Synthesis and hypoxia-selective cytotoxicity of a 2-nitroimidazole mustard

A four-step synthesis of 5-[N,N-bis(2-chloroethyl)amino]-1-methyl-2-nitroimidazole from 1-methyl-2-nitroimidazole is described. This compound showed similar hypoxia-selective cytotoxicity to the dinitrobenzamide mustard SN 23,862 in UV4 cells (ca. 40-fold), and superior selectivity (> 7-fold) in repair-competent AA8 cells.     

Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C

Starting with an extract derived from the stem of Macleaya cordata (Papaveraceae) that was active in the process of inhibiting phorbol 12,13-dibutyrate binding to partially purified protein kinase C (PKC), the benzophenanthridine alkaloid angoline was isolated and identified. This discovery appeared in context, as a related benzophenanthridine alkaloid, chelerythrine, has been reported to mediate a variety of biological activities, including potent and selective inhibition of protein kinase C (PKC). However, in our studies, angoline was not observed to function as a potent inhibitor of PKC. Moreover, we were unable to confirm the reported inhibitory activity of chelerythrine. In a comprehensive series of studies performed with various PKC isozymes derived from a variety of mammalian species, neither chelerythrine nor angoline inhibited activity with high potency. To the contrary, chelerythrine stimulated PKC activity in the cytosolic fractions of rat and mouse brain in concentrations up to 100 microM. In addition, chelerythrine and angoline did not inhibit [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC at concentrations up to 40 microg/ml, and no significant alteration of PKC-alpha, -beta, or -gamma translocation was observed with human leukemia (HL-60) cells in culture. Further, chelerythrine did not inhibit 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity with cultured mouse 308 cells, but angoline was active in this capacity with an IC50 value of 1.0 microg/ml. A relatively large number of biological responses have been reported in studies conducted with chelerythrine, and alteration of PKC activity has been considered as a potential mechanism of action. In light of the current report, mechanisms independent of PKC inhibition should be considered as responsible for these effects.     

Prostaglandin E2 receptor EP3alpha subtype: the role of N-glycosylation in ligand binding as revealed by site-directed mutagenesis

Vesico-ureteral reflux (VUR) is a frequent condition, but in most instances, the precise cause is unknown. We here review the evidence of a genetic aetiology of VUR, inherited as an autosomal dominant trait, with variable expression. We discuss the possible pathogenetic relationship between VUR and other types of uropathies and possible strategies towards the identification of genes underlying VUR are presented. The isolation of the gene(s) responsible for uropathies will not only lead to a better insight into the embryology of the urological system, the pathogenesis of uropathies, but also to a renewed interest from clinicians in congenital uropathies.     

Pulmonary embolism and malpractice claims

BACKGROUND: Deep venous thrombosis (DVT) with pulmonary embolization (PE) often occurs as an unexpected event with fatal consequences. This provides a setting for malpractice claims. METHODS: We reviewed 160 consecutive malpractice claims submitted by attorneys for medical expert review during the 11-year period ending in 1997. Seven cases involved DVT with PE. RESULTS: Alleged failure to anticipate and reduce the chance of PE was the basis for six of the claims. All six patients were at risk for lower extremity DVT, and one had a history of DVT 6 months earlier. The PE was manifested by sudden death in three cases. The seventh case represented a complication of heparin therapy for PE. CONCLUSIONS: We conclude that risk management for PE should focus primarily on DVT. Physicians should perform and document an examination for DVT whenever there is a history of lower extremity stasis or it is likely to occur. They should also consider documenting a concurring second opinion when making anticoagulant-related decisions.     

Occupational exposure to HIV, hepatitis B, hepatitis C, and tuberculosis

This article reviews risk of occupational exposure to bloodborne pathogens, specifically HIV, hepatitis B, and hepatitis C to healthcare workers. Information regarding assessing the risk of exposure, appropriate actions to take if an exposure occurs, the most recent recommendations for treatment and follow-up postexposure, and prevention strategies for avoiding exposure are presented. Additionally, current recommendations for the prevention of the transmission of tuberculosis in healthcare workers and the regulatory guidelines governing this topic are discussed.     

Essential role of the disulfide-bonded loop of chromogranin B for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH2-terminal disulfide- bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Deltacys-hCgB) lacking the 22-amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Deltacys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Deltacys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus-mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.     

TNF-alpha released by comigrating monocytes promotes transendothelial migration of activated lymphocytes

We investigated mechanisms that increase motility and transendothelial trafficking of activated lymphocytes. Freshly isolated lymphocytes stimulated with immobilized anti-CD3 for 2 h migrate into polymerized collagen in 1.99+/-0.25-fold greater numbers and across confluent endothelial monolayers in 4.8+/-0.5-fold greater numbers compared with leukocytes incubated with non-specific IgG. Activated lymphocytes form clusters with monocytes, and their increased motility was dependent on the presence of comigrating monocytes. Five lines of evidence support the idea that monocytes modulate lymphocyte motility through the release of TNF-alpha: 1) flow-cytometric analyses, using highly specific and avid mAbs to probe permeabilized whole blood leukocytes, showed that >80% of circulating monocytes contain intracellular TNF-alpha, whereas <5% contain IL-1 and none contain IL-6; 2) stimulation with immobilized anti-CD3 that was intended to activate lymphocytes also induced monocytes to release increased quantities of TNF-alpha; 3) rTNF-alpha, added in doses of 1 to 20 pg/ml to purified anti-CD3-stimulated lymphocytes, reproduced, in a dose-dependent manner, the motility-enhancing effect of adding monocytes; 4) the transient increase in the expression of TNF R-I on CD3-activated T lymphocytes parallels their transiently increased motility; and 5) addition of anti-TNF-alpha, anti-TNF R-I, anti-TNF R-II, or soluble TNF R-I decreased the motility of stimulated lymphocytes. These results suggest that T lymphocyte stimulation via the CD3-TCR complex signals nearby monocytes to release TNF-alpha, which feeds back on the lymphocytes to increase their locomotor activity.