induced Ca2+-release of 12 calcium ions per photoactivated rhodopsin is coupled in time with the formation of metarhodopsin II.


Molecular mechanism for the initial process of visual excitation. III. Theoretical studies of optical spectra and conformations of chromophores in visual pigments, their analogues and intermdiates based on the torsion model. The torsion model with which we proposed to interpret the specific properties of the photoisomerization reaction of rhodopsin has been developed to apply to isorhodopsin I, isorhodopsin II and some intermediates. Based on this model, optical absorption wavelengths and oscillator strengths, as well as rotational strengths of visual pigments, analogues and intermediates at low temperatures are analyzed by varying twisted conformations of the chromophores. As a result, it was found that most of the optical data could be very well accounted for quantitatively by the torsion model. The twisting characters in the chromophore of rhodopsin are very similar to those of isorhodopsin. The obtained conformations of the chromophores are very similar in rhodopsin and its analogues, and in isorhodopsin and its analogues. Those of the chromophores of bathorhodopsin, lumirhodopsin and metarhodopsin I are similar to one another except that the conjugated chain of metarhodopsin I bends considerably when compared with the other intermediates.


Complex formation of acridine orange with single-stranded polyriboadenylic acid and 5'-AMP: cooperative binding and intercalation between bases. The binding of acridine orange to single-stranded polyribonucleic acid at low polymer to dye ratios exhibits cooperative behavior of the kind observed with other simple polyanions. It is thus attributed to electrostatic interaction between polymer and stacked dye molecules. At higher polymer to dye ratios, however, distinct deviations from the predictions of the basic theory occur. These are interpreted by additional non-cooperative binding of acridine orange to the bases of the polymer subunits owing to dye-base stacking. This effect is studied also with 5'-AMP monomers where it likewise leads to complex formation. Both systems are investigated experimentally by means of the changes produced in the dye spectrum. Based on quantitative analyses the equilibrium constants of both systems are evaluated and discussed. They indicate a sandwich-type of intercalation of dye between two bases of the single-stranded polymer.


Theory of transport noise in membrane channels with open-closed kinetics. A theoretical approach to transport noise in kinetic systems, which has recently been developed, is applied to electric fluctuations around steady-states in membrane channels with different conductance states. The channel kinetics may be simple two state (open-closed) kinetics or more complicated. The membrane channel is considered as a sequence of binding sites separated by energy barriers over which the ions have to jump according to the usual single-file diffusion model. For simplicity the channels are assumed to act independently. In the special case of ionic movement fast compared with the channel open-closed kinetics the results agree with those derived from the usual Master equation approach to electric fluctuations in nerve membrane channels. For the simple model of channels with one binding site and two energy barries the coupling between the fluctuations coming from the open-closed kinetics and from the jump diffusion is investigated.


Soil degradation of trifluralin: mass spectrometry of products and potential products. The examination of soil which has been treated with trifluralin (TREFLAN alpha,alpha,alha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) has resulted in the identification of 28 degradation products. This paper presents the mass spectra of these products and a number of related compounds which were suspected products but were not found in trifluralin-treated soil. The mass spectra of the N,N-dialkylamines, with one exception, show major peaks due to amine alpha-cleavage and due to amine alpha-cleavage followed by carbon-nitrogen bond fission with rearragement of a hydrogen atom. Compounds having nitro groups yield mass spectra containing peaks due to intra-ionic oxidation by the nitro groups. In some cases, propionyl ions formed by this process are major ions in the spectra. Azo and azoxybenzenes resulting from condensation of two trifluraline moieties through nitro-nitrogens fragment mainly through the nitrogen-nitrogen bond.


Active hydrogen by chemical ionization mass spectrometry. A means of determination of a ctive hydrogen of hydroxyl, carboxyl, sulfhydryl, amino, amido and sulfonamido groups by chemical ionization mass spectrometry using ammonia and trideuterioammonia as reagent gases is described. The method is based on exchange of active hydrogen for deuterium occurring during the chemical ionization process using trideuterioammonia, with comparison of m/z for ammonium adduct [M + NH1]+ and trideuterioammonium adduct [M--nH + nD + ND4]+, or of protonated molecular ion [M + H]+ and deuteronated molecular ion [M--nH + nD + D]+ yielding the number of active hydrogens. Applications have been made to several classes of biologically important compounds.


