Balancing oxidative protein folding: The influences of reducing pathways on disulfide bond formation

Oxidative protein folding is confined to few compartments, including the endoplasmic reticulum, the mitochondrial intermembrane space and the bacterial periplasm. Conversely, in compartments in which proteins are translated such as the cytosol, the mitochondrial matrix and the chloroplast stroma proteins are kept reduced by the thioredoxin and glutaredoxin systems that functionally overlap. The highly reducing NADPH pool thereby serves as electron donor that enables glutathione reductase and thioredoxin reductase to keep glutathione pools and thioredoxins in their reduced redox state, respectively. Notably, also compartments containing oxidizing machineries are linked to these reducing pathways. Reducing pathways aid in proofreading of disulfide bond formation by isomerization or they provide reducing equivalents for the reduction of disulfides prior to degradation. In addition, they contribute to the thiol-dependent regulation of protein activities, and they help to counteract oxidative stress. The existence of oxidizing and reducing pathways in the same compartment poses a potential problem as the cell has to avoid futile cycles of oxidation and subsequent reduction reactions. Thus, compartments that contain oxidizing machineries have developed sophisticated ways to spatiotemporally balance and regulate oxidation and reduction. In this review, we discuss oxidizing and reducing pathways in the endoplasmic reticulum, the periplasm and the mitochondrial intermembrane space and highlight the role of glutathione especially in the endoplasmic reticulum and the intermembrane space. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Regulation and Migratory Role of P-Selectin Ligands during Intestinal Inflammation

Dendritic cells from mesenteric lymph nodes (MLN) can convert retinal to retinoic acid (RA), which promotes induction of the gut-specific homing receptor α4β7. In contrast, priming within peripheral lymph nodes leads to upregulation of E- and P-selectin ligands (E- and P-lig). Apart from its α4β7 promoting effect, RA was shown to suppress E- and P-lig induction in vitro. However, enhanced frequencies of P-lig⁺ CD4⁺ T cells were reported during intestinal inflammation. To understand this contradiction, we first determined whether location of intestinal inflammation, that is, ileitis or colitis, affects P-lig induction. Both conditions promoted P-lig expression on CD4⁺ T cells; however, P-lig expressed on T cells facilitated Th1 cell recruitment only into the inflamed colon but not into inflamed small intestine induced by oral Toxoplasma gondii infection. A majority of P-lig⁺CD4⁺ T cells found within MLN during intestinal inflammation co-expressed α4β7 confirming their activation in the presence of RA. Mesenteric P-lig⁺CD4⁺ cells co-expressed the 130 kDa isoform of CD43 which requires activity of core 2 (beta)1,6-N-acetyl-glycosaminyltransferase-I (C2GlcNAcT-I) suggesting that C2GlcNAcT-I contributes to P-lig expression under these conditions. To test whether inflammatory mediators can indeed overrule the inhibitory effect of RA on P-lig expression we stimulated CD4⁺ T cells either polyclonal in the presence of IL-12 and IFNγ or by LPS-activated MLN-derived dendritic cells. Both conditions promoted P-lig induction even in the presence of RA. While RA impeded the induction of fucosyltransferase-VII it did not affect IL-12-dependent C2GlcNAcT-I induction suggesting that C2GlcNAcT-I can support P-lig expression even if fucosyltransferase-VII mRNA upregulation is dampened.     

Mycorrhizae of Entoloma saepium: parasitism or symbiosis?

