Applied Research on Laparoscopic Simulator in the Resident Surgical Laparoscopic Operation Technical Training.

The purpose of this study was to estimate the effects of surgical laparoscopic operation course on laparoscopic operation skills after the simulated training for medical students with relatively objective results via data gained before and after the practice course of laparoscopic simulator of the resident standardized trainees. Experiment 1: 20 resident standardized trainees with no experience in laparoscopic surgery were included in the inexperienced group and finished simulated cholecystectomy according to simulator videos. Simulator data was collected (total operation time, path length, average speed of instrument movement, movement efficiency, number of perforations, the time cautery is applied without appropriate contact with adhesions, number of serious complications). Ten attending doctors were included in the experienced group and conducted the operation of simulated cholecystectomy directly. Data was collected with simulator. Data of two groups was compared. Experiment 2: Participants in inexperienced group were assigned to basic group (receiving 8 items of basic operation training) and special group (receiving 8 items of basic operation training and 4 items of specialized training), and 10 persons for each group. They received training course designed by us respectively. After training level had reached the expected target, simulated cholecystectomy was performed, and data was collected. Experimental data between basic group and special group was compared and then data between special group and experienced group was compared. Results of experiment 1 showed that there is significant difference between data in inexperienced group in which participants operated simulated cholecystectomy only according to instructors' teaching and operation video and data in experienced group. Result of experiment 2 suggested that, total operation time, number of perforations, number of serious complications, number of non-cauterized bleeding and the time cautery is applied without appropriate contact with adhesions in special group were all superior to those in basic group. There was no statistical difference on other data between special group and basic group. Comparing special group with experienced group, data of total operation time and the time cautery is applied without appropriate contact with adhesions in experienced group was superior to that in special group. There was no statistical difference on other data between special group and experienced group. Laparoscopic simulators are effective for surgical skills training. Basic courses could mainly improve operator's hand-eye coordination and perception of sense of the insertion depth for instruments. Specialized training courses could not only improve operator's familiarity with surgeries, but also reduce operation time and risk, and improve safety.

Cost analysis of living donor kidney transplantation in China: a single-center experience.

BACKGROUND: Kidney transplantation is the most cost-effective option for the treatment of end-stage renal disease, but the financial aspects of kidney transplantation have not yet been fully investigated. The purpose of this study was to determine the hospital cost of living donor kidney transplantation in China and to identify factors associated with the high cost. MATERIAL/METHODS: Demographic and clinical data of 103 consecutive patients who underwent living donor kidney transplantation from January 2007 to January 2011 at our center were reviewed, and detailed hospital cost of initial admission for kidney transplantation was analyzed. A stepwise multiple regression analysis was computed to determine predictors affecting the total hospital cost. RESULTS: The median total hospital cost was US $10,531, of which 69.2% was for medications, 13.2% for surgical procedures, 11.4% for para clinics, 3.7% for accommodations, 0.5% for nursing care, and 2.0% for other miscellaneous medical services. A multivariate stepwise logistic regression model for overall cost of transplantation revealed that the length of hospital stay, induction therapy, steroid-resistant rejection, maintenance therapy, infection status and body weight were independent predictors affecting the total hospitalization cost. CONCLUSIONS: Although the cost of living donor kidney transplantation in China is much lower than that in developed countries, it is a heavy burden for both the government and the patients. As medications formed the greater proportion of the total hospitalization cost, efforts to reduce the cost of drugs should be addressed.

Outcome of a solitary kidney transplant into adult recipients from pediatric donors after controlled circulatory death.

OBJECTIVES: We sought to evaluate the outcomes of a single kidney transplant from pediatric donors into adult recipients after controlled circulatory death. MATERIALS AND METHODS: A retrospective, single-center review of all adult recipients who received a single pediatric kidney from controlled-cardiac deceased-donors (aged, < 9 years old) between January 2006 and March 2008 was performed. RESULTS: Eleven adult recipients (aged, 16-41 years) used single renal grafts from the controlled-cardiac deceased-donors (median donor age, 74 months; range, 49-106 months; median donor weight, 20.95 kg; range, 16.6-37.8 kg). The median recipient age was 27 years (range, 16-41 years; median recipient weight, 47 kg; range 39.5-53.6 kg). The patient's serum creatinine level gradually decreased, and the estimated glomerular filtration rate increased stably more than 2 times during follow-up. The graft length increased significantly the first week after transplant compared with that recorded immediately after reperfusion (P < .001) and grew slightly thereafter. Acute rejection occurred in 1 patient. Nine patients had high renal artery blood flow velocity index at 1 to 2 cm. Beside the anastomosis (167-321 cm/s), only 1 patient developed hypertension and slightly increased serum creatinine at 14 weeks after transplant. The 1-year patient/graft survival was 100%. CONCLUSIONS: Use of single kidneys from pediatric donors after controlled cardiac death could expand the donor pool without compromising recipient outcomes.

