Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Trypsin and α-amylase inhibitors are differentially induced in leaves of amaranth (Amaranthus hypochondriacus) in response to biotic and abiotic stress

Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an α -amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and α -amylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect α -amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in α -amylase inhibitor activity and AAI levels were detected in leaves of insect-damaged, MeJA- and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water- or salt-stress. A differential induction of trypsin inhibitor activity and α -amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and α -amylase inhibitors could protect A. hypochondriacus against multiple types of stress.     

Isolation of Corynebacterium diphtheriae from sperm fluid

The isolation of toxigenic Corynebacterium diphtheriae from sperm is reported. The organism was identified through the investigation of fluorescence under the UV light, the presence of pirazinecarboxilamidase enzyme (Pyz), in vitro and in vivo and virulence methods (single radial immunodiffusion, cell culture, guinea pig intradermic test). The strain was initially considered nontoxinogenic by single radial immunodiffusion, but its virulence was observed afterwards, when we applied the tests already mentioned. The strain could be considered a "Diphtheroid" without adequate specification.     

Dual substrate removal by an axenic bacterial culture

A pure bacterial culture capable of utilizing either L-lysine or 2-chlorophenol (2-CP) as sole carbon source was isolated and used in continuous culture experiments to determine its response to dual substrate limitation by those two compounds. Dilution rate and feed composition were each set at three levels in a two factorial experimental design. The total chemical oxygen demand (COD) of the feed was fixed at 225 mg/L and its composition was varied by changing the ratio of lysine to 2-CP. The effects of the two independent variables (dilution rate and feed composition) on the concentrations of cells, lysine, COD, and dissolved organic carbon (DOC) in the reactors were systematic whereas the effects on the 2-CP concentration were less predictable. The concentrations of the two substrates responded to the two independent variables in a complex interactive manner which is not explained by existing models for dual, substitutable substrates. Rather, the results suggested that the prediction of the fate of a single organic component in a reactor receiving a multicomponent feed is a very difficult task.     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Underwater observations of piranhas in western Brazil

Synopsis The behaviour of three piranha species,Serrasalmus marginatus, S. spilopleura, andPygocentrus nattereri, and their prey fishes was studied underwater in the Pantanal region, Mato Grosso, Brazil. General habits, predatory tactics, feeding behaviour, and social interactions while foraging, as well as defensive tactics of prey fishes were observed.S. marginatus is solitary whereas the other two species live in shoals; their agonistic behaviour varies accordingly, the simplest being displayed by the solitary species. Predatory tactics and feeding behaviour also vary:S. spilopleura shows the most varied diet and highly opportunistic feeding strategy, which includes aggressive mimicry. The solitaryS. marginatus, besides fin and scale-eating, occasionally cleans larger individuals ofP. nattereri. Several cichlid species display defensive tactics clearly related to piranha attacks: tail protecting, watching, and confronting the predator are the most commonly observed behaviours. Piranhas seem to strongly influence use of habitat, social structure, and foraging mode of the fish communities.     

Some Characteristics of Threonine Transport Across the Blood-Brain Barrier of the Rat

Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system.