Control of fructose and citrate synthesis in guinea pig seminal vesicle epithelium. Glucose utilization, biosynthesis of fructose and citrate, and certain aspects of energy metabolism were studied in a homogeneous preparation of mating guinea pig seminal vesicle epithelium. Under aerobic conditions, ATP:ADP ratios between 6 and 7 were maintained attesting to the viability and metabolic integrity of the preparation. There were multiple differences between seminal vesicle epithelium and smooth muscle on comparison of concentrations of 19 different metabolites including the adenine nucleotides. In seminal vesicle, glycolysis was rapid under anaerobic conditions (approximately 25 mumol times g-1 times hour-1 during the first 3 min) and also occurred under aerobic conditions (approimately 5 mumol times g-1 times hour-1). Anaerobically, the concentration of fructose diphosphate increased 2-fold and that of fructose 6-phosphate decreased to one-third of control values, consistent with regulation at the level of phosphofructokinase. ATP and total adenine nucleotides decreased rapidly and, by 3 min, had attained steady state values of about one-third and one-half of control values, respectively. Under aerobic conditions, the rate of fructogenesis increased with increasing concentrations of added glucose, reaching maximum (3 mumol times g-1 times hour-1) at 8 to 10 mM glucose and half-maximum at 2 mM glucose. Simultaneously, sorbitol synthesis occurred at rates that were similarly influenced by the concentrations of added glucose. The highest rate of fructogenesis (6.5 mumol times g-1 times hour-1) occurred during aerobic incubation immediately after a period of anaerobic incubation: exogenous substrate was not required. This could be prevented by addition of D-glyceraldehyde which was converted to glycerol stoichiometrically because of its preferential (versus glucose) reduction by aldose reductase. Our data are consistent with the sorbitol pathway of Hers as the major route of fructose biosynthesis. The rate of citrate syntehsis increased with increasing concentrations of added pyruvate. The maximum rate (3.4 mumol times g-1 times hour-1) was achieved with 2 mM pyruvate. Added glucose failed to support citrate synthesis to the same extent as did added pyruvate. The highest rate of citrate synthesis (8.0 mumol times g-1 times hour-1) occurred simultaneously with the highest rate of fructogenesis (after the anaerobic-aerobic transition). Exogenous substrate was not required.


Interrelation between glycosidic torsion, sugar pucker, and backbone conformation in 5'-beta-nucleotides. A 1H and 31P fast Fourier transform nuclear magnetic resonance investigation of the conformation of 8-aza-5'-beta-adenosine monophosphate and 8-aza-5'-beta-guanosine monophosphate. Hydrogen-1 and phosphorus-31 nuclear magnetic resonance spectra of 5'-AMP, 5'-GMP, 8-aza-5'-GMP, adenosine, 8-aza-adenosine, and guanosine were obtained at biological pH values at a concentration sufficiently low to obtain information regarding their intramolecular solution conformation. The spectra were analyzed by computer simulation. It is shown that all of the above nucleosides and nucleotides have a flexible molecular framework in aqueous solution, 5'-AMP and 5'-GMP showing preference for anti(2-E in equilibrium 3-E)gg-g'g' conformations and the 8-aza analogs showing preference for (syn in equilibrium anti)-(2E in equilibrium 3-E) - g/t-g'g' conformations. In addition, aza substitution causes increase in 3-E sugar populations and in the populations of g'/t' conformers. It is argued that in the 8-aza analogs repulsive electrostatic interactions would prevail between --N (see article) at the 8 position and the negatively charged phosphate group, if the molecule existed in the anti-gg orientation. Such electrostatic repulsions can be relieved by rotating the C(4')--C(5') bond from gg to g/t orientations as well as by torsional variation about the glycosidic linkage from anti to syn conformation. Rotation from anti to syn orientation, for steric and electrostatic reasons, necessitates a simultaneous rotation about the C(4')--C(5') bond from gg to g/t conformation. The observation that the 8-aza substitution in 5'-beta-purine nucleotides cause a depopulation of gg and anti conformers with corresponding increase in the population of g/t and syn orientation seems to support the above thesis. The finding that rotational variation about a bond such as C(4')--C(5') is accompanied by torsional variation about C(5')--O(5') and the glycosidic bonds, as well as changes in the endocyclic torsion angles of the ribose moiety, gives important insight into the engineering of nucleic acid components: even though they in general prefer certain conformations, there is enough flexibility present in their molecular framework, that the entire system can undergo conformational adjustment in response to a perturbation. A conformational basis for the antileukemic effects of 8-aza purines is proposed.


Evidence for heterogeneity of ribosomes within the HeLa cell. The proteins of several subsets of ribosomal subunits isolated from HeLa cells were separated by high resolution electrophoresis in polyacrylamide slab gels containing sodium dodecyl sulfate. Comparisons were made among native subunits, and subunits derived from single ribosomes, free polysomes, and membrane-bound polysomes. At least 10 heterogeneously distributed proteins were identified among the 40 S subunit proteins, compared wtih 17 to 20 proteins that occurred in all 40 S subunit classes. All 60 S subunits shared 34 separable proteins. One protein was missing only from membrane-bound 60 S subunits and another protein was present only in membrane-bound 60 S subunits. It is suggested that these two proteins regulate the binding of 60 S subunits to the endoplasmic reticulum.


The characterization of mannan of Micrococcus lysodeikticus as an acidic lipopolysaccharide. Ghosts of Micrococcus lysodeikticus contain a mannan that is not removed by intensive washing procedures. Purified mannan, isolated by extraction of whole cells with hot, aqueous phenol, binds to membranes in vitro. Mannan also binds to DEAE-cellulose and migrates toward the anode in neutral and sodium dodecyl sulfate disc gel electrophoresis. In aqueous solution mannan has an apparent molecular weight of 10-6, but in the presence of sodium dodecyl sulfate its apparent molecular weight is 50,000 to 100,000; removal of the detergent results in reaggregation. Purified mannan contains mannose, succinate, fatty acid, and glycerol in a ratio of 50:4.9:2.1:1.0. Treatment of mannan with mild base produces a neutral, hydrophilic polysaccharide of relatively low molecular weight that has no affinity for membranes. At least 90% of the reducing termini are blocked in a base-stable linkage. Based on these results a tentative structure is proposed for the mannan.


Heterogeneity of human hepatic H-acetyl-beta-D-hexosaminidose. A activity toward natural glycosphingolipid substrates. A crude soluble preparation of human hepatic N-acetyl-beta-D-hexosaminidase was examined for its activities toward three natural glycosphingolipid substrates after fractionation by the isoelectric focusing procedure. Profiles of activities toward N-acetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM2-ganglioside) and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide (globoside) were always identical with that of nonspecific N-acetyl-beta-D-hexosaminidase as determined with artificial substrates. The Component A of the enzyme had the activity peak at an isoelectric point of 5.0 to 5.1. In contrast, hydrolytic activities toward N-acetylgalactosaminyl-[N-acetylneuraminyl]galactosyl- glucosylceramide (GM2-ganglioside) were associated with only the most acidic subfraction of the hexosaminidase A component. The activity to hydrolyze GM2-ganglioside had its peak at an isoelectric point of 4.8 to 4.9. These findings might provide an explanation for the GM2-ganglioside accumulation in juvenile GM2-gangliosidosis (partial deficiency of hexosaminidase A) and in the so-called AB variant of GM2-gangliosidosis (apparently normal hexosaminidase A and B activity).


Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. Gamma-Glutamyl-cysteine synthetase is inhibited by glutathione under conditions similar to those which prevail in vivo, thus strongly suggesting a physiologically significant feedback mechanism. Inhibition by glutathione, which is not allosteric, appears to involve the binding of glutathione to the glutamate site of the enzyme as well as to another enzyme site; the latter binding appears to require a sulfhydryl group since ophthalmic acid (gamma-glutamyl-alpha-aminobutyryl-glycine) is only a weak inhibitor. The finding that glutathione regulates its own synthesis by inhibiting synthesis of gamma-glutamyl-cysteine appears to explain observations on patients with 5-oxoprolinuria, who were shown to have a block in the gamma-glutamyl cycle consisting of a marked deficiency of glutathione synthetase and consequently of glutathione. These patients produce greater than normal amounts of gamma-glutamyl-cysteine, which is converted by the action of gamma-glutamyl cyclotransferase to 5-oxoproline; production of the latter compound exceeds the capacity of 5-oxoprolinase to convert it to glutamate. The apparent Km value for L-cysteine for gamma-glutamyl-cysteine synthetase (0.35 mM) is not far from intracellular concentrations of L-cysteine suggesting that the availability of L-cysteine may also play a role in the regulation of glutathione synthesis.


Incorporation of L-azetidine-2-carboxylic acid into hemoglobin in rabbit reticulocytes in vitro. L-Azetidine-2-carboxylic acid is the naturally occurring lower homologue of L-proline. Reticulocytes from anemic rabbits incubated with DL-[14-C]azetidine-2-carboxylic acid synthesized radiolabeled hemoglobin, which when isolated from cell lysates co-chromatographed with unlabeled hemoglobin on Sephadex G-100 columns. Amino acid analysis of hemoglobin from reticulocytes incubated with DL-[14-C]-azetidine-2-carboxylic acid suggested that the homologue was incorporated into hemoglobin intact and unaltered. Alternatively, another amino acid analogue, 1-aminocyclopentane-[1-14-C]carboxylic acid, which is purported to be a valine antagonist, was not incorporated into hemoglobin under these conditions. Incubation of reticulocytes with 1, 5, and 10 mM L-azetidine-2-carboxylic acid reduced L-[U-14-C]proline (0.10 mM) incorporation into hemoglobin by 25, 58, and 72%, respectively. Conversely, 1.45 and 145 muM L-proline reduced radiolabeled azetidine-2-carboxylic acid (0.8 mM) in corporation into hemoglobin by 45 and 92%, respectively. Incorporation of L-[U-14-C]leucine and L-[U-14-C]lysine (0.1 mM each) into hemoglobin was unaffected at these concentrations of L-azetidine-2-carboxylic acid. These results suggest that L-azetidine-2-carboxylic acid is incorporated into hemoglobin without reducing the rate of globin synthesis in rabbit reticulocytes in vitro. The alpha and beta chains of hemoglobin into which [14-C]azetidine-2-carboxylic acid had been incorporated in rabbit reticulocytes in vitro were resolved electrophoretically on sodium dodecyl sulfate-polyacrylamide gels. The ratio of total radioactivity in the alpha and beta chains separately extracted from gels was in good agreement with the known 7:4 ratio of prolyl residues in the respective chains. Autoradiograms of two-dimensional tryptic peptide maps of rabbit globin into which either [14-C]azetidine-2-carboxylic acid or [14-C]proline had been incorporated showed nearly identical patterns of radioactivity. These results suggest that azetidine-2-carboxylic acid substitutes specifically for prolyl residues during in vitro hemoglobin synthesis in rabbit reticulocytes.


Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems. We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors.


The covalent and three-dimensional structural of concanavalin A. I. Amino acid sequence of cyanogen bromide fragments F1 and F2. Concanavalin A is a lectin composed of identical subunits, each containg 237 amino acid residues. The complete amino acid sequence of the first 129 residues of the polypeptide chain has been determined by analysis of peptides obtained from digests of CNBr Fragments F1 (residues 1 to 42) and F2 (residues 40 to 129). Correlation of the chemical sequence with x-ray crystallographic results indicates that Fragment F1 contains all of the protein ligands for the binding of 2 metal ions, Mn2+ and Ca2+, and that Fragment F2 contains many of the residues involved in the interactions of the subunits to form dimers and tetramers. The site of cleavage of the polypeptide chain to yield the naturally occurring Fragments A1 and A2 has also been identified as the peptide bond between residues 118 and 119.


The covalent and three-dimensional structure of concanavalin A. II. Amino acid sequence of cyanogen bromide fragment F3. The amino acid sequence of the COOH-terminal CNBr fragment, F3 (residues 130 to 237), of concanavalin A has been established, completing the determination of the covalent structure of this lectin. Analysis of the chemical sequence showed that the distribution of charged residues is generally more dense in the NH2-terminal half of the polypeptide chain than in the COOH-terminal portion and that in the latter region there is a linear stretch composed of many hydrophobic residues. Correlation with x-ray crystallographic results indicates that the hydrophobic region is located in the interior of the molecule, and that it forms a part of a deep cavity which is the binding site for the inhibitor, beta-(o-iodophenyl)-D-glucopyranoside. In conjunction with the three-dimensional structure, the amino acid sequence reported here provides new data for analysis of variables involved in predicting the three-dimensional folding of proteins from the primary structure. The sequence of concanavalin A is the first determined for a lectin and it serves as a reference structure for comparisons with other lectins.


The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides. The three-dimensional structure of the lectin concanavalin A (Con A) has been determined at 2.0-A resolution by x-ray diffraction analysis. The protomers are ellipsoidal domes of dimensions 42 times 40 times 39 A. Folding of the polypeptide backbone is dominated by the presence of two antiparallel pleated sheets, a twisted sheet of seven strands passing through the center of the molecule and a bowed sheet of six strands which forms the back surface of the monomer. Manganese and calcium ions bind to the protein at adjoining sites to form a binuclear complex of two octahedra sharing a common edge. The ligands for each metal ion are four groups from the NH2-terminal region of the protein and 2 water molecules. The binding site for the inhibitor beta-(o-iodophenyl)-D-glucopyranoside is in a deep cavity which contains distinct hydrophobic and hydrophilic binding subsites. Studies of the binding of beta-(o-iodophenyl)-D-glucopyranoside to Con A in the crystalline state and in solution have indicated that the binding behavior of the protein is somewhat different in the two states.


The covalent and three-dimensional structure of concanavalin A. IV. Atomic coordinates, hydrogen bonding, and quaternary structure. The coordinates of the individual non-hydrogen atoms of the lectin concanavalin A have been determined from the molecular model at 2.0-A resolution and have been adjusted to make them consistent with the known stereochemistry of the constituent amino acid residues. From the coordinates, an analysis has been made of all intra- and intersubunit interactions in the molecule, yielding a description of the hydrogen-bonded structure of the monomer, including two extensive pleated sheet structures and other features of the folding of the polypeptide chain. The description of the noncovalent bonding is extended to include the interactions involved in stabilization of the dimeric and tetrameric structures of the molecule. The complete description of the molecular structure provides a basis for analysis of the biological activities of concanavalin A.


Study of the loosely bound non-histone chromatin proteins. Stimulation of deoxyribonucleic acid-templated ribonucleic acid synthesis by a specific deoxyribonucleic acid-binding phosphoprotein fraction. The loosely bound chromatin proteins of Ehrlich ascites hyperdiploid cells have been prepared by extraction of chromatin with 0.35 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the 0.35 M NaCl-soluble chromatin proteins reveals high heterogeneity with a molecular weight range of 10,000 to 170,000. The 0.35 M NaCl-soluble chromatin proteins contain many components similar to the more tightly bound non-histone chromatin proteins complex with the loosely bound chromatin proteins by gradient dialysis, the inhibitory effect of histones on transcription of DNA in vitro was reduced. The reconstituted complex manifested a level of template activity similar to that of native chromatin as measured in an Ehrlich ascites tumor RNA polymerase reaction. The loosely bound chromatin proteins contain RNA as well as phosphoproteins. Phenol extraction or DNA affinity chromatography of these proteins yielded fractions enhanced 25- to 30-fold in phosphorus which were capable of stimulating DNA-templated RNA synthesis in vitro. The stimulation of transcription from DNA was template-specific, effective only with a DNA template prepared from Ehrlich ascites tumor, but not from rat liver, calf thymus, or chicken erythrocytes. In addition, the stimulatory effect of the specific DNA-binding proteins appears to be RNA polymerase-specific, the stimulation being manifested with Ehrlich ascites tumor nucleoplasmic RNA polymerase and not with Micrococcus luteus RNA polymerase. Thus, the loosely bound chromosomal proteins from Ehrlich ascites tumor contain a fraction that specifically binds to Ehrlich ascites tumor DNA and exhibits a template- and RNA polymerase-specific stimulatory effect on transcription from DNA.


The structure of dihydrofolate reductase. I. Inactivation of bacterial dihydrofolate reductase concomitant with modification of a methionine residue at the active site. Carboxymethylation by iodoacetate of dihydrofolate reductase from the amethopterin-resistant mutant Streptococcus faecium var. Durans strain A leads to a loss of enzymic activity. Amino acid analysis showed that methionine is the only amino acid residue significantly affected by iodoacetate under the experimental conditions, and this was confirmed by the use of [1-14-C]iodoacetate and ion exchange chromatography of the products obtained by acid hydrolysis of the modified enzyme. During loss of 90% of the activity a total of about 2 of the 7 methionine residues present in the enzymes are carboxymethylated. Over this range of activity loss the decrease is proportional to the number of methionine residues modified. Fluorescence-quenching experiments demonstrated that dissociation constants for complexes of inhibitors with the carboxymethylated enzyme were 20 to 30 times greater than dissociation constants for corresponding complexes with native enzyme. Similarly, equilibrium dialysis studies showed that dihydrofolate binding to the modified enzyme was decreased 10-fold compared with binding to the native enzyme. These data suggest that iodoacetate modifies one or more methionine residues at the binding site for dihydrofolate and inhibitors. In accordance with this view it was shown that enzyme can be protected from inactivation by the folate analogue aminopterin and to a lesser extent by folate and dehydrofolate. Enzyme carboxymethylated in the presence of aminopterin, and subsequently freed of the latter, was found to bind inhibitors and dihydrofolate as tightly as the native enzyme. It is concluded that the loss of enzyme activity is caused by carboxymethylation of at least 1 methionine residue which is at or near the binding site of dihydrofolate.


The site of sialic acid incorporation into thyroglobulin in the thyroid gland. Iodine incorporation into thyroglobulin is known to occur within the lumen of the thyroid follicle. Since incorporation of sialic acid, which occupies a terminal position in the oligosaccharide chains, is also a later event in thyroglobulin synthesis, the possibility that sialic acid might be incorporated after thyroglobulin secretion was investigated. In one experimental approach normal rat thyroid hemilobes were incubated with radioactive precursors. Thyroglobulin, analyzed by equilibrium centrifugation in RbCl, had a median density which varied according to the moiety labeled in the following increasing order: leucine smaller than galactose smaller than sialic acid smaller than iodine. The molecules having the highest density were labeled only with iodine. In the second approach, thyroid hemilobes were taken from rats treated with cycloheximide for 16 hours to stop protein synthesis and allow nascent molecules to be secreted, and incorporation of precursors into thyroglobulin was analyzed by sucrose gradient centrifugation. Leucine incorporation was 6% of control but the amino acid was found in the NH2-terminal position. N-Acetylmannosamine (sialic acid precursor) and galactose incorporation were also completely inhibited whereas iodine incorporation was 10% of control. Incorporation was not restored by thyrotropin treatment, and the sialyltransferase and iodination systems were reduced only to 50 to 70% of control. The results indicate that sialic acid is incorporated only in nascent thyroglobulin and not in thyroglobulin molecules already secreted into the follicular lumen. A large fraction of the iodine incorporation also seems to occur in newly synthesized thyroglobulin.