Detection of the in vivo conversion of 2-pyrrolidinone to gamma-aminobutyric acid in mouse brain. Labeled gamma-aminobutyric acid was detected in mouse brain following intravenous injections of deuterium labeled 2-pyrrolidinone. [2H6]Pyrrolidinone was prepared by the reduction of [2H4]succinimide with lithium aluminum deuteride. Quantification was accomplished by a gas chromatography mass spectrometry assay method. gamma-Aminobutyric acid and internal standard, 5-aminovaleric acid, were converted to volatile derivatives by treatment with N,N-dimethylformamide dimethyl acetal. Quantitative estimates were derived from peak area measurements obtained from monitoring the parent ions of the gamma-aminobutyric acid and internal standard derivatives by repetitive scanning during the GC run. The conversion of pyrrolidinone to gamma-aminobutyric acid may provide a method for labeling central gamma-aminobutyric acid pools.


Quantitative analysis of tiamenidine in human plasma by gas chromatography mass spectrometry of a dibenzyl derivative. A gas chromatography mass spectrometry method has been developed and evaluated for the quantitative analysis of tiamenidine in plasma. Tiamenidine and internal standard are extracted from basified plasma, converted to dibenzyl derivatives by reaction with benzyl bromide and potassium t-butoxide in the presence of 18-crown-6 ether prior to analysis by selected ion monitoring. The method can be used over the range 0.2--10 ng ml-1 with a coefficient of variation of better than 20% at 1 ng ml-1.


The gas chromatographic mass spectrometric analysis of the new antitumor drug indicine-N-oxide utilizing a novel reaction accompanying trimethylsilylation. Indicine-N-oxide was analyzed by gas chromatography mass spectrometry with nanogram sensitivity after trimethylsilylation. Two different products were produced by altering the conditions of this reaction. Mass spectral evidence is presented to show that one of these was the expected trisubstituted pyrrolizidine product while the other was a trisubstituted pyrrole. The latter derivative is useful for distinguishing between indicine-N-oxide and indicine which dies not form this novel product under the same conditions. Analogous pyrrole and pyrrolizidine products were formed from heliotrine-N-oxide, a compound that can serve as an internal standard from measuring indicine-N-oxide and its metabolites in biological samples. A method for purifying such samples by strong cation exchange chromatography prior to derivatization is also discussed.


Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water--organic solvent systems. The hydroxysteroid dehydrogenases: beta-HSDH, 20 beta-HSDH, and 3 alpha-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40--60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, beta-HSDH maintained 60% of its original activity two months after continuous use in the water--ethyl-acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki greater than 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water--organic solvent systems.


Immobilized glucose oxidase--catalase and their deactivation in a differential-bed loop reactor. Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.


Nitrogenase activity of immobilized Azotobacter vinelandii. As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotobacter vinelandii was undertaken. Immobilization was acaccomplished by adsorption onto an anionic exchange cellulose (Cellex E) with loadings as high as 10'' cells/g resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenase activities as high as 4200 nmol/min g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.


High-resolution flow-zonal centrifuge system. A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses.


Mathematical model of cell growth and phosphatase biosynthesis in Saccharomyces carlsbergensis under phosphate limitation. The rate kinetics of growth and acid phosphate formation in the batch culture of Saccharomyces carlsbergensis LAM 1068 was studied under varying degrees of phosphate limitation. The mathematical model that was developed is concerned with the time lag for exponential growth, the biphasic growth on a substrate (glucose) and its product (ethanol), sustained growth on conservative phosphate, and the derepression of acid phosphatase. The numerical calculations using appropriate parametric constants successfully described the variation in the cell mass, glucose, ethanol, and inorganic phosphate concentrations, and the enzyme activity of acid phosphatase during aerobic growth of S. carlsbergensis under five different conditions of phosphate starvation. A simulation study revealed that the optimum initial phosphate concentration in the medium giving a high productivity of acid phosphatase was 2.0 mg phosphorus/g glucose liter.


[Activity in the myelin fibers of the cat cutaneous nerve in response to heating]. The collision method combined with some techniques improving the signal-to-noise ratio showed that the activation in the nerve fiber group A gamma 1, A delta, A delta 2 and in "mixed" fibers group was changed by hairy skin heating. A relatively great number of nerve fibers of these groups were activated and only an insignificant part of them inhibited their activity in response to the cutaneous receptor heating. A spontaneous activity increase and skin relaxation under heating. were shown.


[Participation of the contractile activity of the skeletal musculature in systemic circulatory reactions to orthostatic action]. Under orthostatic test (OT) the elimination of the skeletal muscle contractile activity with a myorelaxant sharply elevated the initial hypotension and decreased considerably the compensatory restoration of the arterial pressure (AP) in anesthetized cats. Reaction of the cardiac output in OT changed insignificantly. The AP elevation synchronous with the motor reactions, eliminated by the myorelaxant, was observed. As revealed by the method of artificial circulation (bypass of the left ventricle), the compensatory vasoconstriction of the resistance vessels was eliminated by the myorelaxant under OT. In some experiments myorelaxation evoked a significant elevation of the blood pooling in the capacitance vessels.


