Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis

We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na⁺ and Mg²⁺) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.     

Evaluation of secondary amide replacements in a series of CCR5 antagonists as a means to increase intrinsic membrane permeability. Part 1: Optimization of gem-disubstituted azacycles

Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and increased intrinsic membrane permeability.     

Sinomenine, theophylline, cysteine, and levamisole: Comparisons of their kinetic effects on mineral formation induced by matrix vesicles

The effects of sinomenine (SIN, an alkaloid extracted from the Chinese medicinal plant Sinomenium acutum used for centuries to treat rheumatic disease, including rheumatoid arthritis) on apatitic nucleation and matrix vesicle (MV)-induced mineral formation were compared with those of cysteine, levamisole, and theophylline. We found that SIN was not an inhibitor of tissue non-specific alkaline phosphatase (TNAP), a marker of biological mineralization, but confirmed that cysteine, levamisole, and theophylline were. Further, none of these four molecules directly affected the nucleation of hydroxyapatite (HA) formation, in contrast to pyrophosphate (PP_i) which did. Incubation of 0.25-1.0 mM cysteine, theophylline, or levamisole with MVs in synthetic cartilage lymph (SCL) containing AMP and Ca²⁺, but not inorganic phosphate (P_i), prolonged the induction time of mineral formation, apparently by inhibiting TNAP activity. SIN at the same levels neither inhibited TNAP activity nor affected the induction time of MV mineral formation. However, SIN did markedly delay MV-induced mineral formation in SCL containing P_i (instead of AMP) in a manner similar to theophylline, but to a lesser extent than levamisole. Cysteine did not delay, in fact it slightly accelerated MV-induced mineral formation in Pi-containing SCL. These findings suggest that levamisole, SIN and theophylline may directly affect Ca²⁺ and/or P_i accretion during mineral formation; however, TNAP was not directly involved. The possible roles of annexins and other ion transporters, such as proteins of the solute carrier family implicated in Ca²⁺ and P_i influx are discussed.     

Mutations in IMPG2, Encoding Interphotoreceptor Matrix Proteoglycan 2, Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases caused by progressive degeneration of the photoreceptor cells. Using autozygosity mapping, we identified two families, each with three affected siblings sharing large overlapping homozygous regions that harbored the IMPG2 gene on chromosome 3. Sequence analysis of IMPG2 in the two index cases revealed homozygous mutations cosegregating with the disease in the respective families: three affected siblings of Iraqi Jewish ancestry displayed a nonsense mutation, and a Dutch family displayed a 1.8 kb genomic deletion that removes exon 9 and results in the absence of seven amino acids in a conserved SEA domain of the IMPG2 protein. Transient transfection of COS-1 cells showed that a construct expressing the wild-type SEA domain is properly targeted to the plasma membrane, whereas the mutant lacking the seven amino acids appears to be retained in the endoplasmic reticulum. Mutation analysis in ten additional index cases that were of Dutch, Israeli, Italian, and Pakistani origin and had homozygous regions encompassing IMPG2 revealed five additional mutations; four nonsense mutations and one missense mutation affecting a highly conserved phenylalanine residue. Most patients with IMPG2 mutations showed an early-onset form of RP with progressive visual-field loss and deterioration of visual acuity. The patient with the missense mutation, however, was diagnosed with maculopathy. The IMPG2 gene encodes the interphotoreceptor matrix proteoglycan IMPG2, which is a constituent of the interphotoreceptor matrix. Our data therefore show that mutations in a structural component of the interphotoreceptor matrix can cause arRP.     

Utilisation de la spectrométrie de masse pour l’identification bactérienne: évaluation du système Axima®/Saramis®/SirWeb MALDI TOF®

Concomitant chemoradiotherapy can constitute an alternative to surgical approach in selected patients with bladder carcinoma. TNM staging, histological grading, renal and performance status represent the main prognostic factors useful to define the therapeutic strategy. Biological and genetic factors are still controversial in medical practice. The activation of oncogenes seems to characterize the initiation of superficial lowgrade tumors and the mutations in the tumor suppressor genes (TEN, TP53 and RB) appear as the most important biological features in the in situ and invasive tumors. The expression of p53, Rb, Bcl-2 has an obvious prognostic role but the published studies on molecular factors predictive of radiosensibility are heterogeneous.     

Decomposition of old organic matter as a result of deeper active layers in a snow depth manipulation experiment

A snow addition experiment in moist acidic tussock tundra at Toolik Lake, Alaska, increased winter snow depths 2–3 m, and resulted in a doubling of the summer active layer depth. We used radiocarbon (?¹⁴C) to (1) determine the age of C respired in the deep soils under control and deepened active layer conditions (deep snow drifts), and (2) to determine the impact of increased snow and permafrost thawing on surface CO₂ efflux by partitioning respiration into autotrophic and heterotrophic components. ?¹⁴C signatures of surface respiration were higher in the deep snow areas, reflecting a decrease in the proportion of autotrophic respiration. The radiocarbon age of soil pore CO₂ sampled near the maximum mid-July thaw depth was approximately 1,000 years in deep snow treatment plots (45–55 cm thaw depth), while CO₂ from the ambient snow areas was ~100 years old (30-cm thaw depth). Heterotrophic respiration ?¹⁴C signatures from incubations were similar between the two snow depths for the organic horizon and were extremely variable in the mineral horizon, resulting in no significant differences between treatments in either month. Radiocarbon ages of heterotrophically respired C ranged from <50 to 235 years BP in July mineral soil samples and from 1,525 to 8,300 years BP in August samples, suggesting that old soil C in permafrost soils may be metabolized upon thawing. In the surface fluxes, this old C signal is obscured by the organic horizon fluxes, which are significantly higher. Our results indicate that, as permafrost in tussock tundra ecosystems of arctic Alaska thaws, carbon buried up to several thousands of years ago will become an active component of the carbon cycle, potentially accelerating the rise of CO₂ in the atmosphere.     

Quantitative proteomics of pomegranate varieties with contrasting seed hardness during seed development stages

In pomegranate (Punica granatum), seed hardness is an important trait directly affecting fruit marketability. However, seed formation in pomegranate has not been well studied. We investigated the genetic mechanism underlying pomegranate seed hardness by comparing protein expression profiles between soft- and hard-seeded varieties 60 and 120 days after flowering. We identified 1940 proteins, of which 399 were differentially expressed. Most of the differentially expressed proteins were involved in posttranslational modification and carbohydrate metabolism. Cell wall biosynthesis, which showed positive correlations with seed hardness, was selected as the candidate pathway. The mRNA levels of 14 proteins involved in cell wall biosynthesis were further analyzed by qPCR. Lignin biosynthesis-related differentially expressed proteins showed lower expression at protein and gene levels in a soft-seeded variety at the early stages. Moreover, cellulose biosynthesis-related differentially expressed proteins showed higher expression levels in the soft-seeded variety at 60 days after flowering. Thus, the soft-seeded variety showed lower lignin but higher cellulose biosynthesis at the early fruit developmental stage, suggesting that lignin and cellulose play opposing roles in cell wall formation in pomegranate seeds. Moreover, differentially expressed proteins involved in cell wall degradation showed higher expression levels in the soft-seeded variety at both developmental stages. These results suggested that differences in seed hardness between soft- and hard-seeded pomegranates might result from cell wall biosynthesis and also be affected by cell wall degradation. The present proteome-wide profiling of pomegranate genotypes with contrasting seed hardness adds to the current knowledge base of the molecular basis of seed hardness development.     

Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley-Liss, Inc.     

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.