Updating the H-antigen classification of Bacillus thuringiensis

The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty-nine serotypes and 13 sub-antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, delta-endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross-reacting H-antigens among B. cereus strains is increasing and may be of biological significance.     

Novel substituted 4-phenyl-[1,3]dioxanes: potent and selective orexin receptor 2 (OX(2)R) antagonists

Orexins, also termed hypocretins, consist of two neuropeptide agonists (orexin A and B) interacting with two known G-protein coupled receptors (OX(1)R and OX(2)R). In addition to other biological functions, the orexin-2 receptor is thought to be an important modulator of sleep and wakefulness. Herein we describe a series of novel, selective OX(2)R antagonists consisting of substituted 4-phenyl-[1,3]dioxanes. One such antagonist is compound 9, 1-(2,4-dibromo-phenyl)-3-((4S,5S)-2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-urea, which is bound by the OX(2)R with a pK(i) of 8.3, has a pK(b) of 7.9, and is 600-fold selective for the OX(2)R over the OX(1)R.     

A transesterification reaction is implicated in the covalent binding of benzo[b]acronycine anticancer agents with DNA and glutathion

The benzo[b]acronycine derivative S23906-1 has been recently identified as a promising antitumor agent, showing remarkable in vivo activities against a panel of solid tumors. The anticancer activity is attributed to the capacity of the drug to alkylate DNA, selectively at the exocyclic 2-amino group of guanine residues. Hydrolysis of the C-1 and C-2 acetate groups of S23906-1 provides the diol compound S28907-1 which is inactive whereas the intermediate C-2 monoacetate derivative S28687-1 is both highly reactive toward DNA and cytotoxic. The reactivity of this later compound S28687-1 toward two bionucleophiles, DNA and the tripeptide glutathion, has been investigated by mass spectrometry to identify the nature of the (type II) covalent adducts characterized by the loss of the acetate group at position 2. On the basis of NMR and molecular modeling analyses, the reaction mechanism is explained by a transesterification process where the acetate leaving group is transferred from position C-2 to C-1. Altogether, the study validates the reaction scheme of benzo[b]acronycine derivative with its target.     

Increased severity of murine lupus in female mice is due to enhanced expansion of pathogenic T cells

A strong female predominance is a well-recognized feature of human lupus. The mechanism by which sex influences disease expression and severity is not fully understood. To address this question, we used the parent-into-F(1) (p-->F(1)) model of chronic graft-vs-host disease (cGVHD) in which lupus-like humoral autoimmunity and renal disease are induced in normal F(1) mice. An advantage of this model is that the pathogenic T cells driving disease (donor strain) can be studied separately from nonspecifically activated T cells (host strain). We observed that lupus-like disease using female donor and host mice (f-->F cGVHD) is characterized by more severe long-term disease (glomerulonephritis) than with male donor and host (m-->M cGVHD). Interestingly, differences in disease parameters could be seen at 2 wk after parental cell transfer, as evidenced by a 2- to 3-fold greater engraftment of donor CD4(+) T cells in f-->F cGVHD mice, which persisted throughout disease course. Enhanced engraftment of donor CD4(+) T cells in f-->F cGVHD mice was not due to differences in splenic homing, alloreactive precursor frequency, initial proliferation rates, or apoptotic rates, but rather to sustained high proliferation rates during wk 2 of disease compared with m-->M cGVHD mice. Crossover studies (m-->F, f-->M) demonstrated that enhanced donor CD4(+) T cell proliferation and engraftment segregate with the sex of the host. These results demonstrate that the sex of the recipient can influence the expansion of pathogenic T cells, thus increasing long-term the burden of autoreactive T cells and resulting in greater disease severity.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer

DNA-protein cross-links form when guanine undergoes a 1-electron oxidation in a flash-quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA-histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a (32)P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash-quench technique as a way to generate physiologically relevant DNA-protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.     

Neutralization of toxic heme by Plasmodium falciparum histidine-rich protein 2

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) has been suggested to be an initiator of the polymerization of heme, which is produced as by-product on the digestion of hemoglobin, and a promoter of the H(2)O(2)-induced degradation of heme in food vacuoles of the malarial parasite. In this work, we have designed PfHRP2 model peptides, R18 and R27 (18 and 27 residues, respectively), and used them for optical and electron spin resonance spectroscopic measurements to confirm that the axial ligands of the heme-PfHRP2 complex are the nitrogenous donors derived from the imidazole moieties of histidine residues of PfHRP2. In addition, we revealed that the affinities of R18 and R27 for heme (K(d) = 2.21 x 10(-6) M and 0.71 x 10(-6) M, respectively) might be as high as that of PfHRP2 (K(d) = 0.94 x 10(-6) M). The R27 peptide can remove heme from membrane-intercalated heme and inhibit heme-induced hemolysis. Therefore, we suggest another function of PfHRP2: it may play an important role in the neutralization of toxic heme in the parasite cytoplasm and infected erythrocytes by removing heme from heme-bound membranes or reducing heme-induced hemolysis.     

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.     

Diagnosis of gastric glomus tumor by endoscopic ultrasound-guided fine needle aspiration biopsy. A case report with cytologic,histologic and immunohistochemical studies

BACKGROUND: The majority of glomus tumor are small, benign neoplasms that arise from modified smooth muscle cells. They usually occur in the dermis or subcutis of the extremities. However, rare cases have been reported in the visceral locations, most often in the stomach. CASE: A 32-year-old woman presented with episodes of right upper quadrant pain. She was found to have a gastric tumor that was biopsied at another hospital, where the diagnosis of gastrointestinal stromal tumor (GIST) was made. Endoscopic ultrasound (EUS) performed at our institution revealed a gastric submucosal tumor that was then biopsied by fine needle aspiration (FNA). Cytology revealed cohesive clusters of uniform, round, small cells with ill-defined cytoplasmic borders and scanty, amphophilic cytoplasm. Nuclei were round, with smooth nuclear membranes and evenly distributed, dusty chromatin. Intermingled with those epithelioid cells were small, short, spindled, normal endothelial cells. Immunohistochemical studies performed on cell block showed that the tumor cells were negative for CD34, CD117, chromogranin, synaptophysin, desmin and AE1/AE3 and were strongly positive for SMA, HHF-35 and collagen type IV. Glomus tumor was diagnosed and later confirmed by histology. CONCLUSION: EUS-guided FNA biopsy is efficient and permits adequate sampling for accurate diagnosis of gastric glomus tumor. Although rare, glomus tumor should be in the differential diagnosis among other gastric lesions, such as well-differentiated adenocarcinoma, epithelioid GIST and carcinoid tumor.