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Objective To explore the role of HCN channels in ureteral peristaltic dysfunction by comparing the changes in HCN channel levels between normal and tuberculous ureters. Methods A total of 32 specimens of human upper ureters were collected by nephrectomy from patients with renal tumor (control group, n = 16) or from patients with renal tuberculosis (experimental group, n = 16); the two groups did not receive radiotherapy, chemotherapy, immunotherapy, or any other special treatment before the surgical procedure. An experimental study on smooth muscle strips of human upper ureters showed variation in contraction amplitude and frequency after adding ZD7288, a specific blocker of HCN channels. The expression of HCN channels in the ureter was confirmed by Western blot (WB) and by confocal analysis of double immunostaining for c-kit and HCN channel proteins. Results Before the addition of ZD7288, the experimental and control groups showed significant differences in the frequency and amplitude of the spontaneous contraction of isolated ureteral smooth muscle strips. After ZD7288 was added, the frequency and amplitude of the contractions of the ureteral smooth muscle strips were significantly lower in both groups. The differences observed before and after ZD7288 treatment in each group were significant (P < 0.001), and the difference in contraction amplitude observed between the two groups before ZD7288 was also significantly different (P < 0.001). By using WB technology, we showed that the expression of HCN channels was present in normal human ureters, with the expression of HCN4 and HCN1 being the highest; the expression of HCN4 and HCN1 in the control and experimental groups were both statistically significant (P < 0.001). HCN4 and HCN1 were expressed in the mucosal and smooth muscle layers of human control ureters and tuberculous ureters, as revealed by a confocal analysis of double immunostaining for c-kit and HCNs proteins; there were significant differences between the two groups (P < 0.001). Conclusion Four HCN channels are expressed in the ureter, mainly HCN4 and HCN1, suggesting that HCN channels are involved in the peristaltic contraction of ureteral ICCs, which may be an important reason for peristaltic dysfunction in ureteric tuberculosis. [ABSTRACT FROM AUTHOR]

Abstract  UBE1 plays an important role in the first step of ubiquitin–proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB–UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies. [ABSTRACT FROM AUTHOR]

Abstract  Tungstophosphoric acid (H3PW12O40) (PW) catalysts supported on mesoporous Mn-MCM-41(n Si/n Mn = 25) with various (10, 20 and 30 wt%) acid loadings were prepared by impregnation, and their physical chemical properties were characterized by powder X-ray diffraction (XRD), N2 adsorption–desorption, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), high resolution transmission electron microscopy (HRTEM), and X-Ray Photoelectron Spectroscopy (XPS). The host material suffers severe structural distortions at higher loading, which lead to a considerable loss of long-range orders. Their catalytic performances were evaluated in the epoxidation of styrene with dilute H2O2 (30%) as an oxidizing agent under liquid phase reaction conditions over PW/Mn-MCM-41 catalysts for selective synthesis of styrene oxide. The influences of various reaction parameters such as temperature, time, solvents, PW loading and catalyst amount, styrene to H2O2 mmol ratios and acetonitrile (MeCN) to N,N-dimethylformamide (DMF) volume ratios on the conversion of styrene and yield as well as selectivity of styrene oxide have also been studied in details. The results revealed that 20% PW/Mn-MCM-41 (n Si/n Mn = 25) was more active than other catalysts. The PW/Mn-MCM-41, moreover, was found to be reusable and environmentally benign for the epoxidation of styrene. [ABSTRACT FROM AUTHOR]

Abstract  A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The K m and V max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination of Mg2+ and Co2+ or Zn2+ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification. [ABSTRACT FROM AUTHOR]

Uracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1- R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase–EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism. [ABSTRACT FROM AUTHOR]