Evaluation of living related kidney donors in China: policies and practices in a transplant center.

BACKGROUND: Rigorous donor evaluation is essential for living related donor kidney transplantation (LRDKT). However, guidelines for living kidney donor evaluation are absent in China. The aim of this study is to describe the initial experience in the living kidney donor evaluation process in a single transplant center in China. METHODS: The evaluation process of our center is sequentially divided into five steps: outpatient consultation and information, preliminary evaluation, comprehensive evaluation, final informed consent, and ethics committee oversight. RESULTS: Between June 2007 and March 2009, 124 potential living donors were evaluated in our center, of whom 82 (66.1%) became effective donors and the remaining 42 (33.9%) were excluded. The exclusion reasons were related to clinical problems in 27 cases, psychosocial problems in seven cases, and suspected organ trading in eight cases. CONCLUSION: Although strongly forbidden by Chinese laws, organ trading remains a threat to the healthy development of LRDKT in China. To prohibit organ trading, the kinship between the donor and recipient should be carefully identified. Guidelines for living donor evaluation appropriate to the actual situation in China should be set up for the sake of safety and to protect the rights and interests of both donors and recipients.

Psychosocial evaluation of Chinese living related kidney donors.

BACKGROUND: Although living kidney transplantation has numerous advantages over cadaveric transplantation, donor apprehension remains a problem. This study investigated psychosocial features and quality of life in Chinese living kidney donors after transplantation procedures. METHODS: Participants were 84 donors interviewed during follow-up after donation. Sociodemographic characteristics were evaluated by a 22-item questionnaire. Social support, quality of life, depression, and anxiety were objectively measured with the Social Support Rating Scale for Chinese, the Short-Form 36 Health Survey, the modified Beck Depression Inventory, and the Self-Rating Anxiety Scale questionnaire, respectively. RESULTS: All donors volunteered without pressure from families or recipients. Altruistic motives were the main impetus for donation, and donors had good social support. Living transplantation affected donors' quality of life, with a slight negative effect on some physical aspects. There were no major depressive or anxiety disorders following donation. CONCLUSIONS: Living kidney donation was a generally positive experience without evidence of major psychological disturbance. However, considering the limited size and duration of our study, we recommend careful follow-up of all donors. We also recommend provision of social support services and removal of financial disincentives for donors.

Experimental study on inhibitory effects of histone deacetylase inhibitor MS-275 and TSA on bladder cancer cells.

OBJECTIVE: To investigate the inhibitory effect of histone deacetylase (HDAC) inhibitors (MS-275 and TSA) on T24 human bladder cancer cells in vitro, and explore the possible mechanism. METHODS: The MTT assay was employed to evaluate the inhibitory effect of MS-275 and TSA on T24 cell growth. FCM was used to analyze the variation of T24 cell cycle distribution and the apoptotic ratio after T24 cells were treated with MS-275 and TSA. Histone acetylation level was detected by Western blot. mRNA expression of p21 WAF1/CIP1, cyclin A, and cyclin E was measured by FQ-PCR. Dynamic changes of Bcl-2 and bax expression were detected by FCM. RESULTS: MS-275 and TSA inhibited T24 cell growth in a concentration and time-dependent manner. Treatment with 4 µmol/l MS-275 or 0.4 µmol/l TSA blocked cell cycling in the G0/G1 phase and induced a significant increase in cell apoptosis. MS-275 and TSA significantly increased the level of histone acetylation, induced p21CIP1WAF1 mRNA expression, and inhibited cyclin A mRNA expression, though no significant effect was observed on cyclin E. Bcl-2 expression was down-regulated, while bax expression was up-regulated. CONCLUSION: HDAC inhibitors can block bladder cancer cell cycle in vitro and induce apoptosis. The molecular mechanism may be associated with increased level of histone acetylation, down-regulation of p21WAF1/CIP1 expression, up-regulation of cyclin A expression, and dynamic change of bcl-2 and bax expression.

A comparison of prediction equations for estimating glomerular filtration rate in Chinese potential living kidney donors.

