Biomedical subjects

P Fu

Publications and source records attributed to P Fu.

At least 19 recordsLinked to original sources

PubMed2004

Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3.

Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.

Amino Acid Sequence↗

PubMed2003

Quantitative analysis of Escherichia coli metabolic phenotypes within the context of phenotypic phase planes.

In silico models of Escherichia coli metabolism have been developed to predict metabolic behavior and propose experimentally testable hypotheses. However, a thorough assessment of the metabolic phenotype requires well-designed experimentation and reproducible experimental techniques. A method for the quantitative analysis of E. coli metabolism in vivo within the framework of in silico phenotypic phase plane analysis is presented. Using this approach, we have quantitatively studied E. coli metabolism in various environmental conditions and nutritional media. Our experimental methodology, in combination with steady-state metabolic models, can be used to study biological properties and evaluate the metabolic capabilities of microbes.

Aerobiosis↗

PubMed2001

Tactic-specific success in sperm competition.

Sperm competition is a major force in sexual selection, but its implications for mating-system and life-history evolution are only beginning to be understood. The well-known sneak-guard model predicts that sneaks will win in sperm competition. We now provide empirical confirmation of this prediction. Bluegill sunfish (Lepomis macrochirus) have both sneak (cuckolder) and guard (parental) males. Guards make nests, court females and provide solitary parental care for the embryos. Sneaks include small cuckolders, which are termed 'sneakers', that dart in and out of nests in order to ejaculate between the spawning pair and larger cuckolders, which are termed 'satellites', that mimic females in order to ejaculate between the spawning pair. Using field behavioural data, genetic data and new mathematical models for paternity analyses, we show, for the first time to the authors' knowledge, that sneaks fertilize more eggs than guards during sperm competition. In addition, we show that sneakers are superior to satellites in sperm competition and, thus, that even among sneaks there are tactic-specific differences in competitive success.

Animals↗

PubMed2001

Liver necrosis induced by thyroid hormone administration in rats fed ethanol.

Recently it was observed that the hypothyroid state induced by propylthiouracil treatment reduced the elimination rate of ethanol. This was based on the fact that hypothyroid rats could only tolerate 10 g/kg/day of ethanol infusion over 24 h. Euthyroid rats tolerate 13 to 14 g/kg/day of ethanol infusion. In the present report it was postulated that thyroid supplements would increase the daily dose of ethanol that the treated rats could tolerate. Daily thyroid supplements, given intraperitoneally or orally, did, in fact, result in an increase in the elimination rate of ethanol. Death from ethanol overdose did not occur until the amount of ethanol given reached 16 to 19 g/kg/day. In addition, thyroxine supplements blunted or completely inhibited the urinary ethanol cycle. Submassive centrilobular liver necrosis was present in 9 of 10 rats given thyroid hormone and fed ethanol. Periportal duct metaplasia and fibrosis were also observed. Thus, thyroid supplements probably enhanced central hypoxia caused by ethanol to the point where ischemic necrosis developed. The latter healed by scar formation. Thyroid treatment completely prevented ethanol-induced steatohepatitis, indicating that the fatty change is responsive to the metabolic rate.

Administration, Oral↗

PubMed2001

Oral low-carbohydrate alcohol liquid diet induces experimental steatohepatitis in the rat.

The intragastric tube feeding model of alcoholic liver disease in the rat induces significant liver histopathology, including steatohepatitis and fibrosis. The question is, if the same low-carbohydrate diet is fed ad lib, will the same pathology develop? Rats were fed a liquid diet with ethanol ad lib that was low in calories derived from carbohydrates for 2 months. The urinary ethanol levels (UALs) were monitored at hourly, daily, and weekly intervals, and the growth of the rats was charted. The liver histopathology and blood transaminase levels were determined. Rats fed ethanol grew 1 g/day, which was 2 g/day less than when they were fed the same diet intragastrically. UALs varied hourly between 150 and 500 mg%, daily between 120 and 360 mg%, and weekly between 0 and 500 mg%. Individual rat UALs showed no predictable pattern. The pair-fed controls ate all of their daily ration within 12 h, then fasted until the next day. The histopathology and blood alanine aminotransferase were similar to those seen with the intragastric tube feeding of the same diet, except that necrosis, inflammation, and fibrosis did not develop. The conclusion was that the oral feeding of a low-carbohydrate diet produces less liver injury than that produced by the same diet fed intragastrically. The UALs varied hourly, daily, and weekly in individual rats, making it difficult to synchronize UALs at the time of sacrifice.

Administration, Oral↗

PubMed2001

Total cavopulmonary connection for complex cardiac anomalies with the functional single ventricle.

