Modulation of antibody-mediated glomerular injury in vivo by bacterial lipopolysaccharide, tumor necrosis factor, and IL-1

We have investigated the effects of LPS, human rTNF (hrTNF) and human rIL-1 beta (hrIL-1 beta) pretreatment on the intensity of antibody-mediated injury in vivo by using a passive model of anti-glomerular basement membrane (GBM) antibody-mediated nephritis in rats. The experiments show that all three pretreatments exacerbate injury in this model whether judged by albuminuria or the prevalence of glomerular capillary thrombi. The effect on albuminuria was dose dependent with all three treatments. The lowest effective dose of LPS was 0.025 microgram while those for hrTNF and hrIL-1 beta were 0.4 microgram and 0.5 microgram, respectively. All three pretreatments also increased the prevalence of glomerular capillary thrombi which were rare in rats injected with anti-GBM antibodies without pretreatment. LPS pretreatment appeared to be more effective in causing glomerular capillary thrombi than hrTNF or hrIL-1 beta and this was reflected in the correlations between albuminuria and the proportion of glomeruli with capillary thrombi. This relation was linear for all three pretreatments but the slope was appreciably greater for rats pretreated with LPS (0.37) when compared with results from rats given either hrTNF (0.22) or hrIL-1 beta (0.29). Pretreatment of nephritic rats with both cytokines increased the slope to 0.42 demonstrating a synergistic effect. The synergism of hrTNF with hrIL-1 beta was also demonstrated by the effective doses needed to induce albuminuria which was evident in rats treated with 0.05 microgram of IL-1 beta and 0.4 microgram of TNF. Neither the cytokines nor LPS caused clinical, morphologic, or biochemical evidence of renal toxicity when given alone in the dose used here but they did cause a transient increase in the number of neutrophils marginated in glomerular capillaries. Pretreatment of rats with LPS or cytokines increased the glomerular neutrophil influx after anti-GBM antibodies by roughly sixfold. Our experiments show that TNF and IL-1 can increase the severity of glomerular injury in nephritis; they may be important in modulating glomerular injury clinically.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

Mutations of immunoglobulin transmembrane and cytoplasmic domains: effects on intracellular signaling and antigen presentation

The membrane-bound form of immunoglobulin serves as an antigen-specific receptor for B cells mediating signal transduction and antigen presentation. We have developed an assay that reconstitutes both these physiologic responses with respect to the antigen phosphorylcholine. By introducing specific mutations in the human Ig mu chain gene, we have shown that certain transmembrane residues and the short cytoplasmic domain are crucial for these two activities. Moreover, elimination of a single transmembrane hydroxyl group severely inhibits antigen presentation without affecting signal transduction, suggesting that these two functions are mediated by different protein interactions.     

A recombination event that redefines the Huntington disease region

We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Vacuole Partitioning during Meiotic Division in Yeast

We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y, were also detected outside spores after meiotic division. Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores. These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores. When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores. Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division.     

Reproductive and neonatal outcomes in captive bred baboons (Papio hamadryas)

Abstract: Baboons are widely used in biomedical research. Although it is widely held that Papio hamadryas breed well in captivity, each established colony has a different reproductive success often hypothesised to be due to husbandry practices. The National Baboon Colony in Australia is a unique colony that houses Papio hamadryas to mimic that structure seen in the wild. In this article; we have analysed their reproductive parameters and neonatal outcomes. The success of the colony husbandry practices was demonstrated by lack of maternal mortality, low foetal morbidity, and known maternal and paternal linage.     

Lactococcus lactis and stress

It is now generally recognized that cell growth conditions in nature are often suboptimal compared to controlled conditions provided in the laboratory. Natural stresses like starvation and acidity are generated by cell growth itself. Other stresses like temperature or osmotic shock, or oxygen, are imposed by the environment. It is now clear that defense mechanisms to withstand different stresses must be present in all organisms. The exploration of stress responses in lactic acid bacteria has just begun. Several stress response genes have been revealed through homologies with known genes in other organisms. While stress response genes appear to be highly conserved, however, their regulation may not be. Thus, search of the regulation of stress response in lactic acid bacteria may reveal new regulatory circuits. The first part of this report addresses the available information on stress response in Lactococcus lactis.Acid stress response may be particularly important in lactic acid bacteria, whose growth and transition to stationary phase is accompanied by the production of lactic acid, which results in acidification of the media, arrest of cell multiplication, and possible cell death. The second part of this report will focus on progress made in acid stress response, particularly in L. lactis and on factors which may affect its regulation. Acid tolerance is presently under study in L. lactis. Our results with strain MG1363 show that it survives a lethal challenge at pH 4.0 if adapted briefly (5 to 15 minutes) at a pH between 4.5 and 6.5. Adaptation requires protein synthesis, indicating that acid conditions induce expression of newly synthesized genes. These results show that L. lactis possesses an inducible response to acid stress in exponential phase.To identify possible regulatory genes involved in acid stress response, we determined low pH conditions in which MG1363 is unable to grow, and selected at 37°C for transposition insertional mutants which were able to survive. About thirty mutants resistant to low pH conditions were characterized. The interrupted genes were identified by sequence homology with known genes. One insertion interrupts ahrC, the putative regulator of arginine metabolism; possibly, increased arginine catabolism in the mutant produces metabolites which increase the pH. Several other mutations putatively map at some step in the pathway of (p)ppGpp synthesis. Our results suggest that the stringent response pathway, which is involved in starvation and stationary phase survival, may also be implicated in acid pH tolerance.