BACKGROUND: Accurate measurement of donor renal function has important long-term implications for both donors and recipients. In clinical practice, renal function may be estimated by using 24-h urinary creatinine clearance (urine-CrCl) and various specifically derived prediction equations. We assessed the suitability of urine-CrCl and prediction equations for evaluating Chinese kidney donors. METHODS: A total of 224 donors who had undergone (99m)Tc-diethylenetriaminepentaacetic acid glomerular filtration rate (GFR) estimation were enrolled. We evaluated the performance of the Cockcroft-Gault equation (CG-CrCl), the CG-CrCl equation corrected for GFR (CG-GFR), the isotope dilution mass spectrometry traceable modification of diet in renal disease (IDMS-MDRD) study equation, the new MDRD study equation for Chinese (C-MDRD 1), the modified MDRD study equation with a Chinese coefficient (C-MDRD 2), the modified IDMS-MDRD study equation with a Japanese coefficient (J-MDRD), and urine-CrCl in predicting GFR before donation. RESULTS: Of 224 donors selected, 68 were male and 156 were female. Of all the prediction equations, C-MDRD 1 was the least scattered and most precise. However, predictive performance was poor for all the equations. CONCLUSION: The performance of urine-CrCl and all the equations was disappointing, and even the best performing equation C-MDRD 1 was unacceptable. Considering the potential risk of living kidney donation, other more accurate methods of GFR measurement should be used in clinical practice.

Bromodomain protein Brd4 regulates human immunodeficiency virus transcription through phosphorylation of CDK9 at threonine 29.

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb.

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.

Modulation of the Brd4/P-TEFb interaction by the human T-lymphotropic virus type 1 tax protein.

Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.

Radiographic determination of tissue thickness in paraffin blocks: application to the construction of tissue microarrays.

The determination of tissue thickness in paraffin blocks in the histology laboratory has been largely based on visual estimates. More accurate methods are required for the construction of tissue microarrays (TMAs) to assure a greater yield of cores in sections through the TMA block. We describe an accurate radiographic method to determine tissue thickness in donor paraffin blocks and have validated its application to TMA construction. Individual radiographic analysis was performed on paraffin donor blocks used for the construction of TMAs for determination of donor block tissue thickness. Consecutive numbered slide sections through the TMA block were then examined for the presence or loss of cores in the 150th TMA slide (from the final third of the TMA block) and correlated with the thickness of the individual donor blocks determined radiographically. At the 150th TMA slide, 202 of 1340 cores (15.1%) were depleted. Radiographic measurement showed a greater thickness of the donor paraffin block tissue (2.02 mm) corresponding to the retained cores as compared with the donor tissue (1.54 mm) of the depleted cores (P < 0.001). With progressive slide sections through a TMA block, the retention of tissue cores shows a significant correlation with donor block tissue thickness. Radiographic determination of tissue thickness in donor paraffin blocks can be used in TMA construction. Prior knowledge of tissue thickness in TMA construction can prompt compensatory steps that can enhance the yield of valuable samples and assure sufficient numbers of adequate cores for statistical analysis in biomarker evaluations.

Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription.

Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by co-immunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.

TAF7: a possible transcription initiation check-point regulator.

Transcription consists of a series of highly regulated steps: assembly of a preinitiation complex (PIC) at the promoter nucleated by TFIID, followed by initiation, elongation, and termination. The present study has focused on the role of the TFIID component, TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to TAF1 and inhibits its intrinsic acetyl transferase activity. We now report that although TAF7 remains bound to TAF1 and associated with TFIID during the formation of the PIC, TAF7 dissociates from the PIC upon transcription initiation. Entry of polymerase II into the assembling PIC is associated with TAF1 and TAF7 phosphorylation, coincident with TAF7 release. We propose that the TFIID composition is dynamic and that TAF7 functions as a check-point regulator suppressing premature transcription initiation until PIC assembly is complete.

Urological malignancy as a complication of renal transplantation: a report of twelve clinical cases.

OBJECTIVE: To analyze the epidemiographic features of urological malignancy in renal allograft recipients (RAR) in a single center METHODS: A retrospective analysis was made on 3150 patients who received renal allografts from June 1978 until the autumn of 2006 and anti-rejection treatment for at least 3 months. RESULTS: Of the 3150 recipients, 33 (1.05%) developed malignancies, including 12 patients (0.38%; eight males and four females) with urological tumors and 21 patients with skin carcinoma, right liver lobular cystic adenocarcinoma, hepatocellular carcinoma, gastric cancer, colorectal carcinoma, ileocecal adenoma, lip cancer, nasopharyngeal carcinoma, Kaposi's Sarcoma, pulmonary lymphoma, and breast cancer. The 12 cases of urological malignancies included one case of renal cell carcinoma, one case of transplanted kidney carcinoma, two cases of bilateral pelvic transitional cell carcinoma (TCC), three cases of unilateral pelvic TCC, one case of bilateral ureter TCC, one case of unilateral ureter TCC, and three cases of bladder TCC. The age at which the diagnosis was made ranged from 48 to 66 years with a mean of 58.3 +/- 4.6 years, and the mean course of immunosuppressive therapy ranged from 26 to 120 months with a mean of 62 +/- 18 months. Of the 12 patients who developed urological malignancies, six had been on a Oyclosporine A + Azaithioprine + Prednisone (OsA + Aza + Pred) protocol; five on a Oyclosporine A + Mycophenolate Mofetil + Prednisone (OsA + MMF + Pred) protocol; and one on a Tacrolimus + Mycophenolate Mofetil + Prednisone (FK506 + MMF + Pred) protocol. One of the 12 patients died soon after the diagnosis was made, and the remaining 11 patients received surgical resection. Of them, 10 patients survived well, and the other one died from cerebral hemorrhage soon after operation. CONCLUSION: Urological malignancies, especially TCC, are an important complication in renal transplanatation found in this center. The incidence of urological malignancy in renal allograft recipients (RAR) is about 10 times that found in the general population of Shanghai versus two times for other malignancies. The occurrence of the malignancies in PAR seems to be closely related to the use of immunosuppressive agents. lmmunosuppression results in the weakening of immnuologic surveillance function, leading to mutation, aberration, and carcinogenesis. The immunological status of patients after renal transplantation should be assessed regularly. Painless macroscopic hematuria should be considered a significant sign in assessing the potential occurrence of urological malignancy in RAR. Treatment includes early diagnosis, timely surgical resection, and reduction of immunosuppressive agents.