From Aug. 1999 to Feb. 2001, 8 patients with complex congenital heart diseases, including 5 cases accompanied by hypoplastic left ventricle and 3 by hypoplastic right ventricle, were subjected to total cavopulmonary connection (TCPC). Eight cases underwent the operation under cardiopulmonary bypass and 7 of whom under no cardiac clamp. Seven cases received cavopulmonary anastomosis by flaring method and one case by end-side anastomosis. All the patients underwent the intracardiac tunnels to drain inferior vena cava and plus 4 mm fenestration except one. The results showed that 6 patients had postoperative oxygen saturation more than 90%, sinus rhythm, no anastomostic stoma obstruction, no flow regurgitation and CVP < 16 cmH2O. Two (25%) patients died postoperatively from high venous pressure of 18-20 cmH2O, finally from cardiac failure and anoxima. It was concluded that TCPC was an effective treatment for complex congenital cardiac diseases, especially with ventricular maldevelopment. Intracardiac tunnel plus 4 mm fenestration and flaring cavopulmonary anastomosis could prevent the postoperative complications. Larger anastomotic stoma, venous pressure less than 16 cmH2O and artery saturation more than 90% might indicate excellent TCPC procedures in our experience.

Child↗

PubMed2001

Neonatal mouse model: review of methods and results.

The neonatal mouse model, in various forms, has been used experimentally since 1959 and a large number of chemicals have been tested. The neonatal model is known to be very sensitive for the detection of carcinogens that operate via a genotoxic mode of action. In contrast, it is known not to respond to chemicals that act via epigenetic mechanisms, commonly observed in the two-year carcinogenicity studies. As such, the model has a high sensitivity and specificity in its response. Dose selection for the neonatal model is based on the maximum tolerated or feasible dose. Traditionally, compounds have been tested via the IP route of administration in this model. In some cases, this has limited the amount of material that can be administered because of the low dosing volumes (10 to 20 microL) that can be administered IP. For the ILSI project, the neonatal model was adapted for oral administration, which has the advantages of being the same route for which most pharmaceuticals are administered. In addition, a 10-fold increase in the volume of administration (100 to 200 microL) and the ability to dose drugs in suspension, permits much higher doses to be used as compared to the IP route of administration. The spontaneous tumors in the neonatal model occurred mainly in the liver of male mice and lung of male and female mice with a few tumors observed in the Harderian gland. The positive control, DEN produced a robust, uniform, and reproducible tumor response with the target organs essentially limited to liver and lung. A total of 13 compounds out of the 21 ILSI ACT compounds were evaluated in the neonatal model involving 18 studies with duplicate studies for some compounds. The genotoxic carcinogens including those used as positive controls were clearly positive (cyclophosphamide, diethylnitrosamine, 6-nitrochrysene). The non-genotoxic rodent carcinogens were clearly negative (chlorpromazine, sulfisoxazole, sulfamethoxazole, clofibrate, DEHP, haloperidol, metaproteranol, and phenobarbital). The non-genotoxic human carcinogen (cyclosporin) was clearly negative. The two other human carcinogens phenacetin and DES were negative and interestingly estradiol was negative in one of the two oral studies, but was clearly positive in the other. Considering the mode of action for three of the human carcinogens (DES, cyclosporin and phenacetin), which were negative in this model, the mode of action in humans is likely to be epigenetic. Overall, for the 3 clearly genotoxic chemicals, all were positive. For the 9 clearly non-genotoxic chemicals, all 9 were negative. The two human carcinogens for which genotoxicity may or may not play a role (DES and phenacetin) were negative and estradiol was positive in I of the two oral studies. Overall, the extensive database for compounds tested in the neonatal mouse model would support its use as an alternative model for the assessment of the carcinogenic potential of a chemical. The model responds to chemicals that act via a genotoxic mode of action that represent a greater concern for human cancer risk.

Administration, Oral↗

PubMed2001

Complement-mediated T-cell depletion of bone marrow: comparison of T10B9.1A-31 and Muromonab-Orthoclone OKT3.

BACKGROUND: T10B9.1A-31 (T(10)B(9)) and Muromonab-Orthoclone OKT3 (OKT3) are both murine MAb with a narrow specificity for T lymphocytes. Over the past 10 years, we have used each for T-cell depletion (TCD) of BM. In this report we describe similarities and differences using these antibodies, as well as their effects on patient outcome. METHODS: We compared BM mononuclear cells (BMMC) prepared using a Cobe Spectra apheresis machine with density gradient (DG) separation to remove RBC and enrich for CD34(+) cells prior to TCD. FACS and limiting dilution assays (LDA) were used to measure the efficiency of TCD, the subsets of cells removed and CD34 content. Univariate statistics were used to assess graft outcome, including GvHD, graft failure, post-transplant lymphoproliferative disease (PTLD), relapse, DFS, and TRM. RESULTS: BMMC preparation on the Cobe Spectra resulted in superior recovery of CD34(+) cells. However, this method could not be used with OKT3 due to inhibition of T-cell lysis. Optimal TCD required two rounds of complement at room temperature for OKT3, compared with one or two rounds for T(10)B(9). TCR(gamma delta)(+) T-cells, but not natural killer cells, were spared to a greater degree with T(10)B(9). Further T-cell loss occurred during culture with T(10)B(9) but not with OKT3. Overall efficiency of TCD was superior using T(10)B(9). The risk of acute GvHD was higher with OKT3-mediated TCD, independent of T-cell content, and may have led to a higher incidence of PTLD. A decreased risk of relapse for patients with high-risk disease was seen with OKT3-treated grafts, but engraftment, TRM and DFS did not significantly differ. DISCUSSION: TCD using OKT3 results in higher T-cell content and higher rates of acute GvHD and PTLD compared with T(10)B(9). Cobe Spectra cannot be used for BMMC processing with OKT3, fewer CD34(+) are therefore infused. Technical, as well as biological, differences between narrow specificity MAbs can affect graft outcome.

