Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

G to C transition at position −173 of MIF gene of the recipient is associated with reduced relapse rates after allogeneic stem cell transplantation

Pro-inflammatory and dendritic cell-activating properties of macrophage migration inhibitory factor (MIF) suggest a potentially important role for MIF in alloantigen-specific immune responses after allogeneic stem cell transplantation (allo-SCT). We tested whether MIF −173 G/C gene polymorphism of donor or patient had impacts on the outcomes after allo-SCT. Four hundred and fifty-four donor–patient pairs were genotyped and mortality, relapse, and development of complications were analyzed. Patient but not donor MIF −173*C allele was associated with improved overall survival (OS) (5 years: 60.8% versus 46.3%, p = 0.042) and disease free survival (DFS) (5 years: 55.4% versus 39.5%; p = 0.014) due to a reduction in relapse (day 2000: 22.8% versus 42.0% p = 0.006) but not due to decreased transplantation-related mortality (TRM) (p = 0.44). Multivariate analysis proved patient −173*C allele as an independent factor for reducing relapse after allo-SCT (p = 0.023). Subgroup analysis showed a clear MIF −173*C allele-related reduction in relapse for those patients who did not receive T cell depleted (TCD) SCT (p = 0.01) in contrast to patients receiving TCD SCT (p = 0.20). In summary, patient MIF −173*C allele may be linked to specific, yet unrevealed functions in tumor biology and graft versus leukemia and lymphoma effects and potentially presents a novel prognostic marker for patient-tailored counseling and therapy in allo-SCT.     

A Novel Epimerase That Converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157

Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-d-PerNAc-α-l-Fuc-β-d-Glc-α-d-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[³H]GalNAc, neither the enzymatic synthesis of [³H]GlcNAc-P-P-Und nor [³H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[³H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [³H]GlcNAc-P-P-Und was converted to [³H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [³H]GlcNAc, both [³H]GlcNAc-P-P-Und and [³H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [³H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [³H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.     

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.     

Immunoelectron microscopic localization of cytochrome P-450 isoenzyme CYP4A1 in liver,ileum and kidney of nafenopin treated male rats.

A monoclonal antibody raised against and specific for cytochrome P-450 isoenzyme CYP4A1 was used to investigate the subcellular distribution of this enzyme in the liver, kidney and ileum of nafenopin treated rats by means of immunoelectron microscopy. In the liver and kidney, labelling was restricted to peroxisomes and mitochondria of hepatocytes and proximal tubular epithelial cells whereas in ileum, immunolabelling was exclusively detected in mitochondria of absorptive cells.     

Rotifers in aging research: use of rotifers to test various theories of aging

Four theories of aging are discussed to examine how effectively they might explain the aging process in rotifers. One of the early theories, the rate of living theory of aging can perhaps be discounted. Although the theory predicts that increased biological energy expenditure, in the form of increased activity or reproduction, would lead to a shorter lifespan, these predictions are not born out by experimental evidence. At the whole animal level, a case can be made for a theory of programmed aging, where the end of reproduction signals the end of the lifespan. Support for this view comes from the observation that lifespan is positively correlated with reproductive parameters, that treatments that extend lifespan usually act to extend the reproductive period, and that the end of reproduction is associated with high mortality and senescent biochemical changes. Two molecular theories of aging are also discussed; the free radical theory of aging and the calcium theory of aging. These theories point to the fact that molecular damage accumulates and that calcium influx increases in the course of aging. When free radical buildup or calcium homeostasis is reduced, lifespan is extended. A molecular explanation of aging does not necessarily exclude the idea of programmed aging. It is probable that an eventual understanding of the aging process will rest on both a physiological and molecular basis.