Biomedical subjects

Xin Li

Publications and source records attributed to Xin Li.

At least 199 records · Page 11Linked to original sources

PubMed2004

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed2004

Effect of erythromycin on metabolism of quetiapine in Chinese suffering from schizophrenia.

OBJECTIVE: To study the effect of erythromycin on metabolism of quetiapine in Chinese suffering from schizophrenia. METHODS: Nineteen patients received multiple doses of quetiapine (200 mg, twice daily) with or without co-administered erythromycin (500 mg, three times daily). Blood samples were collected at specified time intervals for determination of plasma concentrations of quetiapine and some of its metabolites. RESULTS: With erythromycin co-administration: for quetiapine, maximal plasma concentration (Cmax), area under concentration-time curve of 0-infinity h (AUC0-infinity) and terminal-phase elimination half-life time (t1/2) increased 68, 129 and 92%, respectively, and clearance (CL) and terminal elimination rate constant (Ke) decreased 52% and 55%, respectively; for quetiapine sulfoxide (QTP-SF), Cmax, AUC0-infinity and AUC ratio decreased 64, 23, and 70%, respectively, and t1/2 increased 211%; for 7-hydroxy-quetiapine (QTP-H), Ke and AUC ratio decreased 61% and 45%, respectively, and t1/2 increased 203%; for 7-hydroxy-N-desalkyl-quetiapine (QTP-ND), Cmax, AUC0-infinity and AUC ratio decreased 36, 40 and 71%, respectively. CONCLUSION: Erythromycin has a noticeable effect on the metabolism of quetiapine. When quetiapine is co-administered with CYP3A inhibitors such as erythromycin, the dosing regimen should be modified according to quetiapine TDM.

Adolescent↗

PubMed2004

Strategy for analysis and screening of bioactive compounds in traditional Chinese medicines.

Traditional Chinese medicines (TCMs), due to their long time clinic test and reliable therapeutic efficacy, are attracting increased global attention served as excellent pools of bioactive compounds for the discovery of new drugs. However, hundreds or even thousands of components are usually contained in traditional Chinese medicines and only a few compounds are responsible for the pharmaceutical and/or toxic effects. The large numbers of other components in traditional Chinese medicines make the screening and analysis of the bioactive components extremely difficult. By the way, the combination effect of bioactive components on the pharmacological activity makes it very difficult to clear the therapeutic mechanism of TCMs. Therefore, some strategies have to design for screening of bioactive compounds in traditional Chinese medicines, which further leads to disclose the therapeutic mechanism of TCMs in molecular level. The review will summarize the present state of the art of screening strategy for active compounds in traditional Chinese medicines, and the chromatography methods for screening and analysis of bioactive compounds in traditional Chinese medicines will be emphasized.

Animals↗

PubMed2004

Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract.

Limited studies have been carried out on the biotransformation of carboxyl nonsteroidal anti-inflammatory drugs (NSAIDs) in the liver. However, the role of the intestine in NSAID metabolism has not been investigated. In this report, the contribution of UDP-glucuronosyltransferases (UGTs) in the human gastrointestinal (GI) tract from five donors to the glucuronidation of the NSAIDs, RS-ketoprofen, S-naproxen, RS- and S-etodolac, was investigated. UGT activity and, for some donors, mRNA levels were evaluated. All NSAIDs were glucuronidated throughout the GI tract; however, glucuronidation was low in stomach and duodenum as compared to the remainder of the intestine. RT-PCR analysis demonstrated that the UGT1A isoforms, UGT1A3, 1A8, and 1A10, and UGT2B7 were expressed in the GI tract. Human recombinant UGT1A3, 1A9, 1A10 and 2B7 were actively involved in the glucuronidation of all NSAIDs while UGT1A7 and the intestine-specific UGT1A8 had no glucuronidating activity towards those compounds. Despite interindividual variations in both the levels of mRNA and the distribution of activity through the intestine, UGTs in the GI tract may contribute significantly to the first pass metabolism of orally administered NSAIDs.

Administration, Oral↗

PubMed2004

Preparation and characterization of a single-chain calcineurin-calmodulin complex.

Calcineurin (CN), a Ca(2+)/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca(2+)-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM-CNB-CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB-CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn2+, Ni2+ and Mg2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca2+.

Binding Sites↗

PubMed2004

p16INK4A hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma.

