Keratinocytes as Depository of Ammonium-Inducible Glutamine Synthetase: Age- and Anatomy-Dependent Distribution in Human and Rat Skin

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component ß-catenin. Inhibition of, glycogen synthase kinase 3β in cultured keratinocytes and HaCaT cells, however, did not support a direct role of ß-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8–10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia

Bioremediation potential of Cd by transgenic yeast expressing a metallothionein gene from Populus trichocarpa

Cadmium (Cd) is an extremely toxic environmental pollutant with high mobility in soils, which can contaminate groundwater, increasing its risk of entering the food chain. Yeast biosorption can be a low-cost and effective method for removing Cd from contaminated aqueous solutions. We transformed wild-type Saccharomyces cerevisiae (WT) with two versions of a Populus trichocarpa gene (PtMT2b) coding for a metallothionein: one with the original sequence (PtMT2b ‘C’) and the other with a mutated sequence, with an amino acid substitution (C3Y, named here: PtMT2b ‘Y’). WT and both transformed yeasts were grown under Cd stress, in agar (0; 10; 20; 50 µM Cd) and liquid medium (0; 10; 20 µM Cd). Yeast growth was assessed visually and by spectrometry OD600. Cd removal from contaminated media and intracellular accumulation were also quantified. PtMT2b ‘Y’ was also inserted into mutant strains: fet3fet4, zrt1zrt2 and smf1, and grown under Fe-, Zn- and Mn-deficient media, respectively. Yeast strains had similar growth under 0 µM, but differed under 20 µM Cd, the order of tolerance was: WT < PtMT2b ‘C’ < PtMT2b ‘Y’, the latter presenting 37% higher growth than the strain with PtMT2b ‘C’. It also extracted ~80% of the Cd in solution, and had higher intracellular Cd than WT. Mutant yeasts carrying PtMT2b ‘Y’ had slightly higher growth in Mn- and Fe-deficient media than their non-transgenic counterparts, suggesting the transgenic protein may chelate these metals. S. cerevisiae carrying the altered poplar gene offers potential for bioremediation of Cd from wastewaters or other contaminated liquids

INFLUÊNCIA DAS FÁBRICAS DE FERTILIZANTES NA COMPOSIÇÃO DAS ÁGUAS DA CHUVA E SUBTERRÂNEA (RIO GRANDE, RS).

Fluoride concentration was determined by potentiometric method in rainwater and in groundwater at Rio Grande city. In rain water, the range of fluoride concentration was 0.05 – 4,40 ppm, and in ground water it was 0,08 – 2,20 ppm. It was concluded that fluoride concentration in rainwater has relation to wind direction and rain parameters (pH and conductivity). Up until distant places from the factories (10 km), the fluoride concentration in rainwater can be several times higher than those found in seawater, showing a great evidence of the fertilizer factories effect. Groundwater fluoride concentrations demonstrated a tendence of decreasing while the distance from the factories increased.A concentração de fluoreto foi determinada, através do método potenciométrico, com eletrodo íon seletivo, na água da chuva e na água subterrânea, no município de Rio Grande. Os teores de fluoreto na água da chuva variaram de 0,05 a 4,40 ppm, enquanto que na água subterrânea as concentrações de fluoreto variaram entre 0,08 e 2,20 ppm. Os resultados mostram que os teores de fluoreto na água da chuva coletada no ponto de amostragem está relacionada à direção do vento e aos parâmetros de chuva (pH e condutividade). Mesmo em regiões mais afastadas das fábricas (10 km de distância), as concentrações de fluoreto podem atingir valores que superam muitas vezes o teor de background mundial para águas de chuva, apresentando forte evidência do efeito das atividades industriais realizadas nas fábricas de fertilizantes. As concentrações de fluoreto nas águas subterrâneas mostram uma tendência de diminuição com afastamento das fábricas