Asymmetric ketone reduction with immobilized yeast in hexane: biocatalyst deactivation and regeneration

There is a need to develop methods for producing enantiomerically pure pharmaceuticals because the racemic mixtures made today will probably not be allowed in the future. Synthetic chiral catalysts are being developed for this purpose, as well as new product separation techniques. Another possible option is to use biocatalysts, such as purified enzymes or whole microbial cells, since these can result in the production of mostly a single enantiomer. This study emphasizes the use of alginate-entrapped yeast cells to catalyze the reduction of ketones as a model system. The emphasis is on the factors that might limit the reactivity of such cells, such as equilibrium conditions, substrate or product inhibition, solvent toxicity, loss of cell viability, or the degradation of intracellular levels of enzymes or cofactors.It was found that there was a progressive loss of catalytic activity of the immobilized yeast cells, which appeared to be mainly associated with a loss of cell viability and a decline of intracellular NAD(H) levels during the reaction. The other factors investigated did not have a large effect. A regeneration scheme was developed in order to replenish the intracellular NAD(H) lost during the reaction, which involved removing the biocatalyst from the reaction and supplying the cells with a nutrient source. This resulted in an increase in the NAD(H) to initial levels and also resulted in a maintenance of the ketone reduction rate over time.     

Preliminary study of gene expression levels in human T-cells exposed to cosmic radiations

Several experiments demontrated the influence of microgravity on mitogenic activation of T cells at molecular level. To discriminate between cffects of microgravity and cosmic radiations, in this work we studied the effects of high cosmic radiations on the genetic expression in human T cells boarded in a stratospheric balloon (BIRBA-1 mission, 22 hours of flight). The genetic expression was analized by the cDNA microarray hybridization technology, which allows the comparative and simultaneous estimate of hundreds of mRNAs Activated cells react to the ionizing stress by activating genes involved in cell cycle check-point, oxidative stress response, heat shock proteins production or by repressing genes involved in antigen recognition.     

Mechanisms of band 3 oxidation and clustering in the phagocytosis of Plasmodium falciparum-infected erythrocytes

Erythrocytes (RBCs) opsonized by IgG and complement are prevalently recognized and phagocytosed by complement receptor CR1. This mechanism, effective in senescent and damaged RBCs seems to be operative in ring-parasitized RBCs, since infection by Plasmodium falciparum induces stage-dependent binding of auto-antibodies and activated C3 to the RBC membrane. Later, parasite forms are also recognized by non-opsonic receptors, such as scavenger receptor CD36. Malaria parasites induce the oxidative formation of hemichromes which are the trigger for the auto-antigen development. Band 3 protein is the most plausible candidate of the RBC auto-antigen, induced by hemichromes. Auto-antigens isolated from trophozoites were found only in a high-molecular-weight protein aggregates not present in the normal RBC. The immunocomplex was purified by protein-A affinity chromatography, purified proteins digested by trypsin and analyzed by MALDI-TOF. Peptide mapping showed that the main antigen consisted of band 3 protein aggregates that also contained hemichromes, IgGs, complement factor 3 (C3), and traces of spectrin and glycophorin but no parasite proteins. Two cysteines located in the band 3 cytoplasmic domain were found to be particularly reactive to oxidants and mediated band 3 covalent dimerization via disulfide bonds. Thus, parasites promote oxidative alterations in the membrane of the host which lead to exposure of antigenic sites recognized by anti-band 3 auto-antibodies. Formation of band 3 clusters appears to be mediated by cytoplasmic binding of hemichromes and also by direct band 3 oxidation, whereby clustered, oxidized and antigenic band 3 was underglycosylated.     

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.     

Plasma methionine determination by capillary electrophoresis-UV assay: application on patients affected by retinal venous occlusive disease

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.     