[Characteristics of the reflex to stretching in spinal cord injury]. This work is aimed at ascertaining the correlation in the state of spinal cord reflex apparatus in spinal patients with the presence or absence of clonus. The value of electrical response of m. Soleus to passive flexion of the foot at different speed was studied. Clonus was observed in 10 patients (the first group), and absent in 12 (the second group). Ten normal persons served as controls. The curves of the response value distribution displayed a marked difference in individual test groups. The first group was characterized by the mode shift to the right, and the second group--to the left, in comparison with the control. This was true of all angular speeds tested. The threshold speed proved to be statistically significantly increased in the second group as compared with the normal; in the first group it was about the normal. The activity in the shin muscle and the capacity to move and even to walk was observed in the majority of the patients of the first group and only in a few patients of the second group. The data obtained showed that in patients with clonus the state of the spinal cord reflex apparatus was more normalized and the clinical condition was better than in those without clonus.


[Change in the cholesterol and triglyceride content in the blood in self-stimulation and avoidance reactions]. The changes in the blood serum cholesterol and triglyceride content were studied or rabbits of both sexes during reactions of self-stimulation and avoidance. Self-stimulation was accompanied by a significant decrease in the blood serum cholesterol and triglyceride content. During the reaction of avoidance the character of the cholesterol changes varied. The reaction of avoidance of the "agression" type was accompanied by elevated blood serum cholesterol content, whereas the reaction of the "fear" type was associated with a decrease in its content. The maximal changes in the cholesterol and triglyceride content as compared to the initial level were observed in all types of the reactions 15--30 minutes following stimulation.


[Effectiveness of terrilytin in the treatment of experimental pulmonary vascular thrombosis]. The thrombolytic effect of Aspergillus terricola proteinase terrilytin was studied in 24-hour experimental pulmonary vascular thrombosis of rabbits. The drug was dissolved in polyvinylpyrrolidon and injected into the blood flow in doses of 175 and 220 PU/kg by two routes: locally (into the thrombosed vessel) and systemically. The drug produced a high thrombolytic effect irrespective of the administration and dosage; however, the effect was more marked in local infusion of terrilytin in a dose of 220 PU/kg. No side effects were observed.


[Role of vacuolar apparatus overload in lysosomal changes in liver cells in acute toxic hepatitis]. Physical and chemical properties of the rat liver lysosomes with single Triton WR 1339 overloading were studied during the administration of a detergent to intact rats and those with acute toxic hepatitis. Administration of the latter to intact animals was accompanied by a reduction of the floating density of the particles, solubilization of the lysosome enzymes and by increased fragility of the particles in the hypotonic medium. Lysosomes of the hepatocytes in rats with toxic hepatitis also displayed signs of overloading of the vacuolar apparatus with the preparation administered. The most pronounced solubilization of the lysosomal enzymes beta-galactosidase, acid RNA-ase, cathepsin D--was noted in case of combined action of CCl4 and Triton WR 1339 24, 48, 72 hours and 7 days after the CCl4 poisoning. Possible consequences of overloading of the vacuolar apparatus of the rat hepatocytes are discussed.


[Effect of psychotropic substances on dopamine uptake by synaptosomes and glial cells]. Dophamine-H3 uptake by synaptosomes and glial cells of the cerebral cortex in rabbits and by the synaptosomes of the cerebral cortex in rats was studied. KM value of the dophamine-H3 uptake was found to be the same (0.075 +/- +/- 0.01 micrometer) in case of synaptosomes and glial cells. The rate of dophamine-H3 uptake by the rabbit cerebral cortex synaptosomes was twice that of the gial cells (as compared by protein). Among the psychotropic agents studied the most active symaptosome uptake inhibitors were phenamine and cocaine; they were less active in respect to glial uptake. Both types of uptake were inhibited by neuroleptics and antidepressants to the same degree. Results of this work conformed to the view on the participation of the strial dophaminergic link in the action mechanism of psychostimulants.


[Cerebral blood flow reactions to hypo- and hypercapnia during indomethacin inhibition of prostaglandin biosynthesis]. Acute experiments on cats demonstrated a suppression of the cerebral vessels reaction to hypercapnia under condithacin, while the reaction to hypocapnia persisted. It is assumed that the effects of hypo- and hypercapnia on the cerebral vessels were realized by different mechanisms, i. e. reduction of prostaglandin concentration decreased the cerebral vessels sensitivity to hypercapnia and increased their sensitivity to hypocapnia.


