Bacteroides sp. Strain D8, the First Cholesterol-Reducing Bacterium Isolated from Human Feces

The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10⁻⁸ dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 μmol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.     

Environmental regulation of the reproductive system in a flexibly breeding Sonoran Desert bird, the Rufous-winged Sparrow, Aimophila carpalis

We investigated reproductive regulation in male Rufous-winged Sparrows, Aimophila carpalis, a Sonoran Desert passerine that breeds after irregular summer rains. Field and captive data demonstrate that increased photoperiod stimulates testicular development in March and maintains it until early September. Free-living birds caught in July and placed on captive long days (16L: 8D) maintained developed testes for up to 7 months, and free-living birds caught in September, during testicular regression, redeveloped testes when placed on captive long days, indicating that these birds were still photosensitive. Captive birds on long days maintained testicular development when exposed to temperatures mimicking those occurring during regression in free-living birds. In free-living birds, testicular development was observed during spring and summer, but unless this was associated with rainfall, breeding (indicated by juveniles) did not occur. Large increases in plasma luteinizing hormone (LH) in free-living males were correlated with heavy rainfall in July/August, when the birds bred, and in November, when they did not breed. In captive birds, plasma LH concentrations were unresponsive to photoperiodic changes, but may have responded to social cues. Plasma prolactin concentrations were directly correlated with photoperiod in free-living birds, but an effect of photoperiod on prolactin secretion was not seen in captive birds. It is concluded that male Rufous-winged Sparrows use long photoperiods to stimulate and maintain testicular development, but exposure to long photoperiods does not terminate breeding by inducing absolute photorefractoriness. The specific timing of reproductive behaviors is apparently determined by elevated plasma LH coinciding with long day stimulated gonad development.     

Synthesis, structure and magnetic properties of a new family of chiral metal(II) citramalates

A new series of chiral carboxylate-bridged complexes of Mn(II), Co(II), and Ni(II) has been synthesized by reaction of M(II) salts with (S)-2-hydroxy-2-methyl-butanedioic acid ((S)-citramalic acid) under solvothermal conditions. The Mn(II) compound 1 is obtained as a crystalline powder, whereas the Co(II) and Ni(II) compounds (2 and 3 respectively) are obtained as single crystals. All the compounds crystallize in orthorhombic chiral space group P2₁2₁2₁. Compounds 2 and 3 are isostructural, and their structure consists in helicoïdal chains of M(II) centres linked by carboxylate bridges. The magnetic data indicate a rather weak coupling interaction between paramagnetic centres. The Mn(II) compound 1 exhibits antiferromagnetic ordering at T_N = 2.64 K. The Co(II) and Ni(II) compounds show ferromagnetic interactions within the chains. For 3, the chains couple antiferromagnetically, which leads to a metamagnetic behaviour with T_N = 1.69 K.     

A unified probabilistic model of the perception of three-dimensional structure from optic flow

Human observers can perceive the three- dimensional (3-D) structure of their environment using various cues, an important one of which is optic flow. The motion of any point’s projection on the retina depends both on the point’s movement in space and on its distance from the eye. Therefore, retinal motion can be used to extract the 3-D structure of the environment and the shape of objects, in a process known as structure-from-motion (SFM). However, because many combinations of 3-D structure and motion can lead to the same optic flow, SFM is an ill-posed inverse problem. The rigidity hypothesis is a constraint supposed to formally solve the SFM problem and to account for human performance. Recently, however, a number of psychophysical results, with both moving and stationary human observers, have shown that the rigidity hypothesis alone cannot account for human performance in SFM tasks, but no model is known to account for the new results. Here, we construct a Bayesian model of SFM based mainly on one new hypothesis, that of stationarity, coupled with the rigidity hypothesis. The predictions of the model, calculated using a new and powerful methodology called Bayesian programming, account for a wide variety of experimental findings.     

Lysophosphatidic acid induces endothelial cell death by modulating the redox environment

Oxidant stress plays a significant role in hypoxic-ischemic injury to the susceptible microvascular endothelial cells. During oxidant stress, lysophosphatidic acid (LPA) concentrations increase. We explored whether LPA caused cytotoxicity to neuromicrovascular cells and the potential mechanisms thereof. LPA caused a dose-dependent death of porcine cerebral microvascular as well as human umbilical vein endothelial cells; cell death appeared oncotic rather than apoptotic. LPA-induced cell death was mediated via LPA(1) receptor, because the specific LPA(1) receptor antagonist THG1603 fully abrogated LPA's effects. LPA decreased intracellular GSH levels and induced a p38 MAPK/JNK-dependent inducible nitric oxide synthase (NOS) expression. Pretreatment with the antioxidant GSH precursor N-acetyl-cysteine (NAC), as well as with inhibitors of NOS [N(omega)-nitro-l-arginine (l-NNA); 1400W], significantly prevented LPA-induced endothelial cell death (in vitro) to comparable extents; as expected, p38 MAPK (SB203580) and JNK (SP-600125) inhibitors also diminished cell death. LPA did not increase indexes of oxidation (isoprostanes, hydroperoxides, and protein nitration) but did augment protein nitrosylation. Endothelial cytotoxicity by LPA in vitro was reproduced ex vivo in brain and in vivo in retina; THG1603, NAC, l-NNA, and combined SB-203580 and SP600125 prevented the microvascular rarefaction. Data implicate novel properties for LPA as a modulator of the cell redox environment, which partakes in endothelial cell death and ensued neuromicrovascular rarefaction.     

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.     

Identification of a novel series of benzimidazoles as potent and selective antagonists of the human melanocortin-4 receptor

A novel series of benzimidazoles was identified and optimized, leading to the discovery of potent and selective antagonists of the human melanocortin-4 receptor. In addition, compound 5i was shown to cross the blood-brain barrier after intravenous dosing in rats.     

Pyrazolone methylamino piperidine derivatives as novel CCR3 antagonists

The discovery and optimization of a novel class of potent CCR3 antagonists is described. Details of synthesis and SAR are given together with some ADME properties of selected compounds. An optimal balance between activities, physicochemical properties, and in vitro metabolic stability was reached by the proper choice of substituents.     

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.     

Synthesis and in vitro evaluation of targeted tetracycline derivatives: effects on inhibition of matrix metalloproteinases

Among other non-antibiotic properties, tetracyclines inhibit matrix metalloproteinases and are currently under study for the treatment of osteoarthritis. Quaternary ammonium conjugates of tetracyclines were synthesized by direct alkylation of the amine function at the 4-position with methyl iodide. When tested in vitro, they inhibited cytokine-induced MMP expression to a lesser extent than parent tetracyclines. This was compensated by an improved inhibition of MMP catalytic activity. Since inhibition of collagen degradation was maintained these derivatives could be potent drug candidates for cartilage-targeted chondroprotective treatment.