The low molecular weight folate binding protein (FABP) has been purified 1000-fold to a specific activity of 7.2 gamma g of pteroylglutamic acid (PGA) bound per mg of protein. This purified FABP represents two protein bands that bind PGA on polyacrylamide disc gel electrophoreis, elutes from DEAE-cellulose in 0.001 M phosphate buffer, stains positive with PAS, Elutes from concanavalin A Sepharose affinity columns with methyl alpha-mannoside, and shows three major peaks (pl =6.8, 7.5, 8.2) by isotric focusing. The binding of PGA to purified FABP dependent on pH and is inhibited by urea...

The phosphorylated form of liver glycogen phosphorylase (alpha-1,4-glucan : orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) (phosphorylase a) is active and easily measured while the dephosphorylated form (phosphorylase b), in contrast to the muscle enzyme, has been reported to be essentially inactive even in the presence of AMP. We have purified both forms of phosphorylase from rat liver and studied the characteristics of each. Phosphorylase b activity can be measured with our assay conditions. The phosphorylase b we obtained was stimulated by high concentrations of sulfate, and was a substrate for muscle phosphorylase kinase whereas phosphorylase a was inhibited by sulfate, and was a substrate for liver phosphorylase phosphatase. Substrate binding to phosphorylase b was poor (KM glycogen = 2.5 mM, glucose-1-P = 250 mM) compared to phosphorylase a (KM glycogen = 1.8 mM, KM glucose-1-P = 0.7 mM). Liver phosphorylase b was active in the absence of AMP. However, AMP lowered the KM for glucose-1-P to 80 mM for purified phosphorylase b and to 60 mM for the enzyme in crude extract (Ka = 0.5 mM). Using appropriate substrate, buffer and AMP concentrations, assay conditions have been developed which allow determination of phosphorylase a and 90% of the phosphorylase b activity in liver extracts. Interconversion of the two forms can be demonstrated in vivo (under acute stimulation) and in vitro with little change in total activity. A decrease in total phosphorylase activity has been observed after prolonged starvation and in diabetes.

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95% pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 degrees C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10(-4) M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20-30% of the activity could be recovered when the pH was returned to 7.5. In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel electrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.

Histamine has a dual action on the isolated perfused ear preparation of the rabbit. The amine induced a dose-dependent rise in perfusion pressure when the preparation was perfused with Krebs' solution. This pressor response was reversed to a depressor effect when meypramine was added to the perfusion fluid. This depressor effect of the amine was also dose-related. Metiamide competitively inhibited the depressor effect of histamine. Prior treatment of the ear vessels with metiamide alone caused an increase in histamine-induced perfusion pressure. From these results it was concluded that the predominant pressor effect of histamine on the vascular bed of the rabbit ear is mediated through the H1-receptors and the depressor effect of the amine through histamine H2-receptors.

Experiments were conducted on male rats. A study was made of the content of nicotinamide coenzymes in the liver and myocardium 24 hours after the administration of 0.5 ml of dichloroethane into the stomach. In parallel with disturbance of the morphological structure of the liver and of the myocardium, increase in the activity of alanine and aspargic aminotranspherases in the blood serum, dichloroethane reduced the content of nicotinamide coenzymes and deranged the ratio of their oxidized and reduced forms in these organs.

A double-blind study with intra-individual comparisons was carried out to investigate the effects of 15 mg of (8r)-3alpha-hydroxy-8-isopropyl-1alphaH-tropanium bromide(+/-)-tropate (Sch 1000), 15 mg Sch 1000 + 10 mg oxazepam, 10 mg oxazepam and placebo with oral administration in randomized sequence on gastric juice volume, amount of acid, concentration and pH values in 12 healthy volunteers. The secretion parameters were measured during a 1-h basal period and a 2-h stimulation period. The gastric juice was obtained in 15 min portions via stomach tube. Stimulation was effected by 1 mug/kg/h pentagastrin via drip infusion. The Friedman test was used for the comparative statistical evaluation, and individual comparisons were carried out by means of the Wilcoxon test (pair-differences rank). The results show that Sch 1000 and Sch 1000 + oxazepam were equal in effect on basal and stimulated secretion volume. As compared with placebo, it was not possible to establish an effect on secretion volume for oxazepam alone. Sch 1000 and Sch 1000 + oxazepam were found to be equipotent in reducing the amount of basal acid, while oxazepam reduced this quantity only during the first 30 min of basal secretion. None of the three active preparations was capable of inhibiting the stimulated acid, although both Sch 1000 preparations produced a clear trend towards lowered mean values. During the basal secretion period, all three test preparations had an inhibiting action on acid concentration, but none of them had a significant effect during the stimulation period. The pH value was savely increased only by Sch 1000 and Sch 1000 + oxazepam, and this even only during the basal period. The results are discussed.

