Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

[Frusemide plus doxazosin therapy for nocturia in patients with BPH/LUTS].

OBJECTIVE: To determine the efficacy and safety of a diuretic agent, frusemide, combined with doxazosin in the treatment of nocturia in patients with benign prostate hyperplasia / lower urinary tract symptoms (BPH/LUTS). METHODS: Sixty-four BPH/LUTS patients with nocturia were equally randomized into two groups, one treated with doxazosin (4 mg/d), and the other with frusemide (40 mg/d) and doxazosin (4 mg/d), given 6 h before sleep, both for 4 weeks. Urine volume, IPSS, QOL, serum electrolytes, plasma osmolality were recorded and compared between the two groups before and after the treatment. RESULTS: Compared with the doxazosin group, the frusemide plus doxazosin group showed significantly reduced nocturia frequency (P < 0.01), increased daytime urine output (P < 0.01), decreased nocturia urine output (P < 0.01), unchanged total urine output (P > 0.05), improved IPSS and QOL (P < 0.05, P < 0.01), but with no remarkable differences in the levels of serum sodium, potassium, chlorine, and osmotic pressure (P > 0.05). CONCLUSION: Four-week treatment with frusemide plus doxazosin was safe and effective for nocturia in patients with BPH/LUTS.

Postsynaptic calcium pathway contributes to synaptic plasticity between retinal cones and luminosity-type horizontal cells.

It was previously found that the efficacy of synaptic transmission between retinal cone systems and luminosity-type horizontal cells (LHCs) was activity-dependent. Repetitive activation of red-cone pathway increased the LHCos hyperpolarizing response to red light, and the response enhancement was reversible. In this study, intracellular recording and pharmacological method were applied to investigate the mechanism(s) underlying red-flickering-induced response enhancement. Lowering intracellular Ca(2+) in the LHC by intracellular injection of Ca(2+) chelator EGTA prevented the development of red-flickering-induced response enhancement, which implicates the importance of postsynaptic calcium signal. The response enhancement could also be eliminated by a potent antagonist of Ca(2+)-permeable AMPA receptor (CP-AMPAR), which suggests the possibility that Ca(2+) influx via glutamate-gated calcium channels is related to the changes of [Ca(2+)](i). Furthermore, the administration of ryanodine or caffeine also attenuated the phenomenon, which gives evidence that the local calcium signal caused by intracellular calcium-induced calcium release (CICR) may be involved. Taken together, our data implicate that postsynaptic CICR and CP-AMPAR are related to the activity-dependent response enhancement.

Ca2+-permeable and Ca2+-impermeable AMPA receptors coexist on horizontal cells.

Fura-2 fluorescent calcium imaging was used for analyzing the subtype of AMPA receptors in freshly dissociated horizontal cells of carp retina. Exogenous application of AMPA induced an increase of intracellular concentration of free Ca2+ ([Ca2+]i) in horizontal cells, while the [Ca2+]i increase was partly inhibited by nifedipine. The residual [Ca2+]i increase was completely eliminated by joro spider toxin-3, a blocker of Ca2+-permeable AMPA receptors. On the other hand, the application of pentobarbital, which blocked Ca2+-impermeable AMPA receptors, could also partly inhibit the increase of [Ca2+]i, implying that the application of AMPA induced the activation of both Ca2+-permeable and Ca2+-impermeable AMPA receptors and the consequent activation of voltage-gated Ca2+ channels. Taken together, these results suggested that Ca2+-permeable and Ca2+-impermeable AMPA receptors were coexpressed on horizontal cells.

Role of Ca2+ store in AMPA-triggered Ca2+ dynamics in retinal horizontal cells.

Fura-2 fluorescent calcium imaging was applied to measure [Ca(2+)](i) in freshly dissociated horizontal cells of carp retina, and a model containing endoplasmic reticulum (ER) membrane processes and plasma membrane processes was constructed for quantitative analyses of the AMPA-triggered calcium dynamics. A transient increase followed by a sustained steady level of [Ca(2+)](i) was observed when 100 microM AMPA was applied, while the initial transient increase of [Ca(2+)](i) was suppressed by exogenously applied ryanodine. The model analyses results suggest that the AMPA-triggered calcium dynamics involves a number of cytoplasmic and endoplasmic processes that interact with each other. It also suggests that calcium store is an important part contributing to the transient calcium signal.

Growth and expression of rat bone marrow mesenchymal stem cells modified by nerve growth factor in diabetic rat bladders.

The aim of the present study was to determine whether rat bone marrow mesenchymal stem cells (MSCs) transfected with the nerve growth factor (NGF) gene and then transplanted into diabetic rat bladder tissues survive and continue to express NGF. A recombinant lentiviral vector carrying the NGF gene was constructed and transfected into rat bone marrow MSCs. BrdU­labeled immunohistochemistry was used to observe NGF expression in the transfected MSCs. BrdU­labeled and NGF­transfected MSCs were transplanted into diabetic rat bladder tissues. BrdU­labeled immunohistochemistry was used to observe the growth of NGF­transfected MSCs in the tissue samples. NGF mRNA and protein expression levels in MSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) and ELISA, respectively. The recombinant NGF gene lentiviral vector and NGF gene-modified rat bone marrow MSCs were successfully constructed. NGF gene-modified rat MSCs survived in the diabetic rat bladders 4 weeks following injection and NGF gene expression was increased. In the present study, NGF gene-modified MSCs were shown to be capable of survival in diabetic rat bladder tissues and stably expressed NGF.