PURPOSE: The tumor suppressor gene p16INK4A is mainly inactivated by an epigenetic change involving promoter hypermethylation in hepatocarcinogenesis. The possible clinical impact of p16INK4A methylation and the potential risk factors for this epigenetic alteration have not been thoroughly investigated. EXPERIMENTAL DESIGN: We studied the methylation status and mRNA and protein expression of p16INK4A in 50 hepatocellular carcinomas and corresponding nonneoplastic liver lesions using methylation-specific PCR, reverse transcription-PCR, and immunohistochemical techniques. RESULTS: p16INK4A hypermethylation was observed in 58% (29 of 50) of the hepatocellular carcinomas and 16% (6 of 38) of the corresponding chronic hepatitis and cirrhosis tissue samples. p16INK4A methylation was significantly associated with mRNA and protein expression (P <0.001 and P=0.003, respectively). All of the p16INK4A-methylated tumors were positive for hepatitis B virus or hepatitis C virus markers, but none of the virus-negative tumors exhibited p16INK4A methylation (P=0.006). The frequency of p16INK4A hypermethylation tended to be higher in hepatitis C virus-related tumors (23 of 32, 72%) than in hepatitis B virus-related tumors (6 of 13, 46%; P=0.1). Aberrant methylation of p16INK4A was also related significantly to increasing age, female gender, and normal levels of serum PIVKA-II (P=0.02, 0.04, and 0.04, respectively). No statistically significant difference in survival was observed between patients with p16INK4A hypermethylation and those without. CONCLUSIONS: Our observations suggest that p16INK4A hypermethylation may contribute to hepatocarcinogenesis from an early stage and that multiple risk factors, such as viral infections, age, and gender, may be associated with p16INK4A hypermethylation in hepatocarcinogenesis.

Adult↗

PubMed2004

TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.

Amino Acid Sequence↗

PubMed2004

Parathyroid hormone uses multiple mechanisms to arrest the cell cycle progression of osteoblastic cells from G1 to S phase.

Parathyroid hormone (PTH) plays a major role in bone remodeling and has the ability to increase bone mass if administered daily. In vitro, PTH inhibits the growth of osteoblastic cell lines, arresting them in G(1) phase. Here, we demonstrate that PTH regulates the expression of at least three genes to achieve the following: inducing expression of MAPK phosphatase 1 (MKP-1) and p21(Cip1) and decreasing expression of cyclin D1 at both mRNA and protein levels. The induction of MKP-1 causes the dephosphorylation of extracellular signal-regulated kinase and therefore the decrease in cyclin D1. Overexpression of MKP-1 arrests UMR cells in G(1) phase. The mechanisms involved in PTH regulation of these genes were studied. Most importantly, PTH administration produces similar effects on expression of these genes in rat femoral metaphyseal primary spongiosa. Analyses of p21(Cip1) expression levels in bone indicate that repeated daily PTH injections make the osteoblast more sensitive to successive PTH treatments, and this might be an important feature for the anabolic functions of PTH. In summary, our data suggest that one mechanism for PTH to exert its anabolic effect is to arrest the cell cycle progression of the osteoblast and hence increase its differentiation.

Animals↗

PubMed2004

Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation.

Parathyroid hormone (PTH) is the major mediator of calcium homeostasis and bone remodeling and is now known to be an effective drug for osteoporosis treatment. Yet the mechanisms responsible for its functions in bone are largely unknown. Here we report that the expression of amphiregulin (AR), a member of the epidermal growth factor (EGF) family, is rapidly and highly up-regulated by PTH in several osteoblastic cell lines and bone tissues. Other osteotropic hormones (1alpha,25-dihydroxyvitamin D3 and prostaglandin E2) also strongly stimulate AR expression. We found all EGF-like ligands and their receptors are expressed in osteoblasts, but AR is the only member that is highly regulated by PTH. Functional studies demonstrated that although AR is a potent growth factor for preosteoblasts, it completely inhibits further differentiation. AR also strongly and quickly stimulated Akt and ERK phosphorylation and c-fos and c-jun expression in an EGF receptor-dependent manner. Moreover, AR null mice displayed significantly less tibial trabecular bone than wild-type mice. Taken together, we have identified a novel growth factor that is PTH-regulated and appears to have an important role in bone metabolism.

Amphiregulin↗

PubMed2004

The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development.

The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.

Arabidopsis↗

PubMed2004

Expression of plasma vascular endothelial growth factor in patients with hepatocellular carcinoma and effect of transcatheter arterial chemoembolization therapy on plasma vascular endothelial growth factor level.

