Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Extrusion of actin-positive strands from Hep-2 and Int 407 cells caused by outer membrane preparations of enteropathogenic Escherichia coil and specific attachment of wild type bacteria to the strands

Enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea. This nontoxigenic E. coli exhibits a complicated pathogenic mechanism in which its outer membrane proteins and type III secretory proteins damage intestinal epithelium and cause diarrhoea. In accordance with this, our previous study using HEp-2 cells demonstrated cytopathic effects caused by cell-free outer membrane preparations of EPEC. In this study, we report the extrusion of actin-positive strands from HEp-2 and Int 407 cells when treated with outer membrane preparations. An interesting observation of this work, perhaps relevant to the characteristic localized three-dimensional colony formation of EPEC, is the attachment of a wild type EPEC strain to these actin-positive strands.     

Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD

RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161-2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.     

Formation of surfactant-like-particle in rat intestine following chronic ethanol feeding

Ethanol feeding to rats daily for 40 days induces the secretion of surfactant-like-particles in intestine. The isolated lipoprotein particles were enriched with alkaline phosphatase activity and had high phosphatidylcholine content. There was no difference in disaccharidases activities associated with the particles from control and ethanol fed rats. These results suggest that ethanol induced surfactant-like-particles in rat intestine.     

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24)

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.     

Characterization of the Drosophila caspase, DAMM

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced in Escherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels of damm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.     

A conserved alpha-helix at the amino terminus of prosomatostatin serves as a sorting signal for the regulated secretory pathway

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.     

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.     

Fine needle aspiration cytology of foreign bodies presenting as cystic abdominal masses. A report of three cases

BACKGROUND: Foreign body material (gauze sponges) presented as cystic abdominal masses and were confused with malignant tumors. CASES: Two females and one male presented with abdominal masses. They had undergone laparotomy 5-12 years earlier. Clinically the masses were diagnosed as benign or malignant cystic lesions. Fine needle aspiration revealed necrotic material, hemosiderin-laden macrophages, foreign body giant cells, cholesterol crystals and many fragments of birefringent material. The possibility of malignancy was ruled out. Cut sections of the excised cystic lesions revealed gauze sponges surrounded by a thick, fibrotic wall. CONCLUSION: This report underscores the usefulness of fine needle aspiration in ruling out malignancy.