[Antiarrhythmic and electrophysiological properties of etmozin and its diethylamine analog]. Effects of antiarrhythmic drug etmosin and its diethylamine analogue (etmosin DAA) were compared in dogs with the ventricular rhythm disturbances induced by coronary artery ligation according to Harris' method. As demonstrated, both drugs stopped ventricular rhythm disturbances, but etmosin DAA had a more rapid and prolonged effect. Electrophysiological properties of etmosin and etmosin DAA were studied by the method of potential fixation on trabeculae of frog atria. Both drugs proved to reduce rapid sodium inflow, etmosin DAA acting more intensively and longer. Taking into account the high antiarrhythmic activity of etmosin DAA it is believed that this drug had good prospects for further investigation.


[Pharmacogenetic aspects of the immunodepressive action of cyclophosphamide]. The alkylating and immunodepressive activity of the serum of CBA, BALB/c and DBA/2 mice was studied after the cyclophosphamide administration. The interstrain differences between the indices under study were revealed; no direct correlation was shown between them. DBA/2 mice were found to be the most sensitive to the immunodepressive action of cyclophosphamide, and had the highest blood serum immunodepressive activity.


[Pathogenetic significance of the immediate and delayed types of hypersensitivity reactions in the dynamics of nephritis]. Complex study of the immunologic status of rats from the first days after the injection of nephrotoxic serum up to formation of chronic renal failure in comparison with functional and morphological characteristics of the disease confirmed the hypothesis on the phasic development and different pathogenetic role of the hypersensitivity reactions of the immediate and delayed types during nephritis. Signs of the immediate hypersensitivity predominated during the initial stages of the disease; realization of this hypersensitivity is indicated both by the liberation of the mediators of the pathochemical phase and by the change of the renal processes. The development of delayed hypersensitivity reactions plays the basic role in the pathogenesis of chronic nephritis.


[Change in the correlation of 2 RNA precursor synthesis pathways in the tissues of animals with rapidly growing hepatomas]. In the spleen, thymus and liver of mice and rats carrying transplantable rapidly growing hepatomas a shift between the de novo and "salvage" pathways of the pyrimidine RNA nucleotides synthesis in favour of the de novo one was observed. The phenomenon was found to be the manifestation of a drastically diminished uridine utilization by the host tissues, this precursor being preferentially and avidly used by the tumour for its own RNA synthesis. The above findings are regarded as the result of a successful competition of the tumour with the host tissues for vital metabolites representing one of the forms of the systemic effects of malignant neoplasms.


[Lipid peroxide metabolism in chemical carcinogenesis]. Lecithin and kephalin content in the microsomes and mitochondria of the rat liver, and also the activity of enzymatic and nonenzymatic systems of the phospholipid peroxidation showed a sharp change following 3,4-benzpyrene injection. Carcinogenesis is accompanied by significant changes in the lipid peroxides content and in the activity of the enzyme utilizing lipoperoxides (glutathion peroxidase, glutathion reductase). Accumulation of lipid peroxides in the rat liver in carcinogenesis was connected with disturbed balance of the generating systems and detoxication of lipid peroxides in the tumour is attributed to the high activity of the protective enzymatic systems and serves as a reflection of the adaptation mechanisms directed to the maintenance of a high pool of proliferating cells in the tumour.


[In vitro production of hemopoietic stem cells: washings from organ cultures of embryonic liver]. Hemopoietic cells including CFUs could be washed off from the organ culture of fetal liver periodically for 4 weeks. Under the cultivation conditions employed this treatment did not reduce the CFUs content of the culture essentially; thus, the washings off could be used to elevate the CFUs yield per culture.


[Effect of the inhibition of a synthesis processes in the S-period on the subsequent course of mitosis in a synchronized Chinese hamster cell culture]. The transcription of all nuclear RNA and rRNA was inhibited in the synchronized Chinese hamster cell culture by actinomycin D and protein synthesis was suppressed by pyromycin during the first and the second parts of the S period of the mitotic cycle. A decrease of the mitotic cell activity was revealed during the first and second waves of cell proliferation after the synchronization irrespective of the time of the antibiotic administration. At the same time there was noticed an increase of such pathological mitoses as C-mitoses and chromosome scattering in the metaphase, whereas suppression of rRNA synthesis determined the delay of cell going out of mitosis (an increase of the anaphase and telophase cell portion) mostly correlated with such type of pathological mitoses as chromatid and chromosome bridges. The protein synthesis depression during the first half of the S-period failed to influence the mitotic regimen of the cell culture during the first, nearest division, but suppressed considerably the cell proliferation during the distant proliferation wave. It is suggested that the cells entered the S-period having in store the proteins for initiation and supporting the genom reduplication at least during the first stage of this process.


