Characterization of autoreactive T cells. Relative importance of self-peptides versus MHC

The degree to which processed self-peptides contribute to the stimulation of autoreactive T cells has not been determined. In this study we have analyzed a panel of autoreactive T cell hybridomas from normal C57BL/26 mice produced by fusing peripheral lymph node cells with a variant of the BW5147 thymoma line, which does not express endogenous TCR alpha- and beta-chains. All of the autoreactive hybridomas responded to spleen cells expressing the syngeneic I-Ab allele, but not to allogeneic spleen cells. Although all hybridomas were I-Ab restricted, they demonstrated different patterns of reactivity to a panel of APC expressing I-Ab but derived from different genetic backgrounds. In a panel of APC expressing recombinant I-A molecules, changes in the second half of the first domain, which encodes alpha-helix segments that flank the Ag binding site and directly contact the TCR V regions in proposed models, eliminated reactivity of all hybridomas tested. In addition, most of the autoreactive hybridomas also demonstrated inhibition of reactivity to mutations in the amino half of the first domain of the I-A alpha- and beta-chains, which encodes the beta-pleated sheet of the floor of the Ag-binding groove. To confirm the role of processed peptides in the different patterns of reactivity, APC were incubated with competitor Ag and fixed by glutaraldehyde cross-linking before incubation with the autoreactive T hybridomas. The hybridomas were effectively inhibited by exogenous protein and peptide Ag. These results indicate that processed self-peptides are required to activate the autoreactive T cells.     

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.     

Intracellular mechanisms involved in short-term regulation of net protein synthesis in pancreatic acini.

The mechanisms regulating the net synthesis of digestive enzymes during short-term stimulation by agonists were examined in pancreatic acini isolated from the rat. Dispersed pancreatic acini were stimulated for up to 60 min with various concentrations of cholecystokinin octapeptide (CCK-OP), carbachol, A23187, 4 beta-phorbol 12-myristate 13-acetate (PMA). The effects of these agonists on net protein synthesis was determined by measuring the incorporation of [3H]leucine or [35S]methionine into protein. Carbachol, PMA, A23187 and concentrations of CCK-OP of 100 pM and greater caused inhibition of protein synthesis. Fluorography of [35S]methionine labeled acinar cell proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that the agonists inhibited the synthesis of the digestive enzymes. Northern blot analysis using cDNA probes revealed that CCK-OP, carbachol and PMA did not alter the cellular content of amylase, lipase and elastase mRNA. The protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine failed to reverse the inhibitory effects of CCK-OP, carbachol and PMA on protein synthesis. CCK-OP and PMA activated phospholipase A (PLA) which liberated lysophosphatidylcholine (LPC) and free fatty acids from membrane phosphatidylcholine. Exogenously added PLA2 (Naja naja venom) inhibited protein synthesis and increased LPC to a similar extent as CCK and PMA. The results suggest that the inhibitory effects of CCK and carbachol on net protein synthesis are due to their effects on intracellular calcium and PLA-mediated breakdown of phosphatidylcholine rather than protein kinase C activation.     

Metabolic effects of cigarette smoking.

The inverse relationship between cigarette smoking and body weight, a potent obstacle to stopping smoking, may be due in part to effects of smoking on increasing whole body metabolism. Studies examining chronic and acute metabolic effects of smoking, as well as its constituent nicotine, are reviewed. Evidence suggests the absence of a chronic effect; most studies indicate that smokers and nonsmokers have similar resting metabolic rates (RMR) and that RMR declines very little after smoking cessation. Although an acute effect due to smoking is apparent, its magnitude is inconsistent across studies, possibly because of variability in smoke exposure or nicotine intake. In smokers at rest, the acute effect of smoking (and nicotine intake) appears to be significant but small (less than 10% of RMR) and transient (less than or equal to 30 min). However, the specific situations in which smokers tend to smoke may mediate the magnitude of this effect, inasmuch as smoking during casual physical activity may enhance it while smoking after eating may reduce it. Sympathoadrenal activation by nicotine appears to be primarily responsible for the metabolic effect of smoking, but possible contributions from nonnicotine constituents of tobacco smoke and behavioral effects of inhaling may also be important. Improved understanding of these metabolic effects may lead to better prediction and control of weight gain after smoking cessation, thus increasing the likelihood of maintaining abstinence.     

Leukotrienes are active in the rosette inhibition assay mimicking the action of 'early pregnancy factor'

Lipoxygenase metabolites have been found to be active in the rosette inhibition assay by inducing increased rosette inhibition titres. Leukotrienes B4, C4, D4 and E4 were identified as possessing this activity. The cycloxygenase products, prostaglandins E2, D2 and F2 alpha had no such activity; however, prostaglandin E2 and to a lesser degree prostaglandin D2 could counteract the activity of the leukotrienes in this assay. The identified leukotrienes are the first characterised molecules known to display activity in the assay. In this respect they mimic the action of early pregnancy serum, an action ascribed to a so called 'early pregnancy factor'.     

Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function.

