Studies of lipopolysaccharides from Yersinia pseudotuberculosis subtypes VA and VB

Lipopolysaccharides (LPS) from various strains of Yersinia pseudotuberculosis type V have been isolated and characterised. Differences in sugar composition and serological activity of LPS from various strains within the same subtype of Y. pseudotuberculosis have been revealed.     

Equilibration of leucine between the plasma compartment and leucyl-tRNA in the heart, and turnover of cardiac myosin heavy chain.

By 30min continuous infusion of [3H]leucine into rats, the specific radioactivities of plasma leucine and tissue-free and tRNA-bound leucine in heart were equal. The specific radioactivity of leucyl-tRNA in heart therefore follows a time course identical with that of plasma leucine soon after the start of infusion. The half-life of cardiac myosin heavy chain (5.5 days) was the same as that reported by other investigators who used the pulse-labelling protocol.     

Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis

Characterization of a $\text{Salmonella}\text{typhmurium}$ mutant strain (TA98/1,8-DNP₆) resistant to the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitroarenes) revealed that it was also non-responsive to the mutagenic action of nitroso- and N-hydroxylaminoarenes. The mutant strain was fully sensitive to the mutagenic action of the corresponding hydroxamic acid ester. These results suggest that TA98/1,8-DNP₆ is deficient in a specific esterifying enzyme and that esterification of the penultimate mutagenic metabolites of nitro- and aminoarenes ($\text{e.g.}$, arylhydroxylamines) to form potent electrophiles is controlled by a specific gene.     

Lipoamide dehydrogenase from Azotobacter vinelandii: site-directed mutagenesis of the His450-Glu455 diad. Spectral properties of wild type and mutated enzymes.

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active-site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild-type enzyme, whereas considerable changes were observed in the spectra of the two-electron-reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro451----Ala [corrected] showed the greatest deviation from wild type. The enzyme is very easily over-reduced to the four-electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis-trans conversion. From studies on the pH dependence of the thiolate charge-transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450-mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450----Phe, -Ser and -Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge-transfer thiols. The difference in apparent pKa2 values between the His450-mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild-type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852-862]. Based on the results obtained with the His450-mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild-type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455-mutated enzymes essentially follow the model proposed for the wild-type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge-transfer species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

A repetitive DNA element,associated with telomeric sequences in Drosophila melanogaster,contains open reading frames

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.by W. Hennig     

A new role for the transferrin receptor in the release of iron from transferrin

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.     

Study of the interaction of polyols with polymers containing n-substituted [(4-boronophenyl)methyl]-ammonio groups

Polymers, based on dextran and cellulose, having 2-{[(4-boronophenyl)-methyl]-ethylammonio}ethyl and -diethylammonio～ethyl groups were prepared. It was shown that these polymers could be employed for absorption of cis-diol compounds. The polymers were found to be highly specific towards polyols, carbohydrates, nucleosides, and nucleotides over a wide range of pH. The polymer based on DEAE-Sephadex A-25 was used in separating nucleosides, and in fractionating mononucleotides and carbohydrates. The chromatographic behavior of carbohydrates is defined by their structure and conformation, which are also responsible for different stabilities of the boronic complexes generated.