Responses of small rabbit pial arteries in vitro. An in vitro method for measuring changes in isometric force of vessels as small as 100 microns in outer diameter was used to determine whether the adrenergic mechanism of small pial arteries is the same as the large arteries of the circle of Willis. Fluorescence histology demonstrated the rich noradrenergic innervation of small branches of the middle cerebral artery. In spite of this rich innervation, contractile responses to transmural stimulation (TNS) were much smaller than maximum responses to histamine. An insensitivity to exogenous norepinephrine was shown which could account for this small response to TNS. In the presence of low concentrations of histamine, responses to TNS were increased. Thus, small pial arteries have characteristics similar to larger cerebral arteries.


Changes in rat aortic actomyosin content with maturation. It has been reported by several investigators that the maximum active force generating ability (i.e., force/tissue cross-sectional area) of the aorta increases as the animal matures and then declines later in life. The purpose of this work was to determine if changes in the actomyosin content of the aorta occur during maturation which could contribute to the observed changes in force generating ability. The actin and myosin contents of rat thoracic aorta obtained from animals 3, 5, 7, 16 and 43 weeks of age were determined by quantitative gel electrophoresis, and were normalized with respect to tissue mass and protein and DNA contents. The results indicate that the amount of actomyosin (i.e., actin + myosin heavy chains) per tissue mass, protein and DNA increases rapidly during the first 5 weeks after birth and then remains constant. These observations suggest that the increasing actin and myosin heavy chain content of aorta observed early in life could explain in part the increasing force generating ability reported during this period, but that a loss of contractile material is not responsible for the reduction in force generating ability seen later in life.


Comparison of holdings of NLM (CATLINE) with those of resource libraries. The collection development practices of the National Library of Medicine (NLM), with the goal of comprehensive acquisition of biomedical monographs, are compared with those of the resource libraries of the TALON (Region IX) Regional Medical Library. Holdings of two resource libraries in the TALON region, The University of Texas Health Science Center at San Antonio and The University of Texas Medical Branch at Galveston, and of the TALON Union Catalog of Monographs were compared with the NLM CATLINE data base for four subject classes and selected imprint years. Foreign-language coverage is lacking in Region IX, with English-language coverage is lacking in Region IX, with English-language coverage ranging between 70 and 88% of titles listed in CATLINE. Absent English-language material tends to be ephemeral or otherwise out of scope for the resource libraries. Between 7.1 and 18.8% of monographs acquired in each subject class by the two recource libraries are lacking in CATLINE; this represents between 2 and 8% of the CATLINE titles for each class.


Certification of medical librarians, 1949--1977 statistical analysis. The Medical Library Association's Code for Training and Certification of Medical Librarians was in effect from 1949 to August 1977, a period during which 3,216 individuals were certified. Statistics on each type of certificate granted each year are provided. Because 54.5% of those granted certification were awarded it in the last three-year, two-month period of the code's existence, these applications are reviewed in greater detail. Statistics on each type of certificate granted each year are provided. Because 54.5% of those granted certification were awarded it in the last three-year, two-month period of the code's existence, these applications are reviewed in greater detail. Statistics on MLA membership, sex, residence, library school, and method of meeting requirements are detailed. Questions relating to certification under the code now in existence are raised.


Evolution of a veterinary medical library. Planning a new library and developing a book and journal collection for the College of Veterinary Medicine at Iowa State University are described. The Veterinary Medical Library is a self-contained unit (6,800 square feet) for print material within the Veterinary Medical Building. Seating for 140 patrons is available. The collection is designed to provide basic materials for teaching and research in veterinary and comparative medicine. Indexing and abstracting tools permit access to local, state, and national resources as well. At present the collection totals over 17,000 volumes and over 500 serial titles. A working collection of 25,000 volumes will be maintained in the Veterinary Medical Library, and the University Library will continue to function as the principal backup source.


Successful replantation of a totally avulsed scalp. A case of a totally avulsed scalp was replanted successfully by microvascular anastomosis. Though the avulsed part was heavily crushed, which resulted in a central wound, the appearance of a "necrotic" area, haematomas and persistent serous exudation after the restoration of circulation, the whole scalp survived.


