Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.     

Innervation of vas deferens and accessory male genital glands in the water buffalo (Bubalus bubalis). Neurochemical characteristics and relationships to the reproductive activity

Autonomic nerves supplying mammalian male internal genital organs have an important role in the regulation of reproductive function. To find out the relationships between the neurochemical content of these nerves and the reproductive activity, we performed a histochemical and immunohistochemical study in a species, the water buffalo, exhibiting a seasonal sexual behaviour. The distribution of noradrenergic and nitric oxide synthase (NOS)- and peptide-containing nerves was evaluated during the mating and non-mating periods. Fresh segments of vas deferens and accessory genital glands were collected immediately after slaughter and immersed in 4% paraformaldehyde. Frozen sections were obtained and processed according to single and double labelling immunofluorescent procedures or NADPH-diaphorase histochemistry. During the mating period, a dense noradrenergic innervation was observed to supply the vas deferens as well as the accessory genital glands. NOS- and peptide-containing nerves were also observed but with a lower density. During the non-mating period noradrenergic nerves dramatically reduced. In addition, neuropeptide Y (NPY)- and vasoactive intestinal peptide (VIP)-containing nerves were also reduced. These findings suggest the presence of complex interactions between androgen hormones and the autonomic nerve supply in the regulation of male water buffalo reproductive functions.     

Optical single-channel recording: imaging Ca2+ flux through individual N-type voltage-gated channels expressed in Xenopus oocytes

Functional studies of single membrane ion channels were made possible by the introduction of the patch-clamp technique, which allows single-channel currents to be measured with unprecedented resolution. Nevertheless, patch clamping has some limitations: including the need for physical access of the patch pipette, possible disruption of local cellular architecture, inability to monitor multiple channels, and lack of spatial information. Here, we demonstrate the use of confocal fluorescence microscopy as a non-invasive technique to optically monitor the gating of individual Ca2+ channels. Near-membrane fluorescence signals track the gating of N-type Ca2+ channels with a kinetic resolution of about 10ms, provide a simultaneous and independent readout from several channels, and allow their locations to be mapped with sub-micrometer spatial resolution. Optical single-channel recording should be applicable to diverse voltage- and ligand-gated Ca2+-permeable channels, and has the potential for high-throughput functional analysis of single channels.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Vgamma9/Vdelta2 T lymphocytes in Italian patients with Behçet's disease: evidence for expansion,and tumour necrosis factor receptor II and interleukin-12 receptor beta1 expression in active disease

Beh?et's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that gamma/delta T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of gamma/delta T cells with phenotype Vgamma9/Vdelta2, from a group of Italian patients with Beh?et's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vgamma9/Vdelta2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vgamma9/Vdelta2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor beta1 was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor beta1 PE and anti-Vdelta2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vgamma9/Vdelta2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor beta1 expressions were increased in both patients and control individuals. The proportion of Vgamma9/Vdelta2 T cells bearing these receptors was raised in active disease when Vgamma9/Vdelta2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vgamma9/Vdelta2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.     

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27^Kip1 and p16^INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.     

Enantiomerically pure tetrahydroquinoline derivatives as in vivo potent antagonists of the glycine binding site associated to the NMDA receptor

To identify neuroprotective agents after stroke, new substituted tetrahydroquinoline derivatives were designed as antagonists of the glycine binding site associated to the NMDA receptor, satisfying the key pharmacophoric requirements. In particular, the racemate 3c exhibited outstanding in vivo activity in the MCAo model in rats, when given iv both pre- and post-ischemia. Pure enantiomers 3c-(+) and 3c-(-) have been prepared following an original synthetic route. Despite the significant difference of activity observed in vitro, they shown similar neuroprotective profile in the MCAo model in rats.     

Application of an image analysis system to the determination of biomass (ash free dry weight) of pond macroinvertebrates

The absence of a rapid and inexpensive method for estimating macroinvertebrate individual biomass is a limit to the study of freshwater communities, given that traditional methods are time- and money-consuming. We propose an inexpensive method to quickly estimate individual biomass from video-recorded images of pond macroinvertebrates. We used a software that automatically measures several body dimensions (area, perimeter, minor and major axes) on each individual and linear regression to relate these body dimensions to dry weight. Area was found to be the best predictor of dry weight. The method allows individual measures of macroinvertebrates at a reasonable speed and accuracy, and may be useful for biomass size spectra or secondary production studies.     

Fine Exon–Intron Structure of the Fanconi Anemia Group A (FAA) Gene and Characterization of Two Genomic Deletions

Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight genes on the basis of complementation groups (FAAtoFAH). The analysis of theFAAgene in patients suggested the existence of deletions, none of which have thus far been characterized at the genomic level. A detailed restriction map of theFAAgene with the fine localization of its 43 exons is reported in this paper. We also describe the first two genomic deletions, one of 5.0 kb and another of at least 120 kb. The former was likely the result of a recombination between relatedAlusequences. Since these interspersed repeats could generate deletions and insertions by mispairing, rearrangements of this gene are a possibility in those FA families in whichFAAmutations have not been identified.     

Prebiotic chemistry: chemical evolution of organics on the primitive Earth under simulated prebiotic conditions.

A series of prebiotic mixtures of simple molecules, sources of C, H, N, and O, were examined under conditions that may have prevailed during the Hadean eon (4.6-3.8 billion years), namely an oxygen-free atmosphere and a significant UV radiation flux over a large wavelength range due to the absence of an ozone layer. Mixtures contained a C source (methanol, acetone or other ketones), a N source (ammonia or methylamine), and an O source (water) at various molar ratios of C : H : N : O. When subjected to UV light or heated for periods of 7 to 45 days under an argon atmosphere, they yielded a narrow product distribution of a few principal compounds. Different initial conditions produced different distributions. The nature of the products was ascertained by gas chromatographic-mass spectral analysis (GC-MS). UVC irradiation of an aqueous methanol-ammonia-water prebiotic mixture for 14 days under low UV dose (6 x 10(-2) Einstein) produced methylisourea, hexamethylenetetramine (HMT), methyl-HMT and hydroxy-HMT, whereas under high UV dose (45 days; 1.9 x 10(-1) Einstein) yielded only HMT. By contrast, the prebiotic mixture composed of acetone-ammonia-water produced five principal species with acetamide as the major component; thermally the same mixture produced a different product distribution of four principal species. UVC irradiation of the CH(3)CN-NH(3)-H(2)O prebiotic mixture for 7 days gave mostly trimethyl-s-triazine, whereas in the presence of two metal oxides (TiO(2) or Fe(2)O(3)) also produced some HMT; the thermal process yielded only acetamide.