脂质体介导外源基因体外转染牛胎儿成纤维细胞条件的优化

通过脂质体（FuGENE-6）介导,将真核表达载体pEGFP-C1成功导入体外培养的牛胎儿成纤维细胞,探讨影响外源基因转染效率的参数,如DNA和脂质体的用量、转染的细胞数量以及细胞暴露于DNA与脂质体复合物的时间长度。通过实验发现,绿色荧光蛋白（green fluorescent protein,GFP）基因的表达随DNA、脂质体量的增加而增加,延长细胞暴露时间反而使转染效率下降,转染细胞数适当才能得到较高的转化率。 Optimization of Parameters of Exogene Transfection of Bovine Fetal Fibroblasts in vitro Mediated by Liposome LI Yang,WU Kai-feng,GUO Xu-dong,GUO Ji-tong,BOU Shor-gan The Research Center for Laboratory Animal Science of Inner Mongolia University,The Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology,Huhhot 010021,China Abstract:pEGFP-C1 eucaryon expression vector was successfully transfected by liposome into bovine fetal fibroblasts.We investigated the effect of parameter such as the dose of DNA and liposome,number of cell transfected and exposure time of the cell to the DNA-liposome complexes.It was indicated that GFP（green fluorescent protein）expression was enhanced as the dose of DNA and liposome increased and on decline as the exposure time was prolonged.The improvement of transfection efficiency depent on the suitable cell number. Key words：liposome; GFP; bovine fetal fibroblasts; transfection     

In vitro antifungal activity of voriconazole against dermatophytes and superficial isolates of Scopulariopsis brevicaulis

We have studied the in vitro antifungal activity of voriconazole, fluconazole and itraconazole against 252 clinical isolates of dermatophytes and Scopulariopsis brevicaulis by a standardized agar diffusion method (NeoSensitabs). Several important factors such as temperature (28 degrees C vs. 35 degrees C) and incubation time (2-10 days vs. 18-74 h) were adapted to dermatophytes and Scopulariopsis requirements. Voriconazole showed an excellent activity against most species of dermatophytes, higher than itraconazole and fluconazole. However, S. brevicaulis isolates were highly resistant to all azoles used in this study. Voriconazole might be an interesting antifungal alternative to refractory superficial mycoses.     

A novel approach for the management of the chalkbrood disease infesting honeybee Apis mellifera L. (Hymenoptera: Apidae) colonies in Egypt

Except for, very few articles regarding the influence of some organic acids on the causative pathogen, Ascosphaera apis Maassen, no other studies pertaining to the management of the chalkbrood disease were performed, so far in Egypt. Laboratory investigations indicated that the fungicides, i.e (Galben C 46%, Radomil gold pluse WP 42.5% and Daconil 2787) at their recommended rates did not exert any effect on the mycelical growth of the fungus. Therefore, these fungicides were completely excluded from the subsequent apiary trials. As to the Mycostatin, it was found clearly that this mycostatic compound was effective at the rates of 50.000 and 100.000 IU. Regarding the essential oils (ceder, clove, peppermint, parsley, black cumin, garden rocket, and ricin), ceder oil surpassed the other oils and materials in controlling the subject disease. It is peculiar that no studies on the efficacy of ceder are available in the literature, so the present work using ceder oil is recorded for the first time worldwide. Thymol substance at the rate of 2% showed also a great success in managing the CHB disease. Baised on the obtained results, the promising materials in controlling the disease could be arranged according to their efficacy in a descending order as follows: ceder oil>thymol>mycostatin and oxalic acid, so these highly effective materials were again tested under the apiary conditions. Outdoors (apiary) studies revealed that ceder oil 4% gave 100% reduction in mummies numbers. Reductions in number of fallen mummies ranged from 63.22 to 96.94, 18.93 to 81.74, and 10.11 to 68.16%, on average, for thymol, mycostatin, and oxalic acid, respectively. From the practical point of view, thymol could be recommended for controlling the CHB disease, as it is the cheapest material and proved to increase the brood nest as well. In addition, thymol has other uses in the field of apiculture.     

The distribution and density of ET-1 and its receptors are different in human right and left ventricular endocardial endothelial cells

Evidence suggests that endocardial endothelial cells (EECs) may play a role in the regulation of cardiac function by releasing ET-1. Furthermore, reports in the literature suggested that differences may exist in peptide receptor distribution between the left and right EECs. In this study, we verified if the distribution and density of ET-1 and its receptors could be different in right as compared to left ventricular EECs, and whether this difference may affect ET-1-induced increase of intracellular calcium. Using immunofluorescence and 3D confocal microscopy, our results showed that in both cell types, the ET(A) receptor is present and is homogeneously distributed throughout the two cell types. The relative density of the ET(A) receptor is similar in both right and left ventricular EECs. The ET(B) receptor is also present in right and left ventricular EECs, however, the relative density of the ET(B) receptor is higher in the nucleus as compared to the cytosol. In addition, the ET(B) receptor density was found to be higher in left EECs as compared to right EECs. In addition, our results showed that ET-1 is present in the cytosol and the nucleus of both types of cells and that the relative density of ET-1 is higher in right as compared to left ventricular EECs. Moreover, using the Fura-2 calcium measurement technique, our results showed that in left ventricular EECs, both ET(A) and ET(B) receptor activation mediated the effect of ET-1 on intracellular calcium, whereas in right ventricular EECs, this effect was solely mediated by the ET(A) receptor. In conclusion, our results showed that ET-1 and its receptors are present in both right and left ventricular EECs. However, the distribution and relative density of ET-1 and its receptors seem to be different in right EECs as compared to left EECs.     

Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif (249GTTRD253), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly249 and Thr251. On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.     

Insulin and angiotensin II induce the translocation of scavenger receptor class B, type I from intracellular sites to the plasma membrane of adipocytes

Scavenger receptor class B, type I (SR-BI) mediates the selective uptake of lipids from high density lipoproteins and is expressed in several types of tissues. However, to date little is known about its role in adipocytes. In this study, we investigated the cellular distribution of SR-BI in 3T3-L1 adipocytes and its regulation by hormones known to increase lipid storage such as angiotensin II (Ang II) and insulin. SR-BI was mainly distributed in the cytoplasm as determined by laser-scanning confocal analysis of the immunofluorescence labeling of SR-BI or the study of an enhanced green fluorescent protein-tagged SR-BI fusion protein. Exposure of cells to either insulin or Ang II (1-2 h) induced the mobilization of SR-BI from intracellular pools to the plasma membrane. This was further confirmed by Western blotting on purified plasma membrane and by fluorescence-activated cell sorter analysis of the SR-BI receptor. Similar results were also observed in primary adipocytes. We also demonstrated that, in the presence of either insulin or Ang II, SR-BI translocation to the cell membrane is functional, because insulin and Ang II induced a significant increase in the high density lipoprotein-delivered 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3-ol uptake and in total cholesterol content. These data demonstrate that SR-BI can be acutely mobilized from intracellular stores to the cell surface by insulin or Ang II, two hormones that exert lipogenic effects in adipocytes. This suggests that SR-BI might participate in the storage of lipids in the adipose tissue.     

Glycosynthase activity of Bacillus licheniformis 1,3-1,4-beta-glucanase mutants: specificity, kinetics, and mechanism

Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2. The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.     

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.