A simple, highly sensitive and reproducible method for the assay of gamma-glutamyl transpeptidase (EC 2.3.2-) activity is introduced, using gamma-glutamyl-p-nitroanilide as a substrate and glycylglycine as an acceptor in 50 g/l of polyoxyethylene nonylphenol. Serum transpeptidase activity was assayed in 1080 healthy adults, the normal mean value being 14.8 mU/ml. The diagnostic evaluation of the enzyme in various hepatobiliary diseases is also discussed.

Lethally irradiated male and female hybrids, parental strains, and the third party strain A mice were inoculated with reciprocal hybrid marrow from donors of the same sex. A graft-versus-host reaction was produced against H-Y antigens of reciprocal hybrids. Although the late mortality in inbred strain males was generally greater than that of females, without further tests this difference was not automatically attributable to an X or a Y chromosome disparity. Hybrids did not recognize the qualitative difference in antigenicity of their reciprocals, but they were able to recognize the quantitatively greater antigenicity of their parental strains, and a hybrid-versus-parental strain reaction occurred. The graft-versus-host reaction in the third party strain A mice was significantly more severe than that produced by hybrid marrow in their reciprocal hybrid recipients, but significantly less severe than that produced in the parental strains. The relationship between antigenicity and responsiveness was such that the severity of the graft-versus-host reactions masked the maternal influences observed with other reciprocal hybrids.

Tne ultrastructural and fluorescence histochemical characteristics of the mature rabbit substantia nigra and neostriatum have been reviewed as a frame of reference for the developmental study. Biochemical investigations were reported on neostriatal dopamine concentrations and the relative uptake and accumulation of 3H-dopamine by this tissue from fetal to adult stages, to provide quantitative data for correlation with the fluorexcence information. The development of the neurons of the substantia nigra and their axons which project to the neostriatum has been presented from their appearance at day 14 of gestation to their maturation in early postnatal life. The initial bipolar neuroblasts, which develop in the midline of the caudal mesencephalon, are fluorescent as soon as they emerge from the ependymal zone. Their fluorescent axons, which form the nigroneostriatal pathway, reach the telencephalon at day 16 of gestation and ramify extensively in the putamen by day 20, but do not enter the caudate nucleus until several days later. Some of the early fluorescent axonal profiles in the putamen are extremely large. Electron microscopic study of theis stage suggests that the large fluorescent profiles may correspond to axonal growth cones or early synapses. A distinct substantia nigra, pars compacta and reticulata, can be recognized by fluorescence microscopy by day 20 of gestation. Electron microscopy reveals that the young neurons are multipolar with numerous developing dendrites, some of which exhibit early synaptic junctions. The subsequent maturaition of these cells and the neuropil is described. The fluorescent axons of the substantia nigra grow into the putamen and caudate nucleus in a nonuniform manner forming fluorescent islands throughout the neostriatum in late fetal life. Occasionally, minute beaded fluorescent axons are found. These profiles might correspond to some of the axons with varicosities "en passage" revealed by electron microscopy. In an attempt to identify further the dopamine-containing axon, the ultratructure of adult neostriatum incubated in 5-hydroxydopamine was reported. Axonal varicosities "en passage" containing a dense "tag" in the vesicles were found. Most of the tagged boutons did not exhibit synaptic contacts. The possible significance of these finding s as related to dopamine secretion are discussed.

Poly(8-aminoguanylic acid) has in neutral solution a novel ordered structure of high stability. The 8-amino group permits formation of three hydrogen bonds between two residues along the "top", or long axis, of the purines. The usual hydrogen bonding protons and Watson-Crick pairing sites are not involved in the association. The bonding scheme has a twofold rotation axis and is hemiprotonated at N(7). Poly(8NH2G) is converted by alkaline titration (pK = 9.7) to a quite different ordered structure, which is the favored form over the range approximately pH 10-11. The bonding scheme appears to be composed of a planar, tetrameric array of guanine residues, in which the 8-amino group does not participate in interbase hydrogen bonding. Poly (8NH2G) does not interact with poly(C) in neutral solution because of the high stability of the hemiprotonated G-G self-structure. Titration to the alkaline plateau, however, permits ready formation of a two-stranded Watson-Crick helix. In contrast to the monomer 8NH2GMP, poly(8NH2G) does not form a triple helix with poly(C) under any conditions. The properties of the ordered structures are interpreted in terms of a strong tendency of the 8-amino group to form a third interbase hydrogen bond, when this possibility is not prevented by high pH.

A method was developed which involved electroimmunoassay and crossed immunoelectrophoresis of subtilopeptidase A (EC 3.4.21.14). Initial trials with unfractionated antiserum were not successful and interaction of the enzyme with non-immunoglobulin serum components were shown to be the cause of the failures. Quantitative immunoelectrophoresis was possible when purified immunoglobulins were used. A pH of 6.5 (lower than the usual pH 8.6) was necessary to obtain a proper baseline definition. Subtilopeptidase A was confirmed as a multiple isoenzyme system. Qualitative inter-batch variations were detected. Di-isopropyl phosphorofluoridate inhibition altered the electrophoretic pattern, but no loss of antigenic determinants was observed.