Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Different relative abundance of neurotensin and neuromedin N in bovine ocular tissues.

Neurotensin (NT) and neuromedin N (NN) are regulatory peptides encoded by the same gene and located in tandem within a common precursor. Using specific radioimmunoassays for both peptides, their relative abundance in extracts of bovine ocular tissues has been examined. Within the retina, the molar concentration of NN was significantly higher (P less than 0.001) than that of NT. In contrast, within both choroid/sclera and iris/ciliary bodies, the molar concentration of NT was significantly higher (P less than 0.001) than that of NN. These data demonstrate that the theoretical molar ratio of 1:1, which would result from complete processing of both peptides from the common precursor, does not occur in any of the ocular tissues examined. Reverse phase HPLC of extracts of each ocular tissue confirmed the differential abundance of NT and NN. These data would suggest that the common NT/NN precursor is differentially-processed within bovine ocular tissues, a finding which may be of physiological significance.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3 untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.     

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.     

A reexamination of the properties of spinach nitrite reductase: protein and siroheme content heterogeneity in purified preparations

Recent preparations of nitrite reductase do not display the heterodimeric quaternary structure obtained previously (total molecular weight 85,000; subunit molecular weights 24,000 and 61,000), but rather yield only the 61,000 molecular weight subunit, even when buffers containing the protease inhibitor phenylmethylsulfonyl fluoride are used. Nevertheless, such preparations retain the high ratio of ferredoxin-linked to methyl viologen-linked enzyme activity which has been previously taken as a characteristic of only the heterodimeric form. These preparations display a siroheme prosthetic group to protein ratio of 1.1. When nitrite reductase samples are frozen during the purification scheme, even though the ferredoxin-linked specific activity does not significantly decrease, enzyme activity-stained native gel electrophoresis of the subsequently purified protein reveals that gels with several bands of activity can be obtained. Further evidence of protein heterogeneity in these preparations comes from N-terminal amino acid analysis which reveals that even nonfrozen preparations contain two major peptides with valine and cysteine as the N-termini. Formation of complexes of purified nitrite reductase with ferredoxin resulted in siroheme difference electronic spectra which resembled those observed previously for monomeric preparations. However, the siroheme midpoint potential of recent preparations of nitrite reductase (-287 mV) is close to that of the heterodimeric preparations. Ultrafiltration studies of crude extracts of the enzyme indicate that, at least at certain stages of the preparation, higher molecular weight forms of the enzyme may exist. We conclude that the 24,000 molecular weight polypeptide is a contaminant and that the heterodimeric quaternary structure model for spinach nitrite reductase is incorrect. Furthermore, the monomeric preparations we do obtain display both significant protein heterogeneity and facile loss of siroheme upon gel filtration.     

Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase

Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic fluorescence of chloramphenicol acetyltransferase: responses to ligand binding and assignment of the contributions of tryptophan residues by site-directed mutagenesis

Replacement by tyrosine or phenylalanine was used to assign the additive contributions of each of the three tryptophan residues of chloramphenicol acetyltransferase (CAT) to its intrinsic fluorescence on excitation at 295 nm. During the assessment of the fluorescence responses of the wild-type enzyme to the binding of ligands, it was found that the overlapping absorption spectra of chloramphenicol and tryptophan, with an attendant inner filter effect, required the use of a displacement technique involving an alternative substrate (the p-cyano analogue of chloramphenicol) without significant absorption at 295 nm. By the use of two-Trp, one-Trp, and Trp-less variants, in combination with this displacement technique, it was possible to demonstrate that Trp-86 and Trp-152 are involved in the fluorescence quenching associated with the binding of chloramphenicol, most likely via nonradiative energy transfer from these residues to the bound substrate. Trp-152 is mainly responsible for the fluorescence enhancement accompanying the binding of acetyl-CoA (and CoA) through proximity effects and solvent exclusion on substrate association.     

The distribution of neuropeptides in the ocular tissues of several mammals: a comparative study.

1. The distribution of several neuropeptides (vasoactive intestinal peptide, substance P, somatostatin and neurotensin) was assessed in ocular tissues from the cow, sheep, rabbit and rat. 2. Vasoactive intestinal peptide was most abundant in the choroid and sclera in all species except the rat. Substance P was most abundant in the retina of cow and rat and in the iris/ciliary body of sheep and rabbit. Somatostatin and neurotensin were most abundant in the retina of all species examined. 3. Regulatory peptides thus display distinct regional distributions within the ocular tissues of a single species of mammal and, in addition, exhibit interspecific variation.