The ineffectiveness of magnesium sulphate and heparin in preventing thrombosis following microvascular anastomoses. Neither magnesium sulphate nor heparin was found to have a significant local antithrombotic effect on microvascular anastomoses.


Patency rates in end to side anastomoses in the rabbit. It is suggested that more widespread use of the end to side microvascular suturing technique should be made, especially in arteries. A simple experimental practice model in the rabbit is suggested. Patency rates comparable to end to end anastomosis can be achieved.


Nasal injuries: their pathogenesis and treatment. The treatment of a fractured nose by manipulation is criticised. A "perfect" result was achieved in only one-third of 40 cases, a finding mirrored in the literature. The anatomy of nasal fractures produced experimentally in 15 cadavers is described. A recurring pattern of septal fracture and displacement is identified as the cause of the poor results of manipulation. Release of locked septal displacement by a defined local submucous resection, as part of the primary treatment, has given greatly improved results.


Hot water burn of the abdomen after lipectomy. Abdominal lipectomy results in a zone of impaired sensation below the level of the umbilicus. Patients should be warned of the significance of this sensory loss.


Release of prostaglandins during contraction of the human umbilical vein on reduction of temperature. Flow rate was measured through the vein of the human isolated umbilical cord perfused at constant pressure (40 mmHg) at 37.5 degrees C and 20 degrees C. At the latter temperature the flow was decreased by 50.9% when compared with a mean of 201 ml/min at 37.5 degrees C indicating venospasm. Indomethacin (10 microgram/g) effected a highly significant reduction in the venous spasm caused by lowering the temperature. After indomethacin pretreatment, changing the cord temperature from 37.5 degrees C to 20 degrees C caused a mean decrease in flow of only 3.1%. When the effluent from the vein was passed over rat isolated stomach fundus and colonic strips, cooling of the cord was accompanied by contractions of the isolated tissues characteristic of prostaglandins. These results suggest that prostaglandins are involved in temperature-induced closure of the human umbilical vein after birth.


Role of 5-hydroxytryptamine in the vasoconstrictor action of compound 48/80 in the rat femoral vasculature. 1. The effect of compound 48/80 on the rat femoral vasculature was examined by means of a cross-circulation technique. 2. Intra-arterial injection of increasing doses (1.5 to 15 microgram) of compound 48/80 caused dose-dependent vasoconstriction preceded by transient vasodilatation. The vasoconstriction was significantly reduced in reserpine-treated preparations and converted to vasodilatation by methysergide. 3. When the vasoconstrictor response to a large dose (300 microgram) of adenosine was abolished by repeated administration, the response to compound 48/80 remained unaltered. 4. The present results indicate that the femoral arterial vasoconstriction by compound 48/80 in the rat is mediated by the release of 5-hydroxytryptamine (5-HT); this 5-HT may be liberated from a different storage site from that released by adenosine.


Comparative pharmacological and histochemical evidence for purinergic inhibitory innervation of the portal vein of the rabbit, but not guinea-pig. 1 Intramural nerve stimulation elicited a powerful relaxation of the longitudinal muscle of the rabbit portal vein in the presence of atropine and guanethidine, but not of the guinea-pig portal vein.2 Intramural nerve stimulation of the rabbit portal vein produced a 13 fold increase in release of (3)H-adenyl compounds after preloading with [(3)H]-adenosine. About 50% of this release was abolished by guanethidine. All release was abolished by tetrodotoxin. No significant release of radioactive compounds was observed during intramural nerve stimulation of the guinea-pig portal vein in the presence of guanethidine, although there was a 6 fold increase in release of radioactivity in the absence of drugs.3 Histochemical studies using quinacrine, which binds ATP showed a fine fluorescent nerve plexus, nerve bundles, and ganglion cells in the rabbit portal vein, but not in the guinea-pig portal vein. This plexus was still present after chemical sympathectomy with 6-hydroxydopamine.4 Adenosine 5'-triphosphate (ATP) relaxed the rabbit portal vein, but usually produced a biphasic response, consisting of a contraction followed by a relaxation, of the guinea-pig portal vein.5 Prostaglandins E(1) and E(2) caused contraction of the rabbit portal vein. Indomethacin, a prostaglandin synthesis inhibitor, potentiated the relaxations of the rabbit portal vein produced by both non-adrenergic, non-cholinergic nerve stimulation and ATP.6 High concentrations of antazoline and phentolamine, which antagonize purinergic responses in the guinea-pig taenia coli, caused a loss of basal tone so that it was not possible to assess their effects on the responses of the portal vein to either non-adrenergic, non-cholinergic nerve stimulation, or ATP.7 Comparison of the results on the portal vein of the rabbit and guinea-pig provides support for the view that: (i) quinacrine fluorescence can be used to localize purinergic nerves and that the rabbit portal vein is supplied by these nerves; (ii) ATP is released from adrenergic nerve fibres, although, based on histochemical analysis, about 3 to 7 times less than is released from purinergic nerve fibres.


