Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from<Emphasis Type="Italic"> Alicyclobacillus acidocaldarius</Emphasis> and<Emphasis Type="Italic"> Archaeoglobus fulgidus</Emphasis> by random mutagenesis and screening

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.     

The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem-loop structured probes

Enzyme-mediated reactions are a useful tool in mutation detection when using a microarray format. Discriminating probes attached to the surface of a DNA chip have to be accessible to target DNA and to the enzyme (ligase or polymerase) that catalyses the formation of a new phosphodiester bond. This requires an appropriate chemical platform. Recently, an oligonucleotide hairpin architecture incorporating multiple phosphorothioate moieties along the loop has been proposed as an effective approach to solid-phase minisequencing. We have explored in depth several variables (stem length, number of phosphorothioates, stem-loop architecture versus linear structure) involved in this strategy by using a solid-phase ligation reaction. Microarrays were fabricated either from aminosilyl-modified glass or from aminated polymeric surfaces made of poly-lysine. Both platforms were bromoacetylated and reacted with thiophosphorylated oligonucleotides. The resulting microarrays were tested using either a synthetic template or a PCR-amplified 16S rRNA genomic region as the target sequence. Our results confirm the robustness of the proposed chemistry. We extend its range of application to solid-phase ligation, demonstrating the effectiveness of multiple anchors and suggest that linear oligonucleotides incorporating multiple phosphorothioates are equivalent to their hairpin-structured counterparts.     

Enhanced activity of the plasma membrane oxidoreductase in circulating lymphocytes from insulin-dependent diabetes mellitus patients

Circulating human lymphocytes contain a transmembrane oxidoreductase (PMOR) capable of reducing dichlorophenol indophenol (DCIP) by endogenous reductants, presumably NADH. Membranes from lymphocytes obtained from buffy coats contain a NADH DCIP reductase having a K(m) of about 1 microM and almost insensible to dicoumarol. The PMOR of lymphocytes from insulin-dependent diabetic patients is higher than that from age-matched controls and, in addition, has a dicoumarol-sensitive component, lacking in most controls, presumably due to membrane association of DT-diaphorase. The increase of PMOR in diabetes is likely due to overexpression of the enzyme, in view of the very low K(m) for NADH indicating that, in intact cells, the enzyme is practically saturated with the reductant substrate.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

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.     

Leech responses to tissue transplantation

The aim of the present work is to describe histologically, histochemically and immunocytochemically, the sequence of events that lead to first and second set rejection of allo- or xenograft in leeches. Graft responses of leeches are comparable and are described following specific steps: inflammatory phase, rejection phase and granulation tissue formation (including re-epithelialisation, angiogenesis and fibroplasia).The responses to first and second graft in first set graft rejection as well as to the first transplant in second set graft experiments are identical and in the time span of a week all grafts are destroyed and disappear. In the second set graft rejection experiments the responses against the second transplant are markedly accelerated. The second graft shows massive structural alterations and it is rapidly rejected, within 3-4 days.Our results permit to highlight that in leeches there is a specific responsiveness of immune system similar to those described in highly divergent phyla.     

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.