The human immunodeficiency virus rev gene product regulates the expression of viral structural genes. It was recently shown that Rev regulates the export of viral structural mRNAs from the nucleus to the cytoplasm. Analysis of Rev subcellular localization reveals marked accumulation in the nucleolus, suggesting a role for the nucleolus in this export process. We report here the identification of amino acid residues critical to the nucleolar localization of Rev. Consistent with this finding, a Rev/beta-galactosidase fusion protein, harboring this region of Rev, localized entirely within the nucleolus. Of most significance, mutations that eliminated nucleolar localization markedly diminished Rev function, even though accumulation in the nucleoplasm was retained. These findings support a model whereby Rev-induced export of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm is likely to involve nucleolar events.     

Prostaglandin E specifically upregulates the expression of the mannose-receptor on mouse bone marrow-derived macrophages.

The macrophage mannose receptor (MMR) facilitates the binding and internalization of microorganisms and glycoproteins with terminal mannose residues. The receptor is progressively upregulated as bone marrow precursor cells mature into macrophages and thus may serve as a marker of differentiation. Prostaglandins of the E series (PGE) are known inhibitors of monocyte and macrophage precursor proliferation, an effect often associated with cellular maturation. MMR expression was therefore assessed after exposure of bone marrow macrophage precursor (BMMP) cells to these prostanoids. Receptor expression was determined by ligand binding and via immunoprecipitation of newly synthesized receptor molecules. PGE1 and PGE2 at 10(-9)-10(-6) M upregulated MMR surface expression and biosynthesis four- to sixfold in a dose-dependent manner. BMMPs responsive to prostaglandins were characterized by plastic adherence, F4/80 antigen expression, and nonspecific esterase activity. Prostaglandins accelerated the expression of the MMR in cells by 48-72h, with maximal levels of receptor expression being identical in control or treated cells. Thus, prostaglandins enhanced mannose receptor expression in adherent but not fully differentiated macrophage precursors. This effect is specific for PGE and is mimicked by dibutyrl cyclic AMP. These results indicate that prostaglandins accelerate MMR expression and hence the differentiation of macrophage precursor cells. Cells resident in the bone marrow secrete abundant prostaglandins, suggesting that a paracrine mechanism may exist to regulate MMR expression and function.     

Amphotericin B-phospholipid interactions responsible for reduced mammalian cell toxicity.

When interacting with phospholipid in an aqueous environment, amphotericin B forms unusual structures of markedly reduced toxicity (Janoff et al. (1988) Proc. Natl. Acad. Sci. USA 85, 6122-6126). These structures, which appear ribbon-like by freeze-fracture electron microscopy (EM), are found exclusively at amphotericin B to lipid mole ratios of 1:3 to 1:1. At lower mole ratios they occur in combination with liposomes. Circular dichroism (CD) spectra revealed two distinct modes of lipid-amphotericin B interaction, one for liposomes and one for the ribbon-like structures. In isolated liposomes, amphotericin B which comprised 3-4 mole percent of the bulk lipid was monomeric and exhibited a hemolytic activity comparable to amphotericin B suspended in deoxycholate. Above 3-4 mole percent amphotericin B, ribbon-like structures emerged and CD spectra indicated drug-lipid complexation. Minimal inhibitory concentrations for Candida albicans of liposomal and complexed amphotericin B were comparable and could be attributed to amphotericin a release as a result of lipid breakdown within the ribbon-like material by a heat labile extracellular yeast product (lipase). Negative stain EM of the ribbon-like structures indicated that the ribbon-like appearance seen by freeze-fracture EM arises as a consequence of the cross-fracturing of what are aggregated, collapsed single lamellar, presumably interdigitated, membranes. Studies examining complexation of amphotericin B with either DMPC or DMPG demonstrated that headgroup interactions played little role in the formation of the ribbon-like structures. With these results we propose that ribbon-like structures result from phase separation of amphotericin B-phospholipid complexes within the phospholipid matrix such that amphotericin B release, and thus acute toxicity, is curtailed. Formation of amphotericin B-lipid structures such as those described here indicates a possible new role for lipid as a stabilizing matrix for drug delivery of lipophilic substances, specifically where a highly ordered packing arrangement between lipid and compound can be achieved.     

Cholecystokinin-induced changes in polysome structure regulate protein synthesis in pancreas.

Translational regulation of digestive enzyme synthesis during short-term stimulation by cholecystokinin-octapeptide (CCK-OP), was examined in minced rabbit pancreas by measuring protein synthesis and monitoring alterations in the size of polysomes attached to the rough endoplasmic reticulum (RER). The effect of CCK-OP on protein synthesis was determined by measuring [3H]leucine incorporation into trichloroacetic-acid-precipitable proteins. Concentrations of CCK-OP that caused maximal enzyme secretion (10 and 30 nM) decreased protein synthesis by approx. 50% compared to control. Protein synthesis returned to the control level 60 min after terminating the action of CCK-OP. Autoradiography of [35S]methionine-labeled proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that CCK-OP reversibly inhibited the synthesis of all of the major groups of digestive enzymes. Northern blot analysis revealed that CCK-OP did not alter the cellular content of amylase and elastase mRNA. Incubation with CCK-OP caused a decrease in the size distribution of RER-bound polysomes. Polysome profiles returned to the control pattern 60 min following termination of the stimulus. These results suggest that the inhibitory effects of CCK-OP on the synthesis of digestive enzymes is regulated at translation by decreasing the number of RER-bound ribosomes that are actively translating digestive enzyme mRNA.     

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.