Effects of substance P injected into the substantia nigra. 1 Behavioural and biochemical effects of substance P (SP, 1 to 10 mug) administered in a small volume to discrete areas of the rat's brain were studied by means of a refined microinjection technique.2 SP injected unilaterally into the zona reticulata of the substantia nigra elicited dose-dependent contraversive circling and an increase in dopamine turnover in the ipsilateral striatum. SP applied to the zona compacta or zona lateralis, or to the medial lemniscus, evoked ipsiversive turning with a fall in dopamine turnover and a rise in 5-hydroxytryptamine (5-HT) turnover in the corresponding striatum.3 In both cases the onset of turning was immediate, reached a peak at about 5 min and lasted for 10 min. Both types of behaviour were blocked by haloperidol and exaggerated by nialamide.4 Unilateral injections of SP given into the crus cerebri, zona incerta, caudate nucleus, putamen or globus pallidus did not modify the animal's behaviour.5 In rats pretreated with apomorphine or amphetamine, SP induced contraversive circling which was followed by locomotion in the opposite direction.6 Turning responses to a second dose of SP were diminished at 3 h and reproducible at 24 h after the first injection.7 Bacitracin (50 ng) injected into the zona reticulata caused ipsiversive turning. Larger intranigral doses of bacitracin (10 mug), as with intracisternal SP (10 mug), evoked ;barrel rotation'.8 No changes in the free concentrations of aspartate, glutamate, gamma-aminobutyric acid, glycine or alanine were detected in any brain region following an intracisternal injection of 10 mug SP, although glutamine levels were elevated throughout the brain 30 to 60 min later.


Presynaptic and postsynaptic contributions to ontogeny of sympathetic control of heart rate in the pre-weanling rat. 1. The dependence of heart rate on sympathetic nerve development and on ontogeny of cardiac responses to sympathetic neurotransmitters was evaluated in neonatal rats by use of the ganglionic blocking agent, chlorisondamine, and the beta-adrenoceptor agonist, isoprenaline. 2. Basal heart rates of anaesthetized 4, 7 and 11 day old rats were the same as in adult rats, but the heart rate in 16 day old rats (an age at which sympathetic tone appears to be elevated) was significantly higher. 3. Chlorisondamine failed to lower heart rate significantly in 4 or 7 day old rats, but lowered the rate by 50 to 80 beats per min in the older animals and eliminated the difference in rate between 16 day old and the other ages; atropine had little or no effect on heart rate. 4. Heart rate responses to isoprenaline were lower in 4, 7 and 11 day old rats than they were in adults but were indistinguishable from them by 16 days of age, indicating that the heart is less sensitive to beta-adrenoceptor stimulation early in neonatal life. 5. The ontogeny of sympathetic control of heart rate in the rat depends upon maturational changes in both presynaptic and postsynaptic elements.


Prostaglandin biosynthesis by guinea-pig skeletal muscle in vitro and the effect of oestradiol-17 beta and progesterone. 1. The effect of oestradiol and progesterone on prostaglandin biosynthesis by guinea-pig skeletal muscle in vitro has been investigated. 2. At high concentrations, oestradiol-17 beta (100 microgram/ml) and progesterone (500 microgram/ml) increased prostaglandin E and prostaglandin F synthesis (measured by radioimmunoassay). 3. It is suggested that these steroids are unlikely to be found in vivo at levels sufficient to influence prostaglandin synthesis by skeletal muscle in physiological and pathological situations.


[3H]-Quinuclidinyl benzilate binding to muscarinic receptors in rat brain: comparison of results from intact brain slices and homogenates. 1. The binding of [3H]-( +/- )-quinuclidinyl benzilate ([3H]-( +/- )-QNB) to muscarinic sites in rat brain slice and homogenate preparations was compared. 2. Evidence is presented in support of the view that only the (-)-enantiomer of QNB binds with high affinity to muscarinic sites. 3. The Kd value for [3H]-(-)-QNB binding in slices was eight times higher than that measured in homogenates. 4. Similarly, the potencies of various muscarinic ligands as inhibitors of [3H]-(-)-QNB binding were consistently lower in slices than in homogenates. 5. It is proposed that the results may reflect differences in the binding properties of muscarinic receptors in intact tissue slice and homogenate preparations.