Activation of human prothrombin by highly purified human factors V and X-a in presence of human antithrombin. In this communication we describe the first method for isolating human Factor V. The final product contains no other coagulation components as judged by functional assays and is physically homogeneous as shown by isofocusing gel electrophoresis. In addition, we present a means for obtaining intrinsically activated human Factor X-a. This preparation is usually homogeneous as judged by isofocusing gel electrophoresis. However, on occasion, an additional minor electrophoretic species with Factor X-a activity is observed. Furthermore, we describe the use of isoelectric focusing in sucrose density gradients to free human prothrombin from contamination by coagulation factors and other components. These homogeneous human proteins are employed to examine the conversion of prothrombin to thrombin in the presence and absence of human antithrombin. The latter component is responsible for virtually all of the plasm's capacity to neutralize Factor X-a and thrombin. In the absence of antithrombin, prothrombin (67,800) is converted to the precursor P-2 (51,600) and the fragment F-a (19,500). Subsquently, P-2 is cleaved to form the precursor P-3 (37,000), and the fragment F-b (11,500). Finally, P3 IS proteolyzed to form the heavy chain T-h (29,500) and the light chain T-L (6,500) of active thrombin. In the presence of antithrombin, an additional prothrombin conversion pathway is observed in which the zymogen is directly cleaved to form P-3 and F-A + B (30,000) prior to thrombin generation. Trace amounts of free thrombin remain uninhibited by antithrombin and could bias the zymogen activation pathway. Hirudin is known to neutralized thrombin instantaneoulsly. We demonstrate that the purified leech protein also binds to P-3 and prevents thrombin formation. When hirudin is added to activation mixtures at concentrations sufficient to virtually suppress P-3 conversion to thrombin, molecular species from both activation pathways are observed. Thus two human prothrombin conversion sequences appear to be initiated by Factor X-3 and may be of physiological significance.


Concanavalin A: a stopped flow nuclear magnetic resonance study of conformational changes induced by Mn++, Ca++, and alpha-methyl-D-mannoside. The conformational changes induced in concanavalin A by the binding of Mn++, Ca++, and alpha-methyl-D-mannoside have been studied at pH 5.28 by stopped flow nuclear magnetic resonance techniques. Three distinct conformation states of the protein have been kinetically observed and an ordered binding mechanism elucidated from a detailed analysis of the reaction records. In addition, the individual steps of this mechanism are interpreted in terms of molecular parameters characterizing the conformational states involved such as ligand exchange rates to the paramagnetic Mn++.


Lectins from Wistaria floribunda seeds and their effect on membrane fluidity of human peripheral lymphocytes. Two lectins were isolated from Wistaria floribunda seeds. One is a strong mitogen against human peripheal lymphocytes and has been purified in the previous paper (Toyo-Shima S., Y., Nakano K., Tonomura, A., and Osawa T. (1971) Biochemistry 10, 4457). The other, which is a strong hemagglutinin being devoid of mitogenic activity against normal lymphocytes, has been purified in this paper by affinity chromatography on a Sepharose 6B column followed by DEAE-Sephadex column chromatography. Both lectins were found to be glycoproteins and their molecular weights were estimated to be 136,000 for the equilibrium. The hemagglutinin is composed of four apparently identical subunits of a molecular weight of 35,000 and the mitogen is adimer of 32,000 molecular weight subunit. Binding experiments with 125-I-labeled W. floribunda mitogen revealed that the maximal incorporation of [6-3H]thymidine or 32-PO4 occurred when only 5.2% of the avaliable receptor sites on normal lymphocytes were occupied by the mitogen. Furthermore, the mobility of W. floribunda lectins as well as other lectins bound to the cell receptor sites of normal lymphocytes was determined by fluorescence polarization of fluorescein-labeled lectins. The mitogen lectins tested, have high mobility and Lens culinaris hemagglutinin, have high mobility whereas the nonmitogenic lectins, W. floribunda hemagglutinin, Sophora japonica hemagglutinin, and eel serum anti-H hemagglutin show relatively low mobility. However, W. floribunda hemagglutinin bound to neuraminidase-treated lymphocytes showed relatively high mobility in accord with the fact that this hemagglutinin exerted weak but definite mitogenic activity against neuraminidase-treated lymphocytes. The change of membrane fluidity upon binding of the lectins to normal lymphocytes was also measured by fluorescence polarization of fluorescent hydrocarbon, 1,6-diphenyl-1,3,5-hexatriene, embedded in the membrane. The mitogenic lectins, W. floribunda mitogen and L. culinaris hemagglutinin, increased the membrane fluidity upon binding to lymphocyte cell surface within 30 min, whereas the non-mitogenic lectins, W. floribunda hemagglutinin and S. japonica hemagglutinin, did not effect the membrane fluidity. We suggest that the increase of membrane fluidity is one of the common biochemical events in the earliest stage of lymphocytes transformation.


Further studies on acetamidination as a technique for preparation of a biologically valid 3-H-labeled tracer for parathyroid hormone. The technique of acetamidination of amino groups in parathyroid hormone (PTH) for the purpose of preparation of a tritiated, biologically valid tracer for this hormone, has been more extensively studied. It was found that eight of the ten amino groups in PTH are readily reactive but that two appear unreactive. The kinetics of labeling suggest that preparations of PTH which are labeled to this level of 80% of theoretical consist of a homogeneous population of molecules in which each PTH molecule contains eight tritiated acetamidino groups. There is no question of the presence of unlabeled hormone in such preparations. Eighty per cent labeled acetamidino-PTH is identical, qualitatively and quantitatively, in its biological activity with native PTH as shown by three accepted bioassays: serum calcium elevation, urine phosphate excretion, and activation of kidney cortex plasma membrane adenylate cyclase in vitro. The dose-response relationships are identical for labeled and native hormone in all three systems. Tritiated acetamidino-PTH tends to lose biological activity on storage but full activity can be regained by reduction of the hormone with excess cysteine. However, cysteine reduction conducted at 80 degrees for 2 hours causes some loss of tritium from the hormone. Therefore, dithiothreitol reduction at room temperature was utilized to maintain biological activity. Eighty per cent acetamidino-PTH can be further purified by ion exchange chromatography on carboxymethylcellulose using a continuous gradient of sodium acetate in 8 M urea. Such chromatography reveals the presence of isohormones in both cold and radioactive PTH. Co-chromatography of tritiated acetamidino-PTH with a cold isohormone of PTH shows that while the isohormones can be separated from one another, the acetamidino derivatives of each isohormone elute in a virtually identical position with their parent unlabeled PTH molecule.


Effect of insulin on protein turnover in heart muscle. The effect of insulin on turnover of protein was investigated in isolated perfused rat hearts. The hormone lowered intracellular levels of nine amino acids and reduced or abolished net release of 10 amino acids and ammonia. The extent of the insulin effect on protein degradation was investigated by estimating the rate of dilution of the specific radioactivity of the free phenylalanine pool. Insulin concentrations greater than 200 microunits per ml reduced protein degradation and net phenlylalanine release. Protein degradation was estimated more directly by inhibiting reincorporation of nonradioactive phenylalanine from protein with cycloheximide. Addition of the inhibitor increased the estimated rates about 50%, but the magnitude of the hormone effect was similar. The latency of lysosomal enzymes in control and insulin-treated hearts was assessed by measuring activities of beta-acetylglucosaminidase and cathepsin D in heart homogenates in the presence and absence of Triton X-100. Perfusion with insulin-free buffer increased the activities assayable without detergent, but did not change total activities of these enzymes. Insulin decreased activities assayable without detergent and increased activities sedimenting in the 10-5 times g pellet. These studies showed that insulin restricted the rate of protein degradation in the isolated perfused rat heart. Concomitantly, the latency of lysosomal enzymes was increased when the hormone was provided.


Accepton concentration effect in the selectivity of acyl coenzyme A: U aclglycerylphosphorylcholine acyltransferase system in rat liver. In the acylation of 1-acylglycerylphosphorylcholine (1-acyl-GPC) in rat liver, neither the specificity observed with Triton X-100 treated microsomes were consistent with the selectivity observed in vivo. This apparent discrepancy was solved as follows. 1. Crude microsomes or deoxycholate-treated microsomes had approximately the same levels of activities for oleoyl-, linoleoyl- and arachidonoyl-CoAs. Triton X-100 treated microsomes had much higher activity for arachidonate but very little activity for oleate, which was found to be the consequence of selective inactivation by Triton X-100 of oleoyl-CoA:1-acyl-GPC acyl transfer reaction. These observations favor a concept that different enzymes or different sites on a single enzyme exist in rat liver microsomes for the transfers of different acyl-CoAs. 2. Although the maximum velocities for oleoyl-CoA and arachidonoyl-CoA were approximately the same, more arachidonate was incorporated than oleate at very low concentrations of the acceptor even when both acyl-CoAs were present at saturating concentrations. Thus, higher selectivity for arachidonate (and linoleate to a lesser extent) observed with rat liver in vivo could be correlated with relatively higher affinity in this reaction for 1-acyl-GPC observed in vitro, which exerts its effect at low concentrations of the acceptor.


Purification and characterization of bacteriophage gh-I-induced deoxyribonucleic acid-dependent ribonucleic acid polymerase from Pseudomonas putida. Infection of Pseudomonas putida by the bacteriophage gh-L-induced the synthesis of a novel DNA-dependent RNA polymerase. This gh-L-induced RNA polymerase was purified to near homogeneity. It was shown to be distinct from the host RNA polymerase (alpha-2 beta beta sigma) physically and in respect to many of its catalytic properties. The gh-L-induced RNA polymerase was composed of a single polypeptide of approximately 98,000 molecular weight. The divalent metal ion requirement for in vitro RNA synthesis by the gh-L-polymerase could be satisified with Mg-2+, but not with Mn-2+. Rna synthesis by the gh-L polymerase was highly resistant to inhibition by rifampicin and streptolydigin but could be inhibited by relatively low concentrations of KCl or the rifamycin derivative AF/013. The structural analog of ATP, 3'-deoxyadenosine 5'-triphosphate, inhibited both the gh-L-induced and the host RNA polymerases by competing for a single binding site with ATP. The phage polymerase was extremely sensitive to this inhibitor, exhibiting an apparent K-i value (2 times 10-8 M) approximately 100 times lower than that for the host RNA polymerase. The gh-L polymerase had a highly specific template requirement for DNA from the homologous gh-L phage. It would not efficiently utilize denatured DNA templates and had only low levels of activity with pyrimidine-containing polydeoxyribonucleotide homopolymers.