The mycorrhizae of Entoloma saepium on Rosa sp. are comprehensively described and compared to other mycorrhizae of the genus Entoloma, as well as to similar unidentified mycorrhizae known from the literature. E. saepium appears to be more a parasite than a symbiont, as it invades and almost completely destroys the root meristem and young root cells.Considered as part IL of the series Studies on ectomycorrhizae; part XLVIII: Waller and Agerer (1993)     

MORPHOLOGICAL DIFFERENTIATION AND GENETIC COHESIVENESS OVER A MICROENVIRONMENTAL GRADIENT IN THE MARINE SNAIL LITTORINA SAXATILIS

The marine gastropod Littorina saxatilis has different ecotypes in shores only a few meters apart. This has both taxonomic and evolutionary implications. Here we report on an extreme type of within-shore dimorphism in shell characters. In the wave-exposed rocky shores in northwestern Spain, we found one form of L. saxatilis in the upper-level barnacle zone. It had a white, ridged shell, with black bands in the grooves. Another form confined to the lower-shore mussel belt had a smooth shell that was either white and tessellated or darkly colored. These two forms cooccured in a narrow midshore zone together with individuals that had combined characters, but were present in low frequencies (11%–29%). We used principal-component analysis of metric shell characters to study variation in shell size and shape. We found that the upper-shore form was larger than the lower-shore form. We also found small but significant differences in shell shape. Experiments in a common laboratory environment suggested the differences in shell ornamentation and color are inherited, but the individuals did not develop the morph-specific characters until a shell height of about 3 mm. The occurrence of mainly two distinct forms may suggest the presence of two species that hybridize. An analysis of five polymorphic enzyme loci in populations of snails from three geographically separated sites indicated, however, that there was no positive correlation between morphological distances and genetic distances among populations on a geographic scale (tens of kilometers). Thus, we rejected the hypothesis of two species. However, on a microgeographic scale (meters), genetic differentiation between groups with the same form was less than differentiation between forms. This indicated a partial barrier to gene flow between the two forms, and preliminary mate choice data suggested this was caused by nonrandom mating in the midshore zone of overlap.     

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon γ and tumour necrosis factor α treatment of tumour cells potentiates their interaction with autologous blood lymphocytes

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon and tumour necrosis factor elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.     

Radiation induced chromosomal instability in human T-lymphocytes

Chromosomal instability in proliferating mammalian cells is characterized by a persistent increase of chromosomal aberrations and rearrangements occurring de novo during successive cell generations. Recent results from many laboratories using a variety of cells and cytogenetic end points show that this phenotype can be induced by low as well as high LET irradiation. A typical feature of chromosomal instability in primary human G₀-lymphocytes exposed to γ-irradiation at both high dose rate (45 Gy h⁻¹) and low dose rate (0.024 Gy h⁻¹) is the appearance of novel aberrations in the clonal progeny of the irradiated cell, many generations after the exposure. The same phenotype was observed in lymphocytes that were allowed to recover for 5 days in G₀ after the radiation exposure, as well as in hprt-mutant T cell clones. These results demonstrate that neither the acute genotoxic stress caused by high dose rate as compared to low dose rate irradiation, nor a hypothesized conflict between mitogen induced growth stimulation and growth arrest due to radiation damage, seem to be critical conditions for the development chromosomal instability in these cells. In contrast to observations in other cells, no evidence of a persistent decrease of cloning ability was observed in the progeny of radiation-exposed human lymphocytes, and no alteration was observed in their sensitivity to a second radiation exposure. Furthermore, the frequency of CA-repeat length variation at three loci was not increased in the progeny of X-irradiated T cells as compared to non-irradiated cells, which indicates that microsatellite instability is not part of the chromosomal instability phenotype in human T-lymphocytes.     

Aerobic and anaerobic degradation of furan-3-carboxylate by Paracoccus denitrificans strain MK33

An organism identified as Paracoccus denitrificans was isolated from an enrichment culture with furan-3-carboxylate as its sole source of carbon and energy. The organism degraded furan-3-carboxylate under aerobic and — in the presence of nitrate - under anaerobic conditions. The aerobic degradation was initiated by an oxygenase reaction to form probably 2-hydroxy-3-furoate. Under anaerobic conditions the first intermediate was 3-furoyl-CoA which was reduced by NADPH to probably 4,5-dihydro-furoyl-CoA. Succinic semialdehyde was an intermediate in both aerobic and anaerobic mechanism of furan-3-carboxylate.Abbreviations F-3-C furan-3-carboxylate - 3-FCoA 3-furoyl-CoA     

Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.

In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi- and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro.