Mycobacterium parascrofulaceum in acidic hot springs in Yellowstone National Park

Mycobacterium parascrofulaceum was found in Norris Geyser Basin, Yellowstone National Park, in a system composed of two acidic (pH 3.0) springs with temperatures between 56 degrees C at the source and 40 degrees C at the confluence of both springs. Growth and survival assays at 56 degrees C for 60 days were performed, confirming the origin of the strain.     

Street heroin induces mitochondrial dysfunction and apoptosis in rat cortical neurons

Cortical function has been suggested to be highly compromised by repeated heroin self-administration. We have previously shown that street heroin induces apoptosis in neuronal-like PC12 cells. Thus, we analysed the apoptotic pathways involved in street heroin neurotoxicity using primary cultures of rat cortical neurons. Our street heroin sample was shown to be mainly composed by heroin, 6-monoacetylmorphine and morphine. Exposure of cortical neurons to street heroin induced a slight decrease in metabolic viability, without loss of neuronal integrity. Early activation of caspases involved in the mitochondrial apoptotic pathway was observed, culminating in caspase 3 activation, Poly-ADP Ribose Polymerase (PARP) cleavage and DNA fragmentation. Apoptotic morphology was completely prevented by the non-selective caspase inhibitor z-VAD-fmk, indicating an important role for caspases in neurodegeneration induced by street heroin. Ionotropic glutamate receptors, opioid receptors and oxidative stress were not involved in caspase 3 activation. Interestingly, street heroin cytotoxicity was shown to be independent of a functional mitochondrial respiratory chain, as determined using NT-2 rho(0) cells. Nonetheless, in street heroin-treated cortical neurons, cytochrome c was released, accompanied by a decrease in mitochondrial potential and Bcl-2/Bax. Pure heroin hydrochloride similarly decreased metabolic viability but only slightly activated caspase 3. Altogether, our data suggest an important role for mitochondria in mediating street heroin neurotoxic effects.     

RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Identification of a novel potential antigen for early-phase serodiagnosis of leptospirosis

This study examined four genes encoding for predicted membrane proteins selected from the genome sequences of Leptospira interrogans. Genes were cloned and the proteins expressed in E. coli. Immunoblotting analysis of the recombinants with sera from early and convalescent phases of a leptospirosis patient showed that two proteins, namely Lp29 and Lp49, were reactive with serum from both phases of the illness. These data were further confirmed in enzyme-linked immunosorbent assay using sera from both phases of seventeen confirmed leptospirosis specimens, suggesting that these proteins are presented to the host immune system during infection. In the early phase, anti-Lp29 IgM was detected in all sera when microscopic agglutination tests (MAT), the reference method for diagnosis of leptospirosis, were negative. The gene encoding Lp49 is conserved among five tested leptospiral pathogenic serovars, while Lp29 is present in serovars that are predominant in urban settings. These recombinant antigens might be valuable for serodiagnosis of both phases of leptospirosis.     

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat_48-57 and Antp_43-58 cell-permeable peptide carriers. Both Tat_48-57 and Antp_43-58 are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 μM of Tat_48-57 were essentially harmless in all cells tested, whereas Antp_43-58 was significantly more toxic. Moreover, all peptides bound to Tat_48-57 and Antp_43-58 triggered significant and length-dependent cytotoxicity when used at concentrations above 10 μM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat_48-57 should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 μM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat_48-57- and Antp_43-58 bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.     

Genetic structure of the red mangrove (<Emphasis Type="Italic">Rhizophora mangle</Emphasis> L.) on the Colombian Pacific detected by microsatellite molecular markers

Rhizophora mangle, one of the five species of the genus Rhizophora, is found widely distributed along the American and West African coasts. This species is one of the principal constituents of the mangrove ecosystem in Colombia and is also found within the most important economic activities for the communities that inhabit the littoral. In order to assess the degree of genetic diversity of R. mangle in five populations of the Colombian Pacific, nuclear microsatellite molecular markers were used. In 92 individuals sampled, it was found that 100% of the loci were polymorphic , and no private alleles were detected. The population structure of R. mangle in the Colombian Pacific, was highly significant (P < 0.001); however, the greatest differentiation was detected at the within-population level (94.62%). For the populations of La Plata, Virudó and Charambirá, the tendency toward panmixia could be the cause of the low differentiation among these three locations. Within populations, the genetic diversity revealed a deviation from Hardy–Weinberg equilibrium with high significance in Virudó and Tumaco, where it appears the intense anthropogenic activity has exercised strong pressure on the red mangrove, resulting in the possible fragmentation of the local landscape and therefore an increase in the rate of endogamy within these populations. Despite this situation, our study―one of the first developed in genetics of the red mangrove in Colombia―did not show evidence of recent bottleneck effects or deterioration in its genetic composition, which could be exploited to propose management and restoration programs for the zones where the forests of this species are degraded. Handling editor: K. Martens     

Potential mineralization of nitrogen from organic wastes to ryegrass and wheat crops

Two-pot experiments with ryegrass and wheat plants were conducted in a Cambic Arenosol to test the reliability of N fate predicted by incubation experiments previously performed, with the same soil, to assess potentially mineralizable nitrogen from six organic wastes (municipal solid waste compost, secondary pulp mill sludge, horn meal, poultry manure, solid phase from pig slurry and composted pig manure). Two treatments, corresponding to 80 and 160 kgN/ha were tested, with or without mineral N fertilization. Experimental data obtained in the pot trials was consistent with nitrogen net mineralization trend observed in the aerobic incubations with all the wastes tested. Values of potentially mineralizable nitrogen (N(0)) from the equations obtained by model fitting, to the incubation data, were well correlated to ryegrass and wheat N uptake. Poultry manure was the most efficient N supplier to crops.     

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.