Identifying native animals in crossbred populations: the case of the Sardinian goat population

The aim of this work was to develop a strategy for using a genetic analysis for identifying native animals in regions where local breeds have been crossed with improved breeds and then compare that strategy to the overall morphology and breeding histories of the herds for identifying these animals. The experiment included the Sardinian goat population, which is a crossbred of native animals with the Maltese breed. Whole herds were assigned to Maltese (five herds; 49 animals), crossbred (18 herds; 117 animals) or Sardinian (12 herds; 164 animals) groups. For the genetic analysis, genotypes of 22 microsatellites were determined on 330 animals, and basic measurements of genetic diversity were calculated. Genetic variability in the microsatellites was different in the three groups. High positive F _IS showed that inbreeding existed in the subpopulations. The index of genetic differentiation, Nei's standard genetic distance and Reynolds' genetic distance were calculated and found to be significantly different between the three groups. The Sardinian and Maltese groups were the most distant whereas the crossbred group was closer to the Sardinian group. The proportion of the genome derived from two ancestral populations (native Sardinian and Maltese) was assessed using the structure software. Animals were assigned to three clusters on the basis of native Sardinian thresholds. A good correspondence between the empirical (morphology and breeding histories) and the objective genetic analysis was found. Both approaches indicate the presence of three different subpopulations in the Sardinian goat population.     

NF1 gene mutations represent the major molecular event underlying neurofibromatosis-Noonan syndrome

Neurofibromatosis type 1 (NF1) demonstrates phenotypic overlap with Noonan syndrome (NS) in some patients, which results in the so-called neurofibromatosis-Noonan syndrome (NFNS). From a genetic point of view, NFNS is a poorly understood condition, and controversy remains as to whether it represents a variable manifestation of either NF1 or NS or is a distinct clinical entity. To answer this question, we screened a cohort with clinically well-characterized NFNS for mutations in the entire coding sequence of the NF1 and PTPN11 genes. Heterozygous NF1 defects were identified in 16 of the 17 unrelated subjects included in the study, which provides evidence that mutations in NF1 represent the major molecular event underlying this condition. Lesions included nonsense mutations, out-of-frame deletions, missense changes, small inframe deletions, and one large multiexon deletion. Remarkably, a high prevalence of inframe defects affecting exons 24 and 25, which encode a portion of the GAP-related domain of the protein, was observed. On the other hand, no defect in PTPN11 was observed, and no lesion affecting exons 11-27 of the NF1 gene was identified in 100 PTPN11 mutation-negative subjects with NS, which provides further evidence that NFNS and NS are genetically distinct disorders. These results support the view that NFNS represents a variant of NF1 and is caused by mutations of the NF1 gene, some of which have been demonstrated to cause classic NF1 in other individuals.     

Identification of congenital muscular dystonia 2 associated with an inherited GlyT2 defect in Belgian Blue cattle from the United Kingdom

Two newborn Belgian Blue calves from a farm in the United Kingdom exhibited lateral recumbency, low head carriage and transient muscle spasms following tactile or auditory stimulation. DNA sequence analysis indicated that both calves were homozygous for the recessive congenital muscular dystonia type 2 (CMD2) mutation (c.809T>C, p.Leu270Pro) in SLC6A5, encoding the neuronal glycine transporter GlyT2. Further testing of animals from the index farm and a sample of Belgian Blue sires revealed an unexpectedly high frequency of CMD2 carriers. This implies that linked quantitative trait loci may be influencing the prevalence of CMD2 in the estimated 55,000 Belgian Blue cattle in the United Kingdom. We have therefore developed new inexpensive tests for the CMD2 allele that can be used to confirm diagnosis, identify carriers and guide future breeding strategy, thus avoiding animal distress/premature death and minimizing the future economic impact of this disorder.     

New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors with low affinity for the offtarget cytosolic isoform II

Considering phenols and coumarins as lead molecules for obtaining non-sulfonamide inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1), we screened a large number of compounds possessing diverse chemotypes, but structural features which resemble the two chemical classes. Here we report an investigation of such derivatives which do not significantly inhibit CA II, but show interesting inhibition profiles against other isozymes. Pyridine-N-oxide-2-thiophenol, thiobenzoic acid, thimerosal, two oximes derived from a six-membered-ring lactone and from coumarin; 2-hydroxyquinoline and coumaphos, were investigated as inhibitors of CA I-XIV. All these compounds did not inhibit CA II, whereas the two oximes and 2-hydroxyquinoline were low nanomolar inhibitors of CA I, IX, XII, XIII and XIV, showing a very different inhibition profile compared to sulfonamides and sulfamates. Some other compounds showed low micromolar inhibition of other isoforms of interest, such as CA VA/VB, CA VI and VII. This study demonstrates that a rather wide range of structures show low nanomolar-micromolar inhibitory activity against many CA isozymes, without inhibiting significantly the offtarget isoform CA II.