Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation

In this study, glutathione-S-transferase Mu3 (GST) has been reported to play an important role in sperm capacitation, acrosome reaction, and fertilization. The freshly ejaculated buffalo spermatozoa were in vitro capacitated using heparin (10 μg/mL) or cryopreserved in egg yolk citrate extender. Glutathione-S-transferase was identified and characterized in terms of their isozymic forms, tyrosine phosphorylation, and immunolocalization patterns in cryopreserved buffalo spermatozoa in comparison with freshly ejaculated and in vitro capacitated spermatozoa. Two-dimensional gel electrophoresis, immunoblot, immunocytochemistry, and enzyme activity analyses were done to characterize GST in this study. Five and eight isozymic forms of GST were detected in cryopreserved and capacitated spermatozoa, respectively. Differential tyrosine phosphorylation of these enzymes was observed in cryopreserved and capacitated spermatozoa. The tyrosine phosphorylation of this enzyme involved cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent pathways during in vitro capacitation of the spermatozoa. Differential immunolocalization patterns of GST were observed in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Glutathione-S-transferase Mu3 enzyme activity was found to be significantly (P < 0.05) different in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Activity of GST was significantly (P < 0.05) increased with the progression of capacitation. The cryopreserved spermatozoa showed significantly (P < 0.05) greater enzyme activity compared with fresh spermatozoa and was equal to 2-hour capacitated spermatozoa. The cryopreserved spermatozoa showed significant (P < 0.05) loss of GST enzyme protein. Tyrosine phosphorylated GST showed significantly (P < 0.05) greater activity compared with their dephosphorylated forms. The information generated in this study can be used to understand the molecular mechanism of the effects of GST on capacitation. Regulation of GST during sperm cryopreservation could be a good target to improve fertility of cryopreserved spermatozoa for their use in assisted reproductive technologies.     

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae)

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca²⁺-Mg²⁺-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 μl and 1.0 μl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 μl) exposure but decreased at a higher dosage (1.0 μl), while the activities of SOD and Ca²⁺-Mg²⁺-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca²⁺-Mg²⁺-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.     

谷胱甘肽-硫-转移酶M2基因在小鼠生殖器官中的表达

利用半定量RT-PCR和原位杂交的方法检测Gstm2基因在成年雄性和雌性小鼠生殖器官中的表达,并初步评价其在生殖过程中的作用。在雄性小鼠的睾丸、附睾、输精管和雌性小鼠的卵巢、输卵管、子宫、胎盘中,半定量RT-PCR的方法均检测到Gstm2的表达,在胎盘中表达水平较低,其余组织表达水平较高。利用原位杂交的方法在睾丸的间质细胞检测出较强的信号,在附睾中有微弱的信号,而输精管上皮细胞没有检测到信号;在输卵管上皮细胞和妊娠第3d的子宫上皮细胞中检出较强的信号。由于Gstm2在RNA水平在小鼠的生殖器官中广泛表达,因此我们推测Gstm2可能在小鼠精子发生、睾酮合成、精子的成熟和运输、卵子的发生和运输、胚胎着床等生殖过程中发挥作用,此结果为深入研究Gstm2在生殖生理中的功能打下基础。     

Mouse Fem1b interacts with and induces ubiquitin-mediated degradation of Ankrd37

Ankyrin repeat domain 37 (Ankrd37), a protein containing ankyrin repeats (ARs) and a putative nuclear localization signal (NLS), is highly conserved from zebrafish to humans. In mouse testes, Ankrd37 protein was initially present in the cytoplasm of elongating spermatids, and finally restricted to the nuclei of spermatozoa during spermatogenesis. Ankrd37 bound to feminization 1 homolog b (Fem1b) as indicated by yeast two-hybrid screening and co-immunoprecipitation assays. Ankrd37 facilitated the transport of Fem1b protein from cytoplasm to nuclei in co-transfected CHO cells. In addition, the protein level of Ankrd37 was decreased in a Fem1b dose-dependent manner as shown by the transfection experiments, and Ankrd37 was ubiquitinated in the presence of Fem1b. As the nematode Fem-1 has been shown to target its downstream effector TRA-1 for ubiquitin-mediated degradation, we report in the present study that mouse Fem1b targets Ankrd37 for degradation in the same manner.     

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.     

Optical biosensor consisting of glutathione-S-transferase for detection of captan

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 μg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min.     

Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase , which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.