Ventilatory depression in naive and tolerant rats in relation to plasma morphine concentration. 1 The disappearance of morphine from specially formulated pellets containing 75 mg morphine base was measured for 10 days after they were implanted into adult rats; the morphine content decreased at a rate of 5 mg pellet daily.2 From the 2nd to the 6th day of implantation the plasma morphine concentration increased but by the 10th day had declined to only one half the concentration found on day 6.3 Six and 24 h after the pellets were removed from 6 day implanted animals the plasma concentration of morphine amounted to only one quarter to one sixth of the amount in the plasma, respectively, of animals with pellets intact.4 The pulmonary minute volume of naive and implanted rats was depressed by morphine in proportion to the plasma morphine concentration. Less depression was produced by intravenous morphine in the implanted rats than in the naive animals; the greater morphine tolerance displayed by the implanted animals could be shown by the third day of implantation and appeared to be maintained to the 10th day.5 The pulmonary minute volume of implanted rats on the 6th day was much less than the pulmonary minute volume of naive rats. Six and 24 h after the pellets were removed the pulmonary minute volume increased as the plasma morphine concentration decreased.6 The effects on the pulmonary minute volume produced by the slow release of morphine from the implanted pellets was not changed by the development of tolerance while the effects of morphine produced by rapid injection were diminished by the development of tolerance; the different effects of morphine are accordingly linked to the mode of administration.7 We conclude that the action of morphine on the pulmonary minute volume in tolerant rats following rapid injection is fundamentally different from its action following its slow release from implanted pellets, possibly due to differences in access to an undefined neuronal site.


Time-dependent stimulatory and inhibitory effects of prostaglandin E1 on exudative and tissue components of granulomatous inflammation in rats. 1 The effects of prostaglandin (PGE(1)), following local administration during different phases of developing sponge-induced granulomata, were studied in normal and essential fatty acid deficient (EFAD) rats.2 In normal rats, a single dose of 1 mug PGE(1) on implantation (day 1) increased exudate production without altering total leucocyte counts after 6 h and stimulated granulomatous tissue formation after 8 days.3 Repeated daily administration of the same dose of PGE(1) on days 1 to 3 had no effect, while administration on days 4 to 7 (i.e. when tissue growth is already in progress) inhibited granuloma formation.4 In EFAD rats, which are known to produce only very small amounts of endogenous prostaglandins, acute (6 h) exudate formation was unaffected by 0.05 mug PGE(1). However, early stimulatory and later inhibitory effects of 0.05 mug PGE(1) per day were obtained on the granulomatous tissue, similar to those obtained with the 20 fold higher dose in normal rats.5 The early stimulatory action of PGE(1) on granulomatous tissue formation was enhanced, in normal rats, by concomitant administration of 10 mug theophylline. This latter compound did not influence the later inhibitory effect of PGE(1).6 These results indicate that PGE(1) exerts either pro- or anti-inflammatory actions on the proliferative (tissue) component of the inflammatory process, depending on the time of administration. While the stimulatory effect following early administration may have been secondary to an initial cyclic adenosine 3',5'-monophosphate-mediated, vascular response, such a mechanism is unlikely to have been responsible for the later anti-inflammatory action of PGE(1).7 The implications of these results are discussed in relation to the postulated negative-feedback role of endogenous PGE in chronic inflammation.


On the mechanism of pancuronium-induced supersensitivity to noradrenaline in rat smooth muscle. 1. Pancuronium bromide (5 x 10(-5) M) caused supersensitivity to noradrenaline in the rat isolated vas deferens and hepatic portal vein. This supersensitivity was manifest as a parallel leftward shift of the dose-response curve for noradrenaline with no alteration of the maximum response to the agonist. 2. Pancuronium did not potentiate the response of the vas and portal vein to St 91, an alpha-adrenoceptor agonist which is not a substrate for Uptake1. 3. Pancuronium did not potentiate the response of the vas to CaCl2. 4. In vasa deferentia made supersensitive to noradrenaline by treatment with cocaine (1 x 10(-5) M) pancuronium induced no further potentiation of the response to noradrenaline. However, the supersensitivity to noradrenaline induced by pancuronium alone was augmented by the addition of cocaine. 5. Histofluorescence studies showed that pancuronium inhibited neuronal uptake of alpha-methyl noradrenaline in various noradrenergically-innervated tissues from reserpine-treated rats. 6. This study, with support from the literature, suggests that pancuronium induces noradrenaline supersensitivity by blockade of Uptake1.