Effects of tumour necrosis factor-alpha on BrdU incorporation in cultured human enterocytes

Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFalpha in organ culture. TNFalpha caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G(0), the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFalpha. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFalpha on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFalpha. Exposure to TNFalpha alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFalpha there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFalpha and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFalpha may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.     

Anaerobic Granular Sludge Bioreactor Technology

Anaerobic digestion is a mature wastewater treatment technology, with worldwide application. The predominantly applied bioreactor designs, such as the upflow anaerobic sludge blanket and expanded granular sludge bed, are based on the spontaneous formation of granular sludge. Despite the exploitation of granular reactors at full-scale for more than two decades, the mechanisms of granulation are not completely understood and numerous theories have been put forward to describe the process from a biological, ecological and engineering point of view. New technological opportunities are emerging for anaerobic digestion, aided by an improved understanding of microbiological and environmental factors affecting the formation and activity of anaerobic granular sludge.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.     

Cell-mediated cytotoxicity against HLA-D-region products expressed in monocytes and B lymphocytes. I. Blocking by Unlabelled cells and inhibition by antisera

Cytotoxic effector cells against HLA-D-region produts were generated in cultures with HLA-A- and B-matched, HLA-D-mismatched stimulating cells. Monocytes from unrelated donors sharing HLA-D/DR antigens with the primary stimulator were used as target cells. Lysis of target cells was inhibited by addition of unlabelled monocytes having the same HLA-D/DR antigens. Inhibition of cytotoxicity was also observed with unlabelled B cells, but T lymphocytes had little effect. The distribution of the target antigens, therefore, fits the known distribution of the products of HLA-D. In other experiments, a human alloantiserum specific for HLA-DRw3 was found to inhibit cellular cytotoxicity specific for HLA-D/DRw3. Lysis by HLA-D/DRw3-specific effector cells was not inhibited by sera against HLA-DRw2 or DRw7 or by antibodies against HLA-B8 using HLA-B8 positive, DRw3 positive targer cells. A xenogeneic serum against human Ia antigens, produced in rabbits, blocked cytotoxicity directed at DRw2, DRw3 and DRw4. These results suggest that cell-mediated cyctotoxicity was directed against HLA-D/DR or very closely associated determinants.     

Seed dispersal networks in tropical forest fragments: Area effects,remnant species,and interaction diversity

Seed dispersal interactions involve key ecological processes in tropical forests that help to maintain ecosystem functioning. Yet this functionality may be threatened by increasing habitat loss, defaunation, and fragmentation. However, generalist species, and their interactions, can benefit from the habitat change caused by human disturbance while more specialized interactions mostly disappear. Therefore, changes in the structure of the local, within fragment, networks can be expected. Here we investigated how the structure of seed dispersal networks changes along a gradient of increasing habitat fragmentation. We analyzed 16 bird seed dispersal assemblages from forest fragments of a biodiversity-rich ecosystem. We found significant species–, interaction–, and network–area relationships, yet the later was determined by the number of species remaining in each community. The number of frugivorous bird and plant species, their interactions, and the number of links per species decreases as area is lost in the fragmented landscape. In contrast, network nestedness has a negative relationship with fragment area, suggesting an increasing generalization of the network structure in the gradient of fragmentation. Network specialization was not significantly affected by area, indicating that some network properties may be invariant to disturbance. Still, the local extinction of partner species, paralleled by a loss of interactions and specialist–specialist bird–plant seed dispersal associations, suggests the functional homogenization of the system as area is lost. Our study provides empirical evidence for network–area relationships driven by the presence/absence of remnant species and the interactions they perform.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.