AIM: To investigate the expression level of plasma vascular endothelial growth factor (P-VEGF) in patients with hepatocellular carcinoma (HCC) and its relationship with the clinicopathologic characteristics, and to examine the changes of P-VEGF in the course of transcatheter arterial chemoembolization (TACE). METHODS: Peripheral blood samples were taken from 45 HCC patients before and 1, 3, 7 d, and 1 mo after TACE. Plasma VEGF level was measured with the quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Twenty patients with benign liver lesions and 17 healthy control subjects were also included in this study. RESULTS: Plasma VEGF levels in HCC patients were significantly elevated as compared to those in patients with benign liver lesions (P = 0.006) and in the normal controls (P = 0.003). Significant differences were observed when P-VEGF was categorized by tumor size (P = 0.006), portal vein thrombosis (P = 0.011), distant metastasis (P = 0.017), arterial-portal vein shunting (P = 0.026), and International Union Against Cancer (UICC) TNM stage (P = 0.044). There was no correlation between plasma level of VEGF and the level of alpha fetoprotein (alpha-FP) (r = 0.068, P = 0.658) and weakly correlated with the number of platelets (r = 0.312, P = 0.038). P-VEGF levels increased significantly and reached the peak value on the first day after TACE, and then decreased gradually. The change rate of P-VEGF concentration (one month post-TACE/pre-TACEX100%) was correlated with the retention rate of lipiodol oil (r s = 0.494, P = 0.001) and the tumor volume change (r s = 0.340, P = 0.034). The patients who achieved a partial or complete response to TACE therapy showed significantly less pre-treatment P-VEGF than those nonresponders (P = 0.025). A high pre-therapeutic P-VEGF level was associated with poor response to treatment (P = 0.018). CONCLUSION: A high pre-treatment P-VEGF level is a useful marker for tumor progression, especially for vascular invasion. TACE increases the level of P-VEGF only temporarily which may be associated with tumor ischemia. P-VEGF may be useful in predicting treatment response, monitoring disease course after TACE and judging the effect of different TACE regimens.

Adult↗

PubMed2004

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: impact on GH signaling and GHR proteolysis.

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

Animals↗

PubMed2004

Antiapoptotic effects of estrogen in normal and cancer human cervical epithelial cells.

The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca(2+)-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nM 17beta-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2',3'-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16-1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17beta-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca(2+)-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

Adenosine Triphosphate↗

PubMed2004

cDNA cloning and mRNA expression of a FTZ-F1 homologue from the pituitary of the orange-spotted grouper, epinephelus coioides.

A FTZ-F1 homologue was cloned from the pituitary cDNA library of the orange-spotted grouper. The full-length cDNA of the orange-spotted grouper FTZ-F1 spanned 1735 bp including a poly (A) tail. The open reading frame encodes a protein of 468 amino acids. Sequence analysis indicated that it had a structure typical of the orphan nuclear receptor superfamily, and the FTZ-F1 box, a characteristic of the FTZ-F1 family. Phylogenetic analysis indicated that the orange-spotted grouper FTZ-F1 was closely related to medaka FTZ-F1 and did not belong to either the SF-1/Ad4BP group or the LRH-1/FTF group. Virtual Northern Blot detected a major transcript of about 1.7 kb and a minor transcript of 2.2 kb of FTZ-F1 in the orange-spotted grouper pituitary gland. The expression of FTZ-F1 homologue gene in different tissues and during embryonic development of the orange-spotted grouper was determined using one-step RT-PCR coupled with Southern blot analysis. In addition to the pituitary gland, the orange-spotted grouper FTZ-F1 was also expressed in the hypothalamus, forebrain, heart, liver, kidney, and ovary. The stronger signal from the gel image indicated that the expression level of FTZ-F1 homologue gene was higher in the ovary of stage 3 than stage 2. During embryonic development, mRNA for the orange-spotted grouper FTZ-F1 homologue was present in newly fertilized eggs, but disappeared in embryos at 50 min post fertilization. The orange-spotted grouper FTZ-F1 homologue mRNA reappeared in embryos at 1.5 hr post fertilization. Its expression level was increased from late blastula to neurula stages. Taken together, results of the current study suggest that the orange-spotted grouper FTZ-F1 homologue exhibits characteristics indicative of both the LRH-1/FTF- and the SF-1/Ad4BP-like genes, and may also play important roles in the hypothalamus-pituitary-gonadal axis, cholesterol metabolism, and embryogenesis.

Amino Acid Sequence↗

PubMed2004

P2X7 receptor-mediated apoptosis of human cervical epithelial cells.

