Rhinella marina oocytes: a suitable alternative expression system for functional characterization of aquaglyceroporins.

Amphibian oocytes have been extensively used for heterologous expression of membrane proteins for studying their biochemical and biophysical properties. So far, Xenopus laevis is the main amphibian used as oocytes source to express aquaglyceroporins in order to assess water and solutes permeability. However, this well-established amphibian model represents a threat to the biodiversity in many countries, especially in those from tropical regions. For that reason, the import of Xenopus laevis is subjected to strict control, which essentially has restricted its use in these regions. Therefore, a wider variety of expression systems for aquaglyceroporins is needed. Rhinella marina is extensively distributed in the Americas and its native range spreads from South America to Texas, US. Here we report the use of Rhinella marina oocytes as an alternative expression system for aquaglyceroporins and demonstrated its suitability to determine the permeability to water and non-ionic solutes. Rhinella marina oocytes were able to functionally express channels from human and the protozoan pathogen Trypanosoma brucei, two very distant organisms on the evolutionary scale. Permeability values obtained from Rhinella marina oocytes expressing members of aquaporin family were similar and comparable to those values reported in the literature for the same channels expressed in Xenopus laevis oocytes.

Overexpression of an aquaglyceroporin gene from Trichoderma harzianum improves water-use efficiency and drought tolerance in Nicotiana tabacum.

Aquaporins (AQPs) and aquaglyceroporins (AQGPs) are integral membrane proteins that mediate the transport of water and solutes, such as glycerol and urea, across membranes. AQP and AQGP genes represent a valuable tool for biotechnological improvement of plant tolerance to environmental stresses. We previously isolated a gene encoding for an aquaglyceroporin (ThAQGP), which was up-regulated in Trichoderma harzianum during interaction with the plant pathogen Fusarium solani. This gene was introduced into Nicotiana tabacum and plants were physiologically characterized. Under favorable growth conditions, transgenic progenies did not had differences in both germination and growth rates when compared to wild type. However, physiological responses under drought stress revealed that transgenic plants presented significantly higher transpiration rate, stomatal conductance, photosynthetic efficiency and faster turgor recovery than wild type. Quantitative RT-PCR analysis demonstrated the presence of ThAQGP transcripts in transgenic lines, showing the cause-effect relationship between the observed phenotype and the expression of the transgene. Our results underscore the high potential of T. harzianum as a source of genes with promising applications in transgenic plants tolerant to drought stress.

Expression and clinical significance of aquaglyceroporins in human hepatocellular carcinoma.

Aquaglyceroporins (AQPs) are a subset of the aquaporin family, and are permeable to water and glycerol. The aim of the present study was to determine the expression and clinical significance of three AQPs, AQP3, 7 and 9 in hepatocellular carcinoma (HCC). Fresh HCC and adjacent non­tumorous liver tissues were collected from 68 patients diagnosed with HCC. The expression levels of AQP3, 7 and 9 were detected by reverse transcription­quantitative polymerase chain reaction, western blotting and immunohistochemical analysis. The association between the expression of AQPs and clinicopathological parameters of HCC were investigated. Compared with non­tumorous liver tissue, HCC tissues exhibited a significant (P<0.05) increase in the expression of AQP3 and a concomitant reduction in the expression levels of AQP7 and AQP9, at both the mRNA and protein levels. Immunohistochemistry revealed that AQP9 was dominantly localized on the plasma membrane of hepatocytes, while AQP3 and AQP7 exhibited a predominantly cytoplasmic and nuclear distribution. High expression of AQP3 was significantly (P<0.05) associated with low expression levels of AQP7 and AQP9. High expression of AQP3 was correlated with tumor grade (P=0.017), tumor stage (P=0.010) and lymphatic metastasis (P=0.031). Low expression of AQP7 was correlated with tumor grade (P=0.043). AQP3 was upregulated, and AQP7 and AQP9 were downregulated in HCC. A high expression of AQP3 and low expression of AQP7 was significantly associated with the aggressive features of HCC.

Novel channel enzyme fusion proteins confer arsenate resistance.

Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion.

[The clinical features and colonic epithelium AQP8 expression in diarrhea-irritable bowel syndrome].

OBJECTIVE: To test the expression of AQP8 in ascending and descending colon mucosa of diarrhea-irritable bowel syndrome (D-IBS) patients and to study the relationship between IBS and AQP8 as well as the pathological mechanism for D-IBS. METHODS: The proximal ascending colon and the distal descending colon of 26 D-IBS sufferers were resected. Total RNA was purified from each sample of mucosa and AQP8 mRNA expression was analyzed with fluorescent quantitative RT-PCR. Analysis was conducted with regard to five aspects of the sufferers, i.e. their sex, age at first incidence, duration of illness, frequency of defecation and characteristics of stool. Correlation analysis of the level of AQP8 mRNA expression in the ascending colon and descending colon was made, and relationship between the expression of AQP8 and the incidence and clinical features of D-IBS explored. RESULTS: AQP8 expression was present in specimens of the ascending colon and descending colon of both healthy persons and D-IBS sufferers. Sufferers of D-IBS showed a remarkably lower level of AQP8 expression than the normal controls (Mean 3.1 x 10(4) copies/microg RNA and 2.8 x 10(4) copies/microg RNA vs 8.2 x 10(4) copies/microg RNA and 3.8 x 10(4) copies/microg RNA). The level of AQP8 expression was not correlated with the age of the sufferers or age at first incidence, but was closely correlated with the duration of illness, frequency of defecation and characteristics of stool. The longer the illness lasted, the higher the frequency of defecation and the larger the amount of water was found in stool, the lower the level of AQP8 mRNA expression in colonic mucosa. CONCLUSIONS: The decrease in AQP8 expression in sufferers of D-IBS indicates that colonic absorption function is disordered. As a result, absorption of water is reduced, thus loose stool and diarrhoea occur. There may be some correlation between the variation in AQP8 and the incidence of D-IBS.