The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription.

Brd4 is a mammalian bromodomain protein that binds to acetylated chromatin. Proteomic analysis revealed that Brd4 interacts with cyclinT1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). Brd4 interacted with P-TEFb in the living nucleus through its bromodomain. About half of P-TEFb was bound to the inhibitory subunit and functionally inactive. Brd4 interacted with P-TEFb that was free of the inhibitory subunit. An increase in Brd4 expression led to increased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII) CTD and stimulation of transcription from promoters in vivo. Conversely, a reduction in Brd4 expression by siRNA reduced CTD phosphorylation and transcription, revealing that Brd4 is a positive regulatory component of P-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitment of P-TEFb to a promoter was dependent on Brd4 and was enhanced by an increase in chromatin acetylation. Together, P-TEFb alternately interacts with Brd4 and the inhibitory subunit to maintain functional equilibrium in the cell.

Coordination of transcription factor phosphorylation and histone methylation by the P-TEFb kinase during human immunodeficiency virus type 1 transcription.

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.

Bacterial biota in the human distal esophagus.

The esophagus, like other luminal organs of the digestive system, provides a potential environment for bacterial colonization, but little is known about the presence of a bacterial biota or its nature. By using broad-range 16S rDNA PCR, biopsies were examined from the normal esophagus of four human adults. The 900 PCR products cloned represented 833 unique sequences belonging to 41 genera, or 95 species-level operational taxonomic units (SLOTU); 59 SLOTU were homologous with culture-defined bacterial species, 34 with 16S rDNA clones, and two were not homologous with any known bacterial 16S rDNA. Members of six phyla, Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, Fusobacteria, and TM7, were represented. A large majority of clones belong to 13 of the 41 genera (783/900, 87%), or 14 SLOTU (574/900, 64%) that were shared by all four persons. Streptococcus (39%), Prevotella (17%), and Veilonella (14%) were most prevalent. The present study identified approximately 56-79% of SLOTU in this bacterial ecosystem. Most SLOTU of esophageal biota are similar or identical to residents of the upstream oral biota, but the major distinction is that a large majority (82%) of the esophageal bacteria are known and cultivable. These findings provide evidence for a complex but conserved bacterial population in the normal distal esophagus.

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA.

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.

[Cadaver renal transplantation and multivariate analysis for graft survival: a clinical review of 2 016 cases].

OBJECTIVE: To review kidney transplantation in the center and analyze the risk factors affecting long-term allograft survival. METHODS: Thirty-two relative variables were analyzed with SAS statistical software. Using Log-rank method, we investigated influence of these variables on short-and long-term survival of grafts. Kaplan-Meier analysis was used to estimate the 1-, 3-, 5-, 10-years graft survival rates and half-life. Proportional hazards regression analysis (Cox model) was used to assess and rank the relative risk of potential variables. RESULTS: The 1-, 3-, 5-, 10-years graft survival rates were 83%, 75%, 66% and 48%. After excluding the patients died with functioning grafts, the 1-, 3-, 5-, 10 years grafts survival rate increased to 89%, 82%, 75% and 69%, respectively. The mean half-life was 8.78 +/- 0.14 and 14.09 +/- 0.20 years, respectively. By Log-rank analysis, factors affecting short- and long-term graft survival were identified as: renal function, duration of graft function became normal, cold-ischemia time, presence of acute rejection, delayed graft function, immunosuppressive regimen, complication, infection, anti-rejection therapy. Cox model multivariate analysis showed that there were 18 factors affecting graft survival. CONCLUSIONS: New immunosuppressive agents not only significantly increase short-term graft survival, but also have the better long-term outcome tendency. Making assurance to get high quality donor organ and minimizing the death with graft function may be the most feasible way to prolong graft survival at present.

HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.