Normal human ectocervical epithelial (hECE) cells undergo apoptosis in culture. Baseline apoptosis could be increased by shifting cells to serum-free medium and blocked by lowering extracellular calcium. Treatment with the ATPase apyrase attenuated baseline apoptosis, suggesting that extracellular ATP and purinergic mechanisms control the apoptosis. Treatment with ATP and the P2X7 receptor analog 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased apoptosis significantly, in a time- and dose-related manner. The threshold of ATP effect was 0.5 microM in hECE cells and approximately 1 microM in CaSki cancer cells. The apoptotic effect of BzATP was additive in part to that of tumor necrosis factor (TNF)-alpha, and it could be attenuated by lowering extracellular calcium and by treatment with the caspase-9 inhibitor Leu-Glu-His-Asp-O-methyl-fluoromethylketone (LEHD-FMK). Treatment with BzATP activated caspase-9, and, in contrast to TNF-alpha, it had only a mild effect on caspase-8. Both BzATP and TNF-alpha activated caspase-3, suggesting that BzATP activates predominantly the mitochondrial apoptotic pathway. Both hECE and CaSki cells secrete ATP into the extracellular fluid, and mean ATP activity in conditioned medium was approximately 0.5 microM, which is in the range of values that suffice to activate the P2X7 receptor. On the basis of these findings we propose a novel autocrine-paracrine mechanism of cervical cell apoptosis that operates by P2X7 receptor control of cytosolic calcium and utilizes the mitochondrial apoptotic pathway.

Adenosine Triphosphate↗

PubMed2004

Combinatorial selection and edited combinatorial selection of phosphorothioate aptamers targeting human nuclear factor-kappaB RelA/p50 and RelA/RelA.

Nuclear factor-kappaB (NF-kappaB) transcription factors are important in regulating the immune response and play critical roles in the pathogenesis of chronic inflammatory diseases and a variety of human cancers. Agents that target specific NF-kappaB dimers may serve as therapeutic agents for the prevention of pathogenic immune responses. We have selected monothiophosphate-modified aptamers, or "thioaptamers", to the NF-kappaB p50/RelA heterodimer using combinatorial selection techniques. We also utilized a "double sieve" or editing approach for the generation of thioaptamers with enhanced selectivity to the RelA/RelA homodimer. The thioaptamers from these selections and our previous selections on the p50/p50 and RelA/RelA homodimers all had unique sequences and bound tightly to the recombinant NF-kappaB dimers against which they were selected. The selected thioaptamers also appear to maintain their selectivity and specificity among other cellular proteins, because they have the ability to bind NF-kappaB proteins within nuclear extracts from lipopolysaccharide (LPS)-induced macrophages and B cells.

B-Lymphocytes↗

PubMed2004

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system.

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.

Animals↗

PubMed2004

[Treatment techniques of harvesting injury of donor renal blood vessels].

OBJECTIVE: To study the treatment technique for harvesting injury of donor blood vessels for the clinic application. METHODS: The data of 32 renal transplantation patients with injury of graft blood vessels were retrospectively reviewed. 60 renal transplantation patients with non-injury during the same term were selected as the control group. The treatment techniques for harvesting injury of graft blood vessels mainly includes end-to-end anastomosis of graft artery, side-to-side anastomosis of branch artery, end-to-side anastomosis of branch artery to the main renal artery, reconstruction of multiple segmental arteries by using iliac arterial grafts from cadaveric donors or recipients on the workbench, repairs of injuries for the smaller segmental/polar arteries by using inferior epigastric artery, end-to-end anastomosis of the lower thick segmental/polar arteries with the iliac internal arterial by placing kidney upside down. RESULTS: Those injured included 28 arterial and 4 venous. Average bench surgery time was 42 minutes. Mean warm ischemic time was 31 minutes. No death occurred at an average follow-up of 3.5 years (1 - 5 years). There was no statistical difference in the 1-year graft survival, postoperative 1-year acute rejection, delayed graft function (DGF) and the incidence of constriction of vascular anastomosis rate (96.9%, 12.5%, 21.9%, 3.1%, respectively) compared with non-reconstructed kidneys during the same term (98.3%, 11.7%, 18.3%, 1.7%, P > 0.05, respectively). CONCLUSION: The flexible and appropriate application of different vascular reconstruction means and satisfactory surgery techniques play an important role in assuring quality of kidney with harvesting blood vessels injury and donor kidney availability.

Adolescent↗