Inhibition of amino acyl tRNA synthetase activity by copper complexes of two metal binding ligands. N-Methyl isatin beta-thiosemicarbazone and 8-hydroxyquinoline.

Copper complexes of N-methyl isatin beta-thiosemicarbazone, 1-formyl isoquinoline thiosemicarbazone and thiosemicarbazide inhibit amino acyl tRNA synthetase activity. Copper complexes of 8-hydroxyquinoline and 8-mercaptoquinoline also inhibit. The 1 : 1 ligand-metal complex is significantly more active than the 2 : 1 complex. The free ligand alone and copper sulfate alone have little, if any, effect. These complexes have no effect on the ATP-PPi exchange reaction and do not cause deacylation of amino acyl tRNAs. This indicates that the process inhibited by these complexes is the amino acylation reaction. This is the first report that these copper binding ligands can inhibit enzymatic processes which involve nucleic acids but which are not viral, bacterial or mammalian cell polymerases.     

Ehrlich ascites tumor cell surface labeling and kinetics of glycocalyx release

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (³H₂DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of ³H₂DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the ³H₂DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ_½ = 360 min) component representing 33% of the radioactivity, and a fast (τ_½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ_½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither ³H₂DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.     

Interaction of tritium-labeled H2DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′disulfonic acid) with the ehrlich mouse ascites tumor cell

Summary The experiments reported in this paper were undertaken to explore the interaction of tritiated H₂DIDS (4,4-diisothiocyano-1,2,diphenyl ethane-2,2-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (³H)H₂DIDS to tumor cell suspension at 21°C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H₂DIDS, i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H₂DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H₂DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H₂DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H₂DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H₂DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H₂DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H₂DIDS into the intracellular phase.     

Abnormal regulation of de novo purine synthesis and purine salvage in a cultured mouse T-cell lymphoma mutant partially deficient in adenylosuccinate synthetase.

The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.     

Alternate pathways of l-rhamnose transport in Arthrobacter pyridinolis

Phosphoenolpyruvate: hexose phosphotransferase-negative mutants of Arthrobacterpyridinolis fail to grow on l-rhamnose. Although phosphoenolpyruvate: l-rhamnose phosphotransferase activity could not be consistently demonstrated in extracts of rhamnose-grown cells, low levels of phosphoenolpyruvate-dependent uptake of rhamnose were found using isolated membrane vesicles from rhamnose-grown cells. This uptake was not inhibited by uncoupling agents or an inhibitor of the respiratory chain. Phosphotransferase-negative mutants could grow on l-rhamnose if l-malate was also present in the medium. l-Malate- and succinate-dependent uptake of rhamnose was found in membrane vesicles. Either of the two oxidizable substrates caused a 5-fold stimulation of the rate of l-rhamnose uptake over that observed in the absence of additions. Malate-dependent l-rhamnose uptake had a K_m for rhamnose of 2.9 × 10^?6m. It was inhibited by uncoupling agents, inhibitors of the respiratory chain, and sulfhydryl reagents.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Disability Mediates the Impact of Common Conditions on Perceived Health

Background

We examined the extent to which disability mediates the observed associations of common mental and physical conditions with perceived health.

Methods and Findings

WHO World Mental Health (WMH) Surveys carried out in 22 countries worldwide (n = 51,344 respondents, 72.0% response rate). We assessed nine common mental conditions with the WHO Composite International Diagnostic Interview (CIDI), and ten chronic physical with a checklist. A visual analog scale (VAS) score (0, worst to 100, best) measured perceived health in the previous 30 days. Disability was assessed using a modified WHO Disability Assessment Schedule (WHODAS), including: cognition, mobility, self-care, getting along, role functioning (life activities), family burden, stigma, and discrimination. Path analysis was used to estimate total effects of conditions on perceived health VAS and their separate direct and indirect (through the WHODAS dimensions) effects.Twelve-month prevalence was 14.4% for any mental and 51.4% for any physical condition. 31.7% of respondents reported difficulties in role functioning, 11.4% in mobility, 8.3% in stigma, 8.1% in family burden and 6.9% in cognition. Other difficulties were much less common. Mean VAS score was 81.0 (SD = 0.1). Decrements in VAS scores were highest for neurological conditions (9.8), depression (8.2) and bipolar disorder (8.1). Across conditions, 36.8% (IQR: 31.2–51.5%) of the total decrement in perceived health associated with the condition were mediated by WHODAS disabilities (significant for 17 of 19 conditions). Role functioning was the dominant mediator for both mental and physical conditions. Stigma and family burden were also important mediators for mental conditions, and mobility for physical conditions.

Conclusions

More than a third of the decrement in perceived health associated with common conditions is mediated by disability. Although the decrement is similar for physical and mental conditions, the pattern of mediation is different. Research is needed on the benefits for perceived health of targeted interventions aimed at particular disability dimensions.