Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Myelin Gangliosides in Vertebrates

Abstract: A phylogenetic survey of brain myelin ganglioside patterns and concentrations has been carried out on 16 vertebrate species. Gangliosides were isolated from purified myelin and found to vary in concentration from 25 μg of sialic acid per 100 mg of myeh for goldfish to a value of 395 for turkey. The latter species had approximately equivalent amounts of G_M1 and G_M4 as the two major gangliosides. The 11 mammals studied all had G_M1 as the major ganglioside, with variable amounts of G_M4; rhesus monkey and human had 20-25% G_M4, whereas the others had less than 10%. Amphibia and fish myelin contained the least total ganglioside, with patterns that showed relatively little G_M1 and no detectable G_M4. Alligator myelin was unique in having a total concentration as high as the avian species, but a pattern with predominantly diand trisialo gangliosides.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem- brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter")membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of ¹²⁵I-labeled cell surface proteins. Their specific (Na⁺+ K⁺)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo- plasmic reticulum, as judged from the activity 0: NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.     

Competitive Inhibition of γ-Aminobutyric Acid Synaptosomal Uptake by 4-(4'-Azidobenzoimidylamino)butanoic Acid

Abstract: 4-(4'-Azidobenzoimidylamino)butanoic acid (ABBA) is a potent inhibitor of rat brain synaptosomal [³H]γ-aminobutyric acid uptake. K₁ values were calculated to be 8 μM and 16 μM with respect to the high-affinity and the low-affinity uptake processes. These values are of the same order as those reported for nipecotic acid and guvacine, which until now have been the most potent uptake inhibitors available. Since ABBA contains a phenyl group, it might be capable of penetrating the blood-brain barrier, thus becoming a useful GABA mimetic.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

Catastrophe theory of dopaminergic transmission: a revised dopamine hypothesis of schizophrenia

Mathematical modeling of experimentally observed parameters of dopaminergic neuronal activity suggests the occurrence of multiple equilibrium states in neurons characterized by certain precisely defined properties of the tyrosine hydroxylating system. These equilibria may become unstable under certain conditions and transitions between multiple states are predicted. In addition, modeling of the spatial interactions of dopamine neurons within a neural net leads to domain wall soliton-like solutions of neuronal firing. In the discrete spatial case, these equations are isomorphic to those of the Ising model of phase transitions in lattice spins.The hypothesis is proposed that the occurrence of multiple stable equilibrium states rather than excessive dopaminergic transmission forms the pathophysiological basis of the schizophrenic thought disorder.The model is internally consistent with known clinical effects of drugs such as neuroleptics, reserpine and amphetamine. In agreement with postmortem and other studies, the theory predicts the lack of increased concentrations of dopamine or its major metabolite, homovanillic acid, in brain and cerebrospinal fluid of schizophrenics.The mathematical model is compatible with the theory that postulates an attention deficit as an underlying mechanism of schizophrenic psychosis and allows for a possible genetic heterogeneity of the disease.     

A Gas chromatographic (GLPC) model for the sense of smell. Variation of olfactory sensitivity with conditions of stimulation

Computer simulation of an olfactory detector has been developed using a chemical kinetic scheme originally proposed by McNab and Koshland for bacterial chemotaxis. This model describes response as a function of two opposed reactions, both of which are activated by odorant. One reaction turns on response, while its opponent shuts it off. Net response to various stimulus profiles is compared to psychophysical experiments, with particular attention paid to simulating magnitude estimation and odor adaptation results. Effects of the access route to this detector are evaluated. Transport of odorant molecules is treated as having two sequential steps: step (i), airborne odorant is carried parallel to a retentive layer (mucus) into the detector region; step (ii), molecules diffuse through the retentive layer to the detector. Step (i) is represented as analogous to GLPC on an open tubular column. Each step has a characteristic time constant, which is proportional to (distance)²/diffusion coefficient. Response to highly volatile odorants tends to be limited by step (ii), while odorants of low volatility approach the step (i) limit. Sensitivity at both limits is attenuated by increasing the thickness of the retentive layer, but sensitivity at the step (i) limit is also affected by changes in air passageway and airflow characteristics. This picture can be used to explain variations in women's sensitivity to odorants of low volatility with the menstrual cycle, while their detection of volatile odorants fluctuates to a much lesser extent.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.