Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Purification and characterization of two forms of hydrogenase isoenzyme 1 from Escherichia coli.

A hydrogenase associated with dihydrogen uptake (HUP hydrogenase) was purified from an Escherichia coli mutant (strain SE1100) defective in utilization of molybdate and thus fermentative dihydrogen production. This protein had two subunits with apparent molecular weights of 59,000 and 28,000 (form 1). An immunologically cross-reactive hydrogenase was also purified from E. coli K10 grown in glucose-minimal medium and harvested at the mid-exponential phase of growth. Upon purification to homogeneity, this hydrogenase contained only one subunit with an apparent molecular weight of 59,000 (form 2). The two forms of the HUP hydrogenase exhibited similar kinetic characteristics. The electrophoretic properties of the enzyme and its response to pH suggest that this HUP hydrogenase is the HYD1 isoenzyme. The HYD1 isoenzyme was the only hydrogenase detectable during the stationary phase of growth in E. coli grown in Mo-deficient medium.     

Solution structure of the nogalamycin-DNA complex

The nogalamycin-d(A-G-C-A-T-G-C-T) complex (two drugs per duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Two equivalents of nogalamycin binds to the self-complementary octanucleotide duplex with retention of 2-fold symmetry in solution. We have assigned the proton resonances of nogalamycin and the d(A1-G2-C3-A4-T5-G6-C7-T8) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site on duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the nogalamycin and the DNA in the complex. The molecular dynamics calculations were guided by 274 intramolecular nucleic acid distance constraints, 90 intramolecular nogalamycin distance constraints, and 104 intermolecular distance constraints between nogalamycin and the nucleic acid protons in the complex. The aglycon chromophore intercalates at (C-A).(T-G) steps with the long axis of the aglycon approximately perpendicular to the long axis of the flanking C3.G6 and A4.T5 base pairs. The aglycon selectively stacks over T5 and G6 on the T5-G6-containing strand with the aglycon edge containing OH-4 and OH-6 substituents directed toward the C3-A4-containing strand. The C3.G6 and A4.T5 base pairs are intact but buckled at the intercalation site with a wedge-shaped alignment of C3 and A4 on the C3-A4 strand compared to the parallel alignment of T5 and G6 on the T5-G6 strand in the complex. The nogalose sugar in a chair conformation, the aglycon ring A in a half-chair conformation, and the COOCH3-10 side chain form a continuous domain that is sandwiched within the walls of the minor groove and spans the three base pair (G2-C3-A4).(T5-G6-C7) segment. The nogalose ring is positioned in the minor groove such that its nonpolar face is directed toward the G6-C7 sugar-phosphate backbone while its polar face containing OCH3 groups is directed toward the G2-C3 sugar-phosphate backbone in the complex. The intermolecular contacts include a nonpolar patch of aglycon (CH3-9) and nogalose (CH3-3') methyl groups forming van der Waals contacts with the base-sugar residues in the minor groove and intermolecular hydrogen bonds involving the amino groups of G2 and G6 with the ether oxygens OCH3-3' and O7, respectively, on the nogalose sugar.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR studies of an exocyclic 1,N2-propanodeoxyguanosine adduct (X) located opposite deoxyadenosine (A) in DNA duplexes at basic pH: simultaneous partial intercalation of X and A between stacked bases

The NMR parameters for the 1,N2-propanodeoxyguanosine (X) opposite deoxyadenosine positioned in the center of the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) X.A 9-mer duplex are pH dependent. A previous paper established protonated X5(syn).A14(anti) pairing in the X.A 9-mer duplex at pH 5.8 [Kouchakdjian, M., Marinelli, E., Gao, X., Johnson, F., Grollman, A., & Patel, D. J. (1989) Biochemistry 28, 5647-5657]; this paper focuses on the pairing alignment at the lesion site at pH 8.9. The observed NOEs between specific exocyclic CH2 protons and both the imino proton of G6 and the sugar H1' protons of C13 and A14 establish that X5 is positioned toward the G6.C13 base pair with the exocyclic ring directed between C13 and A14 on the partner strand. The observed NOE between the H2 proton of A14 and the imino proton of G4, but not G6, establishes that A14 at the lesion site is directed toward the G4.C15 base pair. NOEs are detected between all exocyclic CH2 protons of X5 and the H2 proton of A14, confirming that both X5 and A14 are directed toward the interior of the helix. The X5(anti).A14(anti) alignment at pH 8.9 is accommodated within the helix with retention of Watson-Crick pairing at flanking G4.C15 and G6.C13 base pairs. The energy-minimized conformation of the (G4-X5-G6).(C13-A14-C15) segment at pH 8.9 establishes that X5 and A14 are directed into the helix, partially stack on each other, and are not stabilized by intermolecular hydrogen bonds. The X5 base is partially intercalated between C13 and A14 on the unmodified strand, while A14 is partially intercalated between G4 and X5 on the modified strand. This results in a larger separation between the G4.C15 and G6.C13 base pairs flanking the lesion site in the basic pH conformation of the X.A 9-mer duplex. The midpoint of the transition between the protonated X5(syn).A14(anti) and X5(anti).A14(anti) conformations occurs at pH 7.6, establishing an unusually high pKa for protonation of the A14 ring opposite the X5 exocyclic adduct site. Thus, the interplay between hydrophobic and hydrogen-bonding contributions modulated by pH defines the alignment of 1,N2-propanodeoxyguanosine opposite deoxyadenosine in the interior of DNA helices.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.     

Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate.

1. The pH-dependences of the second-order rate constants (k) for the alkylation by chloroacetate of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) were determined over a wide range of pH at 25 degrees C at I 0.1. 2. The main feature of both pH-k profiles is a striking rate maximum at pH6 (characterizing parameters in both cases pKI approx. 3.5, pKII approx. 8.4 and pH-independent rate constant approximately kXH 2.5-3.0 M-1 . s-1). 3. The profile for the ficin reaction contains a plateau at high pH, with approximately kX 0.10 M-1 . s-1; if an analogous plateau exists in the papain reaction, approximately kX ix much lower, less than 0.02 M-1 . s-1. 4. Both enzymes appear to contain closely similar thiolate-imidazolium interactive systems at pH6, but differences in their behaviour in more-acidic media and in alkaline media suggest differences in interaction with the postulated carboxylate component of the putative catalytic triad.     

Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps

The cloned HindIII fragments of human cytomegalovirus (HCMV) strain AD169 DNA were mapped with respect to the BamHI, EcoRI and PstI restriction endonuclease cleavage sites. Composite restriction endonuclease cleavage maps for the entire virus genome were constructed using the previously established linkages between the HindIII fragments.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Improved stability of methanolic Wright's stain solution with dual additives

Degradation of methanolic Wright's stain solutions was greatly diminished with the addition of diethylamine hydrochloride and dimethylamine hydrochloride as costabilizers. Precipitation problems were eliminated by the dual additives. The stabilized stain solutions demonstrated good staining performance on blood smears. Methods for predicting the shelf life using calculated analytical parameters are described. Using these methods, the shelf life of a control stain solution was predicted to be 0.7 years; predicted shelf life was more than tripled with the addition of diethylamine hydrochloride and was increased approximately 27 times with the addition of both diethylamine hydrochloride and dimethylamine hydrochloride.