Primary structural analysis of sulfhydryl protease inhibitors from pineapple stem. Pineapple stem acetone powder provides a rich source of the sulfhydryl protease bromelain and of a family of compositionally similar but chromatographically distinct polypeptide inihibtors of this enzyme. The isoinhibitors have molecular weights of 5600, and they contain five disulfide bonds and about 50 amino acids each (Perlstein, S. H., AND Kezdy, F.J. (1973) J. Supramol. Struct. 1, 249-254). Primary structural analysis of one of the seven inhibitor fractions (VII) revealed extensive microheterogeneity. Each of the inhibitor molecules in Fraction VII was shown to be composed of two peptide chains joined by disulfide bonds. These chains, designated A and B on the basis of size, comprise 41 and 10-11 residues, respectively, and the amino acid sequence of one of each are given below: (see article for formular). On the basis of ionization properties and yields of the A and B chains, it would appear that one of the major inhibitor species in Fraction VII is the covalently linked complex of the two chains shown, namely [A-1, B-2]. The second major inhibitor component of Fraction VII is identical in structure with [A-1, B-2i1 except that residues 1 and 8 in the A chain are pyroglutamate and threonine, respectively, and in the B chain glutamine 11 is replaced by arginine. The third inhibitor in Fraction VII is a minor constituent identical with the second, except that residue 1 in the A chain is glutamate rather than pyroglutamate. This microheterogeneity in the inhibitors of Fraction VII is further increased by the fact that B chains may lack threonine 1, in which case they are decapeptides beginning with alanine. On the basis of the striking homology of the cysteine residues with those of other protease inhibitors, it is proposed that the bromelain inhibitors are generated enzymatically from single chain precursors by excision of a "bridge" paptide which links the NH-2 termal A chain to the COOH-terminal B chain.


Effects of alloxan diabetes, anti-insulin serum diabetes, and non-diabetic dehydration on brain carbohydrate and energy metabolism in young mice. Alloxan-induced diabetes of 4 days duration produced metabolite changes in brain compatible with severe reduction in cerebral metabolism (phosphocreatine increased 70%), and reduced phosphofructokinase activity (fructose diphosphate levels fell 38%). There was a 56% reduction in brain lactate concentration, but pyruvate levels were unchanged. In 5 of 23 animals, brain glycogen levels increased; in the remainder blycogen levels decreased. Brain fructose concentration, 0.4 mmol/kg, was only 1/30 of the glucose concentration. The alloxan-treated animals were also severely dehydrated. Therefore, to determine the casual relation of insulin deficiency to these findings, the effects of chronic dehydration and acute insulin deficiency were investigated. Findings in the brains of severely dehydrated animals (water deprivation and mannitol injections for 4 days) were almost identical with those seen after alloxan treatment. The exceptions were that, in the dehydrated mice, reductions in lactate and pyruvate were proportional, and glycogen levels were consistently reduced. In acute diabetes (6 to 24 hours after repeated anti-insulin serum injections) P-creatine, fructose diphosphate, and lactate levels were normal. Pyruvate levels were normal at 6 hours, but increased 39% by 12 to 24 hours; glycogen was 36% higher at 6 hours and 63% at 12 to 24 hours. Insulin (and glucose) appeared to be specific in correcting the metabolic abnormalities found in the brains of animals with alloxan-induced diabetes. At 4 and one half hours after treatment with insulin and glucose, glucose 6-phosphate levels fell 25%, fructose diphosphate increased 28%, and lactate and the lactate to pyruvate ratio returned to normal; glycogen increased 50%. However, the treatment also had a dramatic clinical effect. Since animals gained 8 to 27% of body weight during therapy, at least some of the improvements in metabolite levels could be related to rehydration.


Immunochemical isolation and characterization of ovalbumin messenger ribonucleic acid. Hen oviduct ovalbumin messenger RNA has been purified to apparent homogeneity and its physical and molecular properties have been examined. Purification was achieved through the use of indirect immunoprecipitation to isolate ovalbumin synthesizing polysomes and the use of poly(U)-Sepharose chromatography to separate quantitatively ovalbumin messenger RNA from ribosomal RNA. Ovalbumin mRNA was purified 90 to 100-fold over oviduct polysomal RNA as judged by both the rate of hybridization to a complementary DNA and by translation in a rabbit reticulocyte lysate protein-synthesizing system. Isolated ovalbumin mRNA migrates as a single sharp symmetrical peak on sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The molecular weight of ovalbumin mRNA determined by sedimentation in denaturing dimethylsulfoxide gradients is 700,000 (equivalent to 2,180 nucleotides). The complexity of purified ovalbumin mRNA determined from the relative rate of hybridization to a complementary DNA is 2,280 nucleotides. Since ovalbumin synthesis requires only 1,161 nucleotides, ovalbumin mRNA appears to contain approximately 1,150 untranslated nucleotides. The average length of the polyadenylate sequence in ovalbumin mRNA is only 44 nucleotides and it does not account for significant fraction of the untranslated nucleotides.


Metabolism of mevalonate in rats and man not leading to sterols. C-5 of mevalonate appears as C0-2 in the breath of rats and men almost immediately after administration either by injection or by mouth. Adult rats exhaled up to 6.5% of a dose of RS-[5-14C]mevalonate (13% of the utilizable R-enantiomer) in the breath in 100 min. The 14-C02 was not derived either from the matabolism of cholesterol biosynthesized from [5-14C]mevalonate or from the metabolism of the unnatural S-enantiomer of mevalonate. The amount of 14-C02 expired in the breath was the same whether the [5-14C]mevalonate was given intravenously or in a drink of water to man. One normocholesterolemic man dissipated 12%, a mildly non-familial hypercholesterolemic man dissipated 10%, and a familial hypercholesterolemic man dissipated 7% of a dose of [5-14C]mevalonate in 24 hours (calculated as a per cent of the R-enantiomer). The observations support the hypothesis of the existence of a metabolic shunt of intermediates of sterol biosynthesis, derived from mevalonate, not leading to sterols.


Laser Raman scattering of neurotoxins isolated from the venoms of sea snakes Lapemis hardwickii and Enhydrina schistosa. The venoms of sea snakes (family: Hydrophiidae) contain potent neurotoxins which bind to the acetylcholine receptor in the neuromuscular junction. A major toxin was isolated from the venoms of the sea snakes Lapemis hardwickii and Enhydrina schistosa according to previously published methods. These pure toxins were studied by laser Raman spectroscopy to elucidate further the structure-function relationship to the neurotoxin. Spectra were obtained from the powder, aqueous solution, and deuterated derivatives of each toxin. The peptide backbone conformation of these neurotoxins was found to be of "anti-parallel beta configuration," as distinct amide I and III bands appeared at 1672 and 1240 cm-1, respectively. No indication of alpha helical structure in the neurotoxins was observed from amide I and III bands. This was further confirmed by the spectra of the neurotoxins after deuterium exchange. The peaks due to a single tyrosine residue appeared at 644, 834, and 846 cm-1. The intensity ratios of the toxin from L. hardwickii venom were 0.92, 1.0, and 0.84 at 644, 834, and 846 cm-1. It is concluded that the tyrosine residue is involved in some unusual intramolecular interactions and not readily accessible to water molecules. Similar results were obtained for the toxin of Enhydrina schistosa (common sea snake). The fact that only 50% of the tyrosine molecule is modified by nitration is in complete agreement with laser Raman result. The lack of a sharp Raman line at 1361 cm-1 suggested that the single tryptophan residue may be "exposed." The previous demonstration that the tryptophan residue can be modified readily with different reagents confirms these results. A relatively symmetrical disulfide peak at 512 cm-1 indicates that the geometry of the C-C-S-S-C-C linkage is nearly identical for all four disulfide bonds in the molecule. The absence of phenylalanine was established by the lack of a phenylalanine peak in the laser Raman spectra and by amino acid analysis.


Lactate dehydrogenase isozymes of salmonid fish. Evidence for unique and rapid functional divergence of duplicated H-4 lactate dehydrogenases. Salmonid fish, as a result of total genome duplication, have two genes, Ldh H and Ldh H', coding for polypeptides H and H', respectively, both of which have been shown in their tetrameric forms to be immunologically related to the classical H-4 lactate dehydrogenase isozyme of higher vertebrates (Bailey, G. S., and Wilson, A. C. (1968) J. Biol. Chem. 243,5843). The H-4 and H'-4 isozymes have now been highly purified from quinnat salmon, and their chemical, physical, immunological, and catalytic properties examined, and compared to the M-4 isozyme of salmon. The two proteins H-4 and H'-4 are shown to be very similar in amino acid composition, but significant differences in a few residues suggest differences in amino acid sequences. This suggestion was born out by quantitative immunological experiments in which the H-4 and H'-4 isozymes were shown to be about as different from each other as are the H-4 lactate dehydrogenases of chicken and duck. This suggests that the gene duplication event in salmon which give rise to two Ldh H genes occurred approximately 80 to 100 million years ago. The H'-4 lactate dehydrogenase which has risen from this duplication in salmon is shown to be somewhat intermediate between H-4 and M-4 in thermal stability, and in all catalytic properties examined, including substrate optima, Michaelis constants, and susceptibility to inhibition by high levels of substrate. In particular the H'-4 isozyme is almost exactly intermediate between H-4 and M-4 in its resistance to product inhibition by lactate, the catalytic parameter suggested to be of major functional importance to M-4 lactate dehydrogenase isozymes (Stambaugh, R., and Post D. (1966) J. Biol. Chem. 241,1462). Further, tissue distribution of these isozymes in salmon and trout are shown to be unusual. The M-4 isozyme salmon and trout are shown to be unusual. The M-4 isozyme occurs in very few tissues in detectable levels. It is the H-4 and H'-4 rather than H-4 and M-4, which occur in independently variable but significant levels in most tissues examined. Thus the H'-4 isozyme, despite its very close structural similarity to H-4 appears to possess functional properties which are different from either H-4 or M-4 in salmon, and some properties are midway between the two. This finding, together with the unusual tissue distribution of these isozymes, suggests that salmon with H'-4 lactate dehydrogenase is evolving to function catalytically in the absence of a balanced H-4-M-4 isozyme complement in most tissues. This balance seems to be met in most tissues by combinations of H-4 and H'-4,


Phage Q-beta ribonucleic acid replicase. Subunit relationships determined by intramolecular cross-linking. Phage Qbeta replicase is composed of four subunits of molecular weights 70,000, 65,000, 45,000, and 35,000. Treatment of the enzyme with protein cross-linking reagents results in formation of three covalently bound complexes of molecular weights 215,000, 135,000, and 80,000. Analysis of the two larger complexes formed by cross-linking with a reversible cross-linker (methyl-4-mercaptobutyrimidate) demonstrates that the 215,000 molecular weight complex is composed of one each of the replicase subunits, while the 135,000 molecular weight complex is composed of the two larger subunits. The 80,000 molecular weight complex was shown to be made up of the two smaller subunits by cross-linking these two subunits in the absence of the larger pair. Increasing ionic strength stabilizes the large complex at the expense of the two smaller complexes. The presence of stoichiometric amounts of Qbeta RNA during cross-linking dramatically reduces formation of the large complex; other natural and synthetic RNAs reduce the formation of this complex to a lesser extent.


