Cytostatic action of methylmercuric chloride on mammalian duodenal cells

Adult male mice of the ICR/Swiss Webster strain received a single intragastric administration of methylmercuric chloride 1,000 ppm, at dose levels of 5,10,15,20,25 and 30 mg/kg of body weight. The animals were killed six hours later. Tissue samples from the duodenum were fixed in 10% neutral buffered formalin for light microscopy. Chromosome clumping was observed in dividing cells at all dose levels, resembling a C-mitotic effect. It would lead to reduced mitotic cell formation on account of the subsequent lysis of the arrested metaphases. The cytostatic effect was brought about by the inactivation of the microtubule spindle fiber polymerization mechanism induced by methylmercuric chloride. There was a direct positive correlation between the varying dose levels of methylmercury and the proportion of cells arrested in metaphase in the crypts of the duodenum.     

The glycosylation of human myelin basic protein at threonines 95 and 98 occurs sequentially

Human myelin basic protein (MBP) was glycosylated by the enzyme, UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase (EC 2.4.2.41). A maximum of 1.7 mol of GalNAc was transferred to basic protein on threonines 95 and 98 of the protein. Proton NMR studies of basic protein glycosylated with 0.48-1.7 mol of GalNAc/mol of MBP showed that the order of addition to the two threonine residues is not random but sequential. The Thr-95 resonances shifted downfield, followed by the downfield shift of the Thr-98 resonances with increasing glycosylation. Since this peptide segment of the molecule is highly structured, conformational factors are probably responsible for this directed addition.     

Reversible deactivation of beta-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex.

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].     

Effect of lead on blood protoporphyrin levels of a group of ring teal ducks (Callonetta leucophrys)

This research determined the relationships between blood lead level and zinc protoporphyrin (ZnPROTO), protoporphyrin IX (PROTO), and free erythrocyte protoporphyrin (FEP) levels in a group of 18 ring teal ducks. Blood samples were drawn from two groups of ring teal ducks as part of the routine health maintenance program of the New York Zoological Park. One group of six teals had been exposed to what is believed to be lead-contaminated dust, while the second group of twelve teals was unexposed. Blood samples were analyzed for lead by flameless atomic absorption and for protoporphyrins by fluorescence. Blood lead level and log blood lead level had positive correlations with each of the protoporphyrins: the logarithmic correlations were better than the nonlogarithmic correlations, and PROTO correlated better than ZnPROTO. With one exception, PROTO levels were higher than ZnPROTO levels. The results suggest that PROTO, FEP, or ZnPROTO could serve as a biological indicator of lead poisoning in ring teal ducks.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.     

Calcium-dependent translocation of cytosolic phospholipase A2 in pancreatic beta-cells

Cytosolic phospholipase A(2)(cPLA(2)), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA(2) distribution in pancreatic beta-cells is different from that of most other mammalian cells: it is evenly distributed throughout the beta-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca(2+) in the MIN6 beta-cell line also stimulated a redistribution of cPLA(2) immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca(2+) being responsible for translocation of cPLA(2). These observations suggest that cPLA(2) may be compartmentalised in unstimulated beta-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca(2+).     

Pair-rule genes cooperate to activate en stripe 15 and refine its margins during germ band elongation in the D. melanogaster embryo

Patterns of gene expression have been well documented during embryogenesis for the Drosophila melanogaster trunk segments. The same is not the case for the terminal segments. Here, gene expression patterns are followed during embryogenesis in the caudal segments (A8-A10 and the anal plate), with special attention paid to the novel regulation of engrailed (en). Chosen for this study are the pair-rule genes even-skipped (eve), fushi tarazu (ftz), runt (run), hairy (h), paired (prd) and odd-skipped (odd), and the segment polarity gene (en). The results demonstrate a progressive and coupled translocation of gene expression distally for all genes studied, suggesting that the most posterior segments are determined later than trunk segments.     

Tool for Rapid & Easy Identification of High Risk Diabetic Foot: Validation & Clinical Pilot of the Simplified 60 Second Diabetic Foot Screening Tool

Background

Most diabetic foot amputations are caused by ulcers on the skin of the foot i.e. diabetic foot ulcers. Early identification of patients at high risk for diabetic foot ulcers is crucial. The ‘Simplified 60-Second Diabetic Foot Screening Tool’ has been designed to rapidly detect high risk diabetic feet, allowing for timely identification and referral of patients needing treatment. This study aimed to determine the clinical performance and inter-rater reliability of ‘Simplified 60 Second Diabetic Foot Screening Tool’ in order to evaluate its applicability for routine screening.

Methods and Findings

The tool was independently tested by n=12 assessors with n=18 Guyanese patients with diabetes. Inter-rater reliability was assessed by calculating Cronbach’s alpha for each of the assessment items. A minimum value of 0.60 was considered acceptable. Reliability scores of the screening tool assessment items were: ‘monofilament test’ 0.98; ‘active ulcer’ 0.97; ‘previous amputation’ 0.97; ‘previous ulcer’ 0.97; ‘fixed ankle’ 0.91; ‘deformity’ 0.87; ‘callus’ 0.87; ‘absent pulses’ 0.87; ‘fixed toe’ 0.80; ‘blisters’ 0.77; ‘ingrown nail’ 0.72; and ‘fissures’ 0.55. The item ‘stiffness in the toe or ankle’ was removed as it was observed in only 1.3% of patients. The item ‘fissures’ was also removed due to low inter-rater reliability. Clinical performance was assessed via a pilot study utilizing the screening tool on n=1,266 patients in an acute care setting in Georgetown, Guyana. In total, 48% of patients either had existing diabetic foot ulcers or were found to be at high risk for developing ulcers.

Conclusions

Clinicians in low and middle income countries such as Guyana can use the Simplified 60-Second Diabetic Screening Tool to facilitate early detection and appropriate treatment of diabetic foot ulcers. Implementation of this screening tool has the potential to decrease diabetes related disability and mortality.