The amino acid sequence of a testis-specific basic protein that is associated with spermatogenesis. The amino acid sequence of the COOH-terminal cyanogen bromide fragment (residues 12 to 54) of the testis-specific basic protein of the rat has been determined. This analysis completes the primary structure of the whole protein by over-lapping the sequence of the 23 residues from the NH-2 terminus previously published (Kistler, W. S., Noyes, C., and Heinrikson, R.L. (1974) Biochem. Biophys. Res. Commun. 57, 341-347). The complete sequence of this small, highly basic protein is: (see article for formular).


Studies on the mechanism of orthophosphate regulation of bovine brain hexokinase. An attempt was made to gain insight into the mechanism of orthophosphate attenuation of glucose-6-P inhibition of bovine brain hexokinase I (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from experiments of ligand binding and initial rate kinetics. Studies of glucose-6-P and phosphate binding to hexokinase reveal one binding site per hexokinase molecule. A model is presented which is consistent with the binding and kinetic data currently available on the alleviation of glucose-6-P inhibition of brain hexokinase by phosphate. The model implies that hexokinase may exist in equilibrium either as a free or phosphate-associated enzyme. The kinetic parameters of the two enzyme forms are similar except in their ability to bind glucose-6-P. It is suggested that the dissociation constant for glucose-6-P is relatively very high for hexokinase to which phosphate is bound. Phosphate appears to bind at an allosteric site on the enzyme, whereas glucose-6-P is associated either at the active site or at an allosteric site which overlaps the catalytic site.


Characterization and tissue distribution of 6-O-beta-D-galactopyranosyl myo-inositol in the rat. A disaccharide was isolated from rat mammary tissue and determined to be 6-O-beta-galactopyranosyl myo-inositol (6-beta-galactinol) on the basis of combined gas-liquid chromatography-mass spectrometry of the permethylated and peracetylated derivatives as well as previously reported chemical and enzymatic evidence. 6-beta-Galactinol, also found in rat milk, increased during lactation, and on the 18th day represented approximately 17% of the total non-lipid neutral myo-inositol. The sugar was absent in all other rat tissues examined, suggesting a unique association with the process of lactation. This novel galactoside of one human subject on the basis of paper and gas-liquid chromatography.


Isolation and characterization of the nervous system-specific protein 14-3-2 from rat brain. Purification, subunit composition, and comparison to the beef brain protein. A procedure is described for the isolation of the nervous system-specific protein designated 14-3-2 from rat brain. The methods utilized are salt precipitation, DEAE-cellulose ion exchange chromatography, Sephadex G-150 gel filtration, and column isoelectric focusing. The native 14-3-2 protein has an isoelectric point of 4.7 in the absence of denaturing agents and 5.0 in the presence of 2.0 M urea. The protein, as isolated, appears homogeneous since it migrates as a single band on Tris-glycine (pH 8.9), sodium dodecyl sulfate (pH 7.2), and 8 M urea (pH 4.0) polyacrylamide gels. Sedimentation velocity and equilibrium data indicate a homogeneous component of molecular weight 78,000. Sedimentation of 14-3-2 in 6 M guanidine HCl containing 0.02% glutathione yielded a molecular weight of 39,000, indicating the dimeric nature of the protein as isolated. The rat brain protein seems to be composed of one subunit type, since polyacrylamide gel electrophoresis in 8 M urea yields a single protein component. Sodium dodecyl sulfate gel electrophoresis of rat brain 14-3-2 produced one sharp band with a relative mobility corresponding to a molecular weight of 48,000. Specific anti-14-3-2 serum has been prepared from both New Zealand white rabbits and goats. Rat 14-3-2 is very similar in amino acid composition to the beef brain protein and to antigen alpha. The antigenic properties of rat and beef 14-3-2 are also similar, since beef 14-3-2 antiserum reacts well with rat 14-3-2 and vice versa. Electrophoretic mobilities of denatured rat and beef 14-3-2 (0.1% sodium dodecyl sulfate and 8 M urea) are identical. Despite these similarities the two proteins are completely resolved on Tris-glycine gels. The sedimentation behavior of the beef and rat proteins are also different, indicating a difference in the association state and conformation of the two preparations.


The synthesis of neurotensin.

The synthesis of neurotensin. A tridecapeptide having the amino acid sequence, less than Glu-Leu-Tyr-Glu-Asn-Pro-Arg-Arg-Pro-Tyr-Iie-Leu-OH, (The nomenclature and symbols follow the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1972) J. Biol. Chem. 247, 977).) has been synthesized by the Merrifield solid-phase procedure. The synthetic scheme chosen involved synthesis of the peptide in the (Gln) form and cyclization to the less than Glu) form. After purification, the (Gln) peptide was obtained in a 7% yield and the (greater than Glu) peptide was obtained in a 35% yield. The (greater than Glu) was found to be chemically and biologically indistinguishable from the tridecapeptide, neurotensin, recently isolated from bovine hypothalami.


Serine transhydroxymethylase. Relaxation and transient kinetic study of the formation and interconversion of the enzyme-glycine complexes. Serine transhydroxymethylase forms three enzyme-glycine complexes which absorb at 3.43, 425, and 495 nm. Temperature-jump studies show three relaxations. Two of the relaxations are observed at both 343 and 425 nm but not at 495 nm. A slower third relaxation is observed only at 495 nm. The absorbance changes for the two relaxations observable at 343 and 425 nm are in opposite directions suggesting that these relaxations are both attributable to the inter conversion of enzyme speiies absorbing at these two wavelengths. The following mechanism is proposed to explain the relaxation effects.  See journal for formula. The forward and reverse rate constants for the first step are 7 X 10-4 M-MINUS1 sminus-1 and 1200 Sminus-1, respectively. The forward and reverse rate constants for the second step are 3200 and 1300 sminus-1, respective.y Stopped flow studies on the rate of formation and breakdown of the complexes absorbing at 343 nm (EX) and 425 nm (EY) are in agreement with the proposed mechanism. Stopped flow studies gave a Kobs of 0.1 Sminus-1 for the formation of the 495-nm absorbing complex. This is compared to the reciprocal relaxation time of 200 Sminus-1 observed in the temperature-jump studies. The addition of tetrahydrofolate to this system increased the Kobs to 275 Sminus-1 in the stopped flow studies and the reciprocal relaxation time to 800 Sminus-1 in the temperature-jump studies. The data do not permit a simple interpretation of the relationship of the 495-nm absorbing complex to those adsorbing at 343 and 425 nm. However, the data do support the interpretation that the ability of tetrahydrofolate to increase by 3 orders of magnitude the enzyme-catalyzed rate of exchange of the alpha-hydrogen of glycine with protons of the solvent is attributable to an increased rate of formation of the 495-nm complex.


Adamantinoma of long bones. A clinicopathological study of fourteen cases with vascular origin suggested. Adamantinoma of long bones is a rare primary bone tumor. Approximately 100 cases have been reported in the literature. In the fourteen cases we studied in detail the ages of the patients ranged from thirteen to sixty-seven years and there were as many males as females. Twelve of the lesions were located in the tibia and two, in the humerus. The roentgenographic findings were sharply defined eccentric lobular lesions in the diaphysis. Some of the lesions were lytic and coarsely trabecular with a honeycomb appearance occasionally associated with periosteal new-bone formation. A sawtooth area of cortical-bone loss was found to be characteristic of this lesion. A microscopic finding is presented to support the theory of angioblastic origin of this tumor. The most successful treatment was amputation. Less often en bloc excision succeeded. Metastases to other bones, regional lymph nodes, and the lung were recorded.


Osteoid-osteoma: diagnostic problems.

Osteoid-osteoma: diagnostic problems. The clinical records and roentgenograms of fifty-four patients had the typical features of osteoid-osteoma, but no histological evidence of a nidus was found at initial surgery. The symptoms of thirty-one patients were relieved by initial operation and those of twenty-three were not. In two cases, Brodie's abscess was identified in the surgical specimen. A second operation in eighteen patients brought relief in thirteen, and a nidus was found histologically in seven of these patients. A parosteal osteogenic sarcoma was found in one of the patients who had no relief of symptoms. Three patients underwent a third operation and were relieved of symptoms, a nidus being found in two. Thus, symptoms were relieved in thirty-six of forty-two patients in whom no nidus or other pathological entity was found, indicating the significance of this symptomatic sclerotic osseous lesion.


Osteoid-osteoma as a cause of scoliosis. In nine cases of osteoid-osteoma causing scoliosis, all lesions were in the spine and several led to significant structural spine changes. One patient had two distinct spine lesions two years apart. Almost all cases were misdiagnosed and improperly treated at first. Prompt recognition should lead to early surgical excision without spine fusion.


Giant-cell tumor of bone. A demographic, clinical, and histopathological study of all cases recorded in the Swedish Cancer Registry for the years 1958 through 1968. All seventy-five cases recorded as giant-cell tumor of bone in the Swedish Cancer Registry for the years 1958 through 1968 were analyzed. At  reexamination, fifty-three cases constituted genuine giant-cell tumor of bone and twenty cases were so-called "giant-cell variants". The genuine giant-cell tumors showed a significantly higher incidence in the urban than in the rural population. The recurrence rate was 42 per cent. Patients under the age of twenty-five rarely had recurrences. A high recurrence rate was found among patients with tumors located in the distal end of the femur and the proximal end of the tibia. Tumors penetrating through the bone cortex were more aggressive than those located entirely within bone, regardless of tumor size and presence or absence of spontaneous fracture. A malignant course was found in 11.3 per cent of cases, predominantly in patients with tumors in the femur. Histopathological grading was of no prognostic value. Primary en bloc resection with or without prosthetic replacement is recommended in patients over the age of twenty-five.


The use of spinal anesthesia for total hip-replacement arthroplasty. Two hundred and thirty-four total hip replacements in 199 patients performed by one surgeon were reviewed to compare the effects of spinal and general anesthesia. The amount of total blood loss was reduced an average of 600 milliliters in patients under spinal anesthesia. The amounts of operative blood loss, postoperative suction drainage, and blood replacement were also reduced very significantly (p smaller than 0.001). The patients in the two anesthesia groups were similar as to sex, age, body weight, pre-existing medical disease, preoperative medications administered, hip disease, type of prosthesis used, position during surgery, and anticoagulation regimen. The postoperative complications were fewer in the spinal anesthesia group. It is concluded from this study that spinal anesthesia is to be preferred over general anesthesia in patients undergoing total hip replacement.


Acquired hallux varus: a preventable and correctable disorder. Acquired hallux varus deformity of either the static or the dynamic type results from surgery to correct hallux valgus deformity and the accompanying bunion. The dynamic type is disfiguring, uncomfortable, and disappointing to the patient and surgeon alike. Linear as well as rotational forces are at work to produce the deformity. When recognized early, before fixation of the first digit has occurred in the deviated and clawed position, resection of the base of the proximal phalanx will correct the deformity. More radical treatment is required in the patient who is treated late. The experience with seven feet of five patients with acquired hallux varus deformity is presented here.


Surgical treatment for absent single-hand grip and elbow extension in quadriplegia. Principles and preliminary experience. A classification of quadriplegic patients based on available sensory receptors (ocular only or both ocular and hand sensibility) and motor function in each upper extremity greatly facilitates the planning and evaluation of surgical treatment. In forty hands of thirty-three patients with post-traumatic quadriplegia and cord lesions higher than those usually thought to be benefited by reconstructive surgery, three or more procedures were performed at one or more sittings to create an active wrist extensor and a thumb flexor grip, a function easier to provide and much more useful to these patients than tripod pinch. In these forty hands no function was lost and in all but two function was improved significantly if not greatly. A useful level of active elbow extension was restored in fifteen of sixteen extremities in quadriplegics by transfer of the posterior half of the deltoid to the triceps aponeurosis, lengthening the deltoid with free grafts from the toe extensors. Each upper extremity of quadriplegic patients of the type under consideration is a highly individualized problem. Successful treatment requires strict attention to every detail of preoperative evaluation, surgical treatment, and postoperative care.


Evolution of treatment of paralytic scoliosis at Rancho Los Amigos Hospital. Between 1954 and 1970, 351 patients with severe paralytic scoliosis were treated at Rancho Los Amigos Hospital. During this time the treatment evolved through five stages: body cast alone, halo cast, halo cast with buttons and traction wires, Harrington instrumentation, and finally preoperative halo-femoral traction and Harrington instrumentation. Coincident with this evolution, correction improved from 20 to 57 per cent, the incidence of curve progression dropped from 38 to 0 per cent, and curve extension decreased from 25 to 0 per cent, while postoperative recumbency was reduced from one year to about three weeks. In addition, complications changed, in general decreasing except for the rate of pseudarthrosis, which remained essentially the same. Clinically significant hyperlordosis involving the thoracic and lumbar spine was seen in sixteen patients who had long fusions from the fourth cervical vertebra and above to the fourth lumbar vertebra or the sacrum.


Blood flow and mineral deposition in canine tibial fractures. Measurement of 125I-labeled 4-iodoantipyrine (I-Ap) washout is a useful method to study changes in bone blood flow because it can demonstrate fine changes in flow, the tracer is not involved in tissue metabolism, and several measurements can be made in the same subject. In the tibiae of twenty-one adult dogs there was a significant (p smaller than 0.001) correlation between I-Ap washout and the deposition of 85Sr, suggesting that the uptake of 85Sr is related to blood flow. After fracture, blood flow at the fracture site reached a maximum on the tenth day and then progressively decreased, but was not back to control values at 112 days. In the proximal part of the diaphysis, away from the fracture site, flow reached a maximum at five to twenty-one days and then progressively decreased to normal.


"Stiction-friction" of total hip prostheses and its relationship to loosening. The static friction, or "stiction-friction", in McKee-Farrar and Charnley-Müller prostheses in a hip joint simulator was compared with dynamic friction determined while the prostheses were oscillated. Under physiological conditions stiction-friction differed little from dynamic friction in both metal-on-metal and metal-on-plastic prostheses, and was affected very little by the lubricant as long as some fluid was present. Stiction-friction increased significantly only after relatively long stationary periods and high loads. However, the frictional forces generated in total hip-replacement prostheses were at least forty times higher than those generated in normal joints and may well be enough to cause late loosening of acetabular components by fatigue failure.


Failed femoral stems in total hip prostheses. A report of six cases. Six femoral stems of total joint prostheses failed and were studied. These included two short-neck Müller, one standard Müller, one new design long-neck Müller, and two Charnley prostheses. In addition, reference is made to another failed Charnley prosthesis which had not required revision at the time of the study. Reoperation and replacement of the femoral component was required in all six cases. Metal fatigue appeared to be the cause of failure in all instances. Metallographic examination of the removed prosthesis revealed no underlying defects in one prosthesis. In the other five prostheses metallurgical defects were found. Varus placement of the femoral prosthesis or loosening of the cement-prosthesis bond, or both, were identified in five of the six and were thought to have led to overload. A combination of metal defects and loosening or malpositioning was thought to be responsible for the failures. It was further indicated that current designs should be regarded as marginal in relation to long service life, until more fatigue information is available on the metallic materials currently in use.


Autosomal dominant inheritance of tibial meromelia. Report of a kindred. Three generations of a family demonstrated an autosomal dominant transmission of congenital absence of the tibia. Nine family members were affected, six in the first generation. Male-to-male, male-to-female, and female-to-female transmission occurred. This is the fourteenth case reported to document familial occurrence of tibial meromelia, and the first to document this mode of inheritance.


Articular damage in familial Mediterranean fever. Report of four cases. Four cases of familial Mediterranean fever have been reported in which the disease produced organic damage to a joint. The diagnosis was confirmed by clinical and family history and a typical course which included attacks of recurrent joint synovitis. The laboratory findings, while typical, were not specific. The main involvement was in the lower limbs. The findings at operation were of a non-specific synovitis with destruction of cartilage. It is emphasized that in the majority of cases of familial Mediterranean fever the joint involvement is transient and only uncommonly does damage to the joint become permanent. The fact that organic joint damage occurs is not widely recognized, which is the reason for our report of these four cases.


Cytoplasmic cleavage during zoosporogenesis in Thraustochytrium sp.: ultrastructure and the effects of colchicine and D2O. Subplasmalemma microtubules were present in the cleavage furrows during zoosporogenesis in Thraustochytrium sp. The microtubules transversed the furrows but were not at right angles to nor circumferentially arranged in the plane of cleavage. Evidence of microtubule-plasmalemma bridges was found. Colchicine and deuterium oxide inhibited cleavage. The data suggested that microtubules had a motive rather than a structural function.


Junctional structures in the midgut cells of lepidopteran caterpillars. The junctional structures present between the midgut cells of 3 lepidopteran caterpillars have been examined using freeze-etching, conventional staining and lanthanum tracer techniques. The bonding junction present in this type of tissue is the so-called continuous junction. Septa have only occassionally been reported in conventionally strained cross-sections of these junctions. During the present study septa have been observed in such sections but were more readily located in tissue treated with lanthanum tracer. Tangential sections of lanthanum-impregnated tissue show that these septa are parallel-sided, in contrast to the honeycomb appearance of the septa in septate junctions. The septa in freeze-etch replicas of glutaraldehyde-fixed tissue often show continuous rods on one membrane face, suggesting that the continuous junction may be more akin to the tight junction than to the normal septate junction. However, freeze-etch replicas of unfixed tissue appear much more like replicas of normal septate junctions. The main differences between septate and continuous junctions appear to be that the inter-membrane septa of the continuous junction are parallel-sided in tangential section, as against the honeycomb appearance of the septate junction, and that the particles which delineate the septa in freeze-etched preparations appear to be both somewhat differentl bonded and closer together in the continuous junction. A diagram is presented showing the internal and inter-membrane structures of the 2 types of junction based on the present study, and suggesting a possible explanation of the fact that septa are more readily seen in cross-sections of septate junctions than in continuous junctions. As septa are present in both types of junction, and because their freeze-etch appearances are not very different, it is suggested that the two types of septate junction be called 'smooth septate' and 'pleated septate' junctions, to indicate their characteristic appearances in tangential section.


Changes in DNA synthesis during cell growth and chloroplast replication in greening spinach leaf disks. Spinach leaf disks grown initially in the dark, show increased cell expansion and chloroplast replication when transferred to the light. These changes are accompanied by increases in the total amount of DNA and the incorporation of [3H]thymidine (3H-TdR). Autoradiography of EDTA-separated cells dried on to glass slides was used to follow changes in 3H-TdR incorporation in both chloroplasts and nuclei. Specificity of incorporation was confirmed by nuclease studies. DNA synthesis occurs in both the chloroplasts and nuclei, and is highest just prior to, and during the period of most rapid cell growth and chloroplast replication which occurs shortly after the transfer to the light. Light, however, appears to have a greater and more immediate effect on nuclear DNA synthesis. Though nuclear and chloroplast DNA syntheses follow similar patterns during disk growth, in a given cell, chloroplast DNA synthesis can be separate in time from nuclear DNA synthesis. The increased nuclear DNA synthesis is possibly required to support the increased population of chloroplasts, while chloroplast DNA synthesis is associated with chloroplast division. If the disks are not transferred to the light but kept in darkness, chloroplast 3H-TdR incorporation remains high, though chloroplast division is reduced. Epidermal cells in light-grown tissue also show 3H-TdR incorporation but low rates of chloroplast division. It would appear that chloroplast DNA synthesis in mesophyll cells from light-grown tissue shows a general relation to chloroplast division, but there does not appear to be an obligatory close coupling between the 2 processes.


Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes. Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.


An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. The dentate gyrus of the rat contains about 600,000 granule cells. These small neurons are generated over a prolonged period from the 14th day of gestation until some time after the second postnatal week. The majority of the cells pass through their last phase of DNA synthesis in the postnatal period, and during the peak period of cell generation, between the fifth and seventh days after birth, up to 50,000 granule cells are formed each day. Contrary to earlier reports, most of the cells pass through their last mitotic division either within the stratum granulosum itself, or within the hilar region of the developing gyrus. The precursor population of cells in the hilar region must therefore constitute a pool of true neuroblasts. The origin of this pool of cells has not been definitely established but it seems probable that its cells are derived from the neuroepithelium lining the lateral ventricle adjacent to the region from which the hippocampal pyramidal cells are generated. Examination of the final location of granule cells labeled at different stages reveals three distinct morphogenetic gradients in the gyrus. The cells in the dorsal blade tend to be formed earlier than those in the ventral blade; cells in the more caudal (or temporal) portions of the gyrus are generated earlier than those in more rostral (or septal) regions; and in all regions the more superficial neurons in the stratum granulosum are formed earlier than the deeper granule cells. The bearing of some of these findings on the development and organization of the connections of the dentate gyrus is discussed.


Origin of specific synaptic types in the motoneuron neuropil of the monkey. An attempt has been made to establish, so far as possible, the specific types of synaptic bulbs on motoneurons which arise from extrinsic (descending and dorsal root) and intrinsic sources (interneurons). This has involved the laborious analysis of thousands of electron micrographs of material from normal motoneuron neuropil, and motoneuron neuropil of animals with lesions of spinal tracts, spinal roots, and motor cortex. Our studies have established that the large synaptic bulbs on dendrites, which are the only type in the spinal cord to possess pre-synaptic synapses (serial synapses), are derived from monosynaptic dorsal root fibers (R bulbs). The presynaptic component of the serial synapse appeared to degenerate at levels below spinal cord transections (P bulbs). By means of transections of spinal cord we have found that descending fibers in the spinal cord terminate as one of at least two distinct classes of synaptic bulbs, one with spheroid synaptic vesicles (S) and one with flattened vesicles (F). There is evidence from several sources that the first type may often be excitatory in function and the second type inhibitory. Only synaptic bulbs with spheroid vesicles show definite signs of degeneration after lesions of the opposite motor cortex. The large (L) synaptic bulbs on motoneuron somata, associated with subsynaptic cisterns, are clearly not derived from the descending systems, or from posterior root fibers.


Demonstration of terminalis, olfactory, trigeminal and perivascular nerves in the rat nasal septum. The innervation of the nasal septum and around the olfactory bulb has been investigated in rats by means of whole-mount preparations and histological sections. Silver staining, OsO4 staining, PAS staining, cholinesterase reaction and fluorescence for catecholamine-containing nerves were used. The nervus terminalis forms on the medial side of the olfactory bulb a ganglionated plexus, from which branches are given off which course peripherally with the vomeronasal nerves. From a dorsal part of the terminalis nerve plexus an anterior branch is given off which runs along the anterior ethmoidal nerve to the nasal vestibule where it connects with a group of ganglia. The peripheral branches of the nerve run from here along two epithelial cristae formed histologically like dermal papillae. Ventrally in the respiratory region at the junction of the nasal cavity and the nasopharynx is a 1 times 2 mm area with olfactory epithelium, glands of Bowman and an independent innervation from the olfactory bulb. This is the so-called septal olfactory organ. Trigeminal nerves form a plexus in the respiratory region and in the vestibule, but do not supply the olfactory region. Catecholamine-containing and cholinesterase-positive nerves run along the meningeal arteries on the cribriform plate and accompany their branches to the vascular plexus in the olfactory and respiratory regions. Double innervation is found not only of this vascular plexus but of the venous sinuses in the swell bodies of the vestibule. The glands of the nose are not surrounded by catecholamine-containing nerves.


Monoamine distribution in primate brain. I Catecholamine-containing perikarya in the brain stem of Macaca speciosa. The distribution of catecholamine-containing cell bodies was examined in the brain stem of Macaca speciosa using the Falck-Hillarp histofluorescence technique. Extensive accumulations of such cells were seen in the ventral tegmental area, locus coeruleus, mesencephalic reticular formation and ventrolateral reticular formation of pons and medulla. This distribution was compared to that previously reported in rat, cat, squirrel monkey and human brain. Apparent species dissimilarities and similarities are reported.


The ventral spinothalamic tract and other ascending systems of the ventral funiculus of the spinal cord. The ascending degeneration resulting from experimental lesions of the ventral funiculus of the spinal cord of Macaca mulatta has been studied using the Nauta technique and its variants. The ventral spinothalamic tract is shown to be an independent entity with respect to the lateral spinothalamic tract; its fibers are widely distributed in the ventral funiculus and it establishes connections with the brain stem and thalamus which are analogous but not identical to those of the latter. Its role in the relay of nociceptive input is discussed in view of the similarity in hodology of the two systems and it is proposed that it may be responsible for the failure of anterolateral cordotomy to control pain on a long term basis. Other ascending systems in the ventral funiculus include the spino-olivary and spino-reticular tracts, as well as minor connections to the N. of Edinger-Westphal, the red nucleus and the superior colliculus. The projections from the ventral quadrant of the spinal cord to the brain stem are almost entirely ipsilateral until the rostral mesencephalon is reached, at which level the N. of Darkschewitz receives both ipsilateral and crossed input; the magnocellular nucleus of the medial geniculate body receives a small contribution which is mainly ipsilateral. In the thalamus the VPL receives predominantly ipsilateral projections while the input to the paralaminar nuclei is only slightly less pronounced contralaterally than ipsilaterally.


The cochlear nuclei of lizards.

The cochlear nuclei of lizards. The cochlear nuclei of 14 lizard species (eight families) were studied in normal animals and in a small series of animals with lesions of the posterior division of the eight nerve. The development of the cochlear nuclei is division of the eighth nerve. The development of the cochlear nuclei is directly related to the length and complexity of the papilla basilaris. The best development of basilar papillae and cochlear nuclei is found in teiid and gekkonid lezards, and an intermediate grade of development in scincid, lacertid, and anguid lizards. A lesser degree of development occurs in the iguanids, and practically no cochlear nuclei differentiation is observed in Chameleo. Two well defined cochlear nuclei are found in most lizard families: nucleus angularis (NA) AND nucleus magnocellularis medialis (NMM). NA is located in the cephalic third of the acoustic tubercle and contains variably sized darkly staining cells. NMM is the most caudally located nuclear group and is characterized by regularly round to ovoid cells. The development of NMM is more closely related to papilla basilaaris length and complexity than is NA. Two less well defined cochlear nuclei are also thought to be present. In the region beteen NA and NMM is a sparsely cell-populated area, nucleus magno-cellularis lateralis (NML), which consits of a variety of small, darkly staining cells and large, pale staining cells which are usually laterally located in the nucleus. Like NMM, Nml is better developed in lizards ith more complex papillae basilares. Nucleus laminaris may be represented by a few fusiform cells in the ventral portion of the NML region.


Deafferentation slows the growth of specific dendrites of identified giant interneurons. The effect deafferentation has on the morphology of giant interneurons was studied in the abdominal nervous system of crickets (Acheta domesticus). The morphology of four uniquely identified giant interneurons was exxamined by iontophoresing cobalt chloride into the neurons of interest. A major source of afferents for these interneurons consists of mechanoreceptors located on paired abdominal sensory appendages -- the cerci. Partial deafferentation of the giant interneurons was obtained by pinching off the cercus at hatching and maintaing the specimen in this deprived condition until adulthood. The interneurons of three groups of animals were examined; control specimens which were not treated surgically, unilaterally treated specimens which had a single cercus removed and bilaterally treated specimens which had both cerci removed. Two types of morphological changes were detected. (1) Chronic removal of a cercus was correlated with a reduction in length of dendrites ipsilateral to the ablated cercus; however, the general form of the dendritic branching pattern remained constant and recognizable. Two dendrites of a single neuron could be influenced independently if they were innervated by separate cerci. Thus deprivation did not have a generalized effect on growth of a neuron, rather it specifically influenced the dendrites deprived of afferents. (2) It was also observed that the projection of cercal sensory fibers in specimens reared with a single cercus differed from normal in that scattered fibers cross the midline in regions of the ganglion where none usually exist. It is suggested that modifications in the response properties of these deprived neurons are based on these two changes in morphology.


Scanning and transmission electron microscope studies of interkinetic nuclear migration in the cerebral vesicles of the rat. A scanning electron microscope (SEM) was used to examine the morphology and surface texture of neuroepithelial cells during interkinetic nuclear migration in the cerebral vesicles of the rat at 12, 13 and 14 days of gestation. Serial sections of embryonic material of the same age were also prepared for the transmission electron microscope (TEM). Particular attention was paid to the SEM and TEM appearance of mitotic neuroepithelial cells which occur exclusively along the ventricular border of the neural epithelium. Three distinctly-shaped classes of mitotic cells were recognised in scanning micrographs. (1) Pyriform cells. This type of mitotic cell was characterised by the presence of very long, fine processes radiating from the tip and shoulders of a short external (basal) process. These fine processes were termed "intramitotic filopodia." Microvilli were found on the surface of most pyriform cells. (2) Conical cells. These lacked an external process but there were large numbers of intramitotic filopodia at the basal pole of the cell body, and the perikaryal surface was rich in microvilli. (3) Globular cells. It was possible to subdivide this class of cell into large and small sizes, but usually a few short intramitotic filopdia were present at the basal pole. The perikaryal surfaces of the globular population were raised in coarse lumps and bubble-like protrusions. By pooling TEM and SEM information we were able to deduce that pyriform cells probably possess a prophase or prometaphase chromosome morphology, while conical cells exhibit a chromosome morphology somewhere between prometaphase and early anaphase. Large globular mitotic cells were found to be between metaphase and late anaphase and small globular cells were identified as early telophase cells. On the basis of these findings we have proposed that as a bipolar neuroepithelial cell rounds up for mitosis it passes first through a pyriform stage during which the external process is retracted or broken down, and then through a conical stage when the cell consolidates its position on the ventricular surface. Finally, the cell enters a large globular stage before dividing into two small globular telophase cells. It is not known what part, if any, the intramitotic filopodia play in this process of rounding up.


The topographical and laminar organization of the presubiculum's projection to the ipsi- and contralateral entorhinal cortex in the guinea pig. This is a detailed report of a previously described projection from the presubiculum to the entorhinal area (Shipley, '74). The terminal degeneration in the entorhinal area is specifically confined to the outer three layers of this cortex. The projection is topogtaphically organized and confined to dorsal and medial parts of the entorhinal area. A similarly organized projection courses via the dorsal psalterium (dorsal hippocampal commissure) to the contralateral entorhinal area. Thus each presubiculum has an organized terminal pathway to the entorhinal cortices of both hemispheres.


Involvement of the avian amygdalar homologue (archistriatum posterior and mediale) in defensively conditioned heart rate change. Archistriatal involvement in visually conditioned heart rate change (established by pairing light and foot-shock) was studied in 198 pigeons by evaluating conditioning performance following lesions of different archistriatal subdivisions or interruption of their subtelencephalic projections. Lesions of the anterior-intermedium archistriatum or interruption of its descending projection (tractus occipitomesencephalicus) had no effect on conditioned response development. In contrast, destruction of the posterior-mediale region or interruption of its descending projection (tractus occipitomesencephalicus, pars hypothalami) produced profound conditioning deficits. The findings are entirely consistent with a recent anatomical analysis (Zeier and Karten, '71) suggesting that only the posterior-mediale archistriatum constitutes the avian amygdalar homologue. It is concluded that the integrity of the posterior-mediale region and its projection to the hypothalamus via the tractus occipitomesencephalicus, pars hypothalami are essential for establishing visually conditioned heart rate change in a defensive conditioning paradigm, and these findings are discussed in the context of structures previously implicated in mediating this conditioning autonomic response.


The role of supraspinal input in embryonic motility: a re-examination in the chick. The present experiments represent an attempt to further clarify the role of the brain in embryonic motility and behavior. By making high chronic cervical transections ("gaps") at early prefunctional stages of incubation (i.e., 40-50 hours) and studying the subsequent emergence of motility in the chick it has been possible to determine that supraspinal input is not functional until about the tenth day of incubation. Acute cervical transection results in a modification of the temporal pattern (rhythm) of motility without affecting the frequency of activity. Qualitatively the movements of spinal embryos are indistinguishable from controls up to 16-17 days. At that time there are detectable differences in the character of spontaneous movements, in reflex responsivity and in hatching behavior; spinal embryos are not able to initiate the coordinated Type III movements necessary for escape from the shell. Injection of strychnine into chronic cervical and control embryos at 10 days and at 16-17 days indicates that certain aspects of the typical strychnine response are lost following removal of brain input to the spinal cord. Finally, chronic thoracic gaps result in a clear modificaect at this age. These data suggest that propriospinal integration is present at least several days prior to the onset of supraspinal input in the chick spinal cord.


Synaptic patterns in the visual cortex of turtle: an electron microscopic study. The part of turtle general cortex that receives afferent fibers from the dorsal lateral geniculate nucleus and that shows evoked potentials to light stimuli has been studied with the electron microscope. This cortex consists of an outer molecular layer, a perikaryal layer, and a subcellular layer lying on a row of ependymal cell bodies. Neurons in the perikaral lamina are characterized by long spine-bearing apical dendrites ascending through the outer molecular layer and short finer basal dendrites in the subcellular zone. Scattered neurons without apical dendrites occur in both the molecular and subcellular zones. Two types of dendritic spines can be distinguished. Some are large, have a complex irregular shape, contain a variety of membranous sacs and mitochondria, and occasionally, a single bundle of microtubules embedded in an electron-dense background [corrected] opacity. These large spines are the most common postsynaptic element in the outer third of the molecular layer, where they are located on the distal enlargement that contains only electron-dense fuzz. They are the most common post-synaptic element in the lower two-thirds of the molecular layer where they arise from the proximal portion of apical dendrites. Most synaptic contacts are found on the dendritic spines and are of the "round-asymmetrical" type. Not infrequently "flat-symmetrical" synapses are coupled to "round-asymmetrical" contacts on individual large spines. The few contacts present on spine-bearing dendritic shafts are of both types. Axo-somatic contacts are mainly of the "flat-symmetrical" variety. Thus the synaptic patterns on the principal cells of turtle visual cortex are remarkably similar to those found on pyramidal cells of mammalian neocortex. In addition, however, axon terminals, dendrites and glial (ependymal) processes were often seen to give rise to membranous pouches containing large vacuoles and invaginating into dendritic shafts or spines. Rarely, axon terminals were seen to form contacts, identical in appearance to synaptic contacts, on cell bodies in the ependymal lining. More frequently, unusual types of membrane differentiations were present at the site of apposition of the membranes of axon terminals and ependymal processes. They are interpreted as functional neuroependymal contacts.


Fine structure and distribution of axon terminals from the cochlear nucleus on neurons in the medial superior olivary nucleus of the cat. The morphology and distribution of axon terminals on central column and marginal neurons of the cat medial superior olivary nucleus (MSO) were analyzed by electron microscopy. Individual neurons or groups of cells oriented such that substantial lengths of their dendrites were within a 5-7 mu thich section were selected for detailed study. Thin sections were cut from remounted thick sections. Boutons with spherical vesicles arise directly from myelinated axons; more than one synaptic region of an axon, each separated by a myelinated segment, may contact a given dendrite. Boutons with flattened and occasionally dense core vesicles arise from both myelinated and unmyelinated portions of axons; these axons may also have more than one synaptic region. Both kinds of synaptic profiles are found on the somata and dendrites of all MSO neurons. To determine which nerve endings are from the cochlear nucleus (CN) lesions were made to produce orthograde degeneration. Following unilateral CN lesions degenerating spherical vesicle terminals were observed on the lateral dendrites and somata of ipsilateral central column cells and the medial dendrites and somata of contralateral neurons. Degenerating terminals were rarely seen on the opposite dendrite (three of 48 cells). In six of seven instances where medial and lateral dendrites of two cells overlapped degeneration was limited to one oriented toward the lesion. Marginal cells examined received virtually all spherical vesicle terminals from only one CN. Terminals with flattened vesicles persisted on the somata and dendrites of all neurons studied including cells from cats with bilateral lesions.


The development of synapses in the visual system of the cat. Synapses have been counted by electron microscopy and neurones by light microscopy through the depth of the visual cortex in a series of cats from 37 days gestation to adulthood. A few definite synapses are present as early as three weeks before birth, but there is then a latent period of four weeks before synapses increase rapidly in number 8-37 days after birth. The synapses occur just above and just below the cell plate at first, but in the adult cat they become evenly distributed in the depth of the cortex. The gradual separation of neurones by neuropil during development precedes a parallel increase in the density of synapses by about one week. The average number of synapses associated with one neurone rises to a peak of about 13,000 at seven weeks after birth. The densities of synapses and of neurones subsequently fall to slightly lower values in adult cats as the glial cells continue to develop. The timing of synaptic development in the visual cortex has been compared quantitatively with that in the L. G. N. and qualitatively with synaptogenesis in the retina. Synapses develop in the L. G. N. and cortex in a parallel fashion, and the L. G. N. precedes the cortex by a short interval of about two days. In the cell plate of the retina a few receptor synapses are present nine days before birth. Inner plexiform synapses are aslo present at this time, but ribbon-containing synapses do not appear until birth. Very few receptors possess outer segments with discs at birth, but five days later disc-bearing outer segments have developed. Thus synaptic development starts before afferent impulses can enter the visual system, but the main increase in synapses in the L. G. N. and cortex takes place four weeks after the start of synapse formation while the visual system is being used.


Receptive fields of single cells and topography in mouse visual cortex. The visual cortex was studied in the mouse (C57 Black/6J strain) be recording from single units, and a topographic map of the visual field was constructed. Forty-five percent of the neurons in striate cortex responded best to oriented line stimuli moving over their receptive fields; they were classified as simple (17%), complex (25%) and hypercomplex (3%). Of all preferred orientations horizontal was most common. Fifty-five percent of recpetive fields were circularly symmetric: these were on-center (25%), off-center (7%) and homogeneous on-off in type (23%). Optimal stimulus velocities were much higher than those reported in the cat, mostly varying between 20 degrees and 300 degrees/sec. The field of vision common to the two eyes projected to more than one-third of the striate cortex. Although the contralateral eye provided the dominating influence on cells in this binocular area, more than two-thirds of cells could also be driven through the ipsilateral eye. The topography of area 17 was similar to that found in other mammals: the upper visual field projected posteriorly, the most nasal part mapped onto the lateral border. Here the projection did not end at the vertical meridian passing through the animal's long axis, but proceeded for at least 10 degrees into the ipsilateral hemifield of vision, so that at least 20 degrees of visual field were represented in both hemispheres. The magnification in area 17 was rather uniform throughout the visual field. In an area lateral to area 17 (18a) the fields were projected in condensed mirror image fashion with respect to the arrangement of area 17. Medial to area 17 a third visual area (area 18) was again related to 17 as a condensed mirror image.