Animals↗

PubMed2001

[The retinal nerve fiber layer defect and its related clinical features in early primary open-angle glaucoma].

OBJECTIVE: To investigate the distribution of diffuse and local retinal-nerve-fiber-layer defects (RNFLD) and their clinical features in early primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). METHOD: The types of RNFLD in 81 cases with POAG and 70 cases with NTG were observed, the distribution of diffuse and local RNFLD were analyzed, and the maximum untreated intraocular pressure (IOP) and the history of optic disc hemorrhage between the two types of RNFLD in the two glaucomatous groups were compared. RESULTS: RNFLD appeared in 78 of 81 cases with POAG, 50 of them were diffuse and 28 were local; 27 of diffuse and 43 of local RNFLD were found in 70 cases with NTG. The IOP was (28.78 +/- 5.41) mm Hg in the diffuse and (26.2 +/- 5.2) mm Hg in the local type in POAG (t = 2.04, P < 0.05), and (19.2 +/- 1.9) mm Hg and (17.8 +/- 2.0) mm Hg, respectively in NTG (t = 2.94, P < 0.05). The incidence of disc hemorrhage was 10/78 and 18/70 in POAG and NTG, respectively (chi(2) = 4, P < 0.05). That was 3/50 in the diffuse and 7/28 in the local type in POAG (chi(2) = 4.22, P < 0.05), and 3/27 and 15/43, in NTG respectively (chi(2) = 4.91, P < 0.05). There were no statistic differences in the diffuse type between the POAG and NTG groups (chi(2) = 0.15, P > 0.05), and neither were in the local type (chi(2) = 1.31, P > 0.05). CONCLUSION: The diffuse RNFLD tends to be distributed in POAG, while the local RNFLD tends to be distributed in NTG. The maximum IOP in the eye with diffuse RNFTD is higher than that in the eye with local RNFLD, either in POAG or NTG group. The occurrence of disc hemorrhage is higher in the eye with local RNFLD than that in the eye with diffuse RNFLD in the two groups respectively. There are differences in clinical features between the two types of RNFLD in early POAG and NTG.

Aged↗

PubMed2001

[Correlation of JNK/SAPK activity and the production of ICAM-1 on renal mesangial cells in vitro].

OBJECTIVE: To observe the activity of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and assess the correlation between JNK/SAPK activity and ICAM-1 expression on mesangial cells in mesangial proliferative glomerulonephritis(MSPGN). METHODS: Seven patients with MSPGN and 6 controls were selected for this study. JNK/SAPK activity was detected by immunoprecipitation and western blotting; RT-PCR was used to detect ICAM-1 mRNA expression, and flow cytometry was used to analyze cell surface ICAM-1 expression. RESULTS: Either in spontaneous condition or under the stimulus of TNF alpha, the activity of JNK/SAPK on mesangial cells in vitro in MSPGN was higher than that of the controls(P < 0.05); the activity of JNK/SAPK in MSPGN was found to have a positive correlation with the levels of ICAM-1 mRNA and protein. The levels of ICAM-1 mRNA and protein were increased by TNF alpha stimulation while the levels ICAM-1 mRNA and protein were decreased by DMAP inhibition (P < 0.01). CONCLUSION: JNK/SAPK may have been aberrantly activated in MSPGN, which may involve in the overexpression of ICAM-1 in MSPGN.

Adaptor Proteins, Signal Transducing↗

PubMed2001

[Lymphocyte subpopulation, interleukin-2 and high affinity interleukin-2 receptor expression in primary nephrotic syndrome].

OBJECTIVE: To understand the cellular immune response during relapse and remission stage in primary nephrotic syndrome (PNS). METHODS: We applied the radioligand binding assay (RBA), the bioactivity measurement of IL-2, and monoclonal antibody sensitized red blood cell method to evaluate the expression of IL-2R, the production of IL-2 and T lymphocyte subsets of PBMC from 22 patients suffering from PNS, 17 patients with PNS in remission, and 25 normal subjects matched age and sex. RESULTS: CD3, CD4, CD8, IL-2, IL-2R in relapse were significantly lower than those in control (P < 0.05); all above except CD8 in remission patients were significantly higher than those in relapse, but lower than those in control (P < 0.05). CONCLUSION: This study suggested that the cell mediated immunity (CMI) during acute nephrotic phase decreased; the deficiency of CMI of PNS in remission improved a lot when compared with the acute phase, but still not recovered completely; the deficiency of CMI with PNS in remission might be one of the causes of easily recurring of PNS.

Adult↗

PubMed2000

Acetylation of HIV-1 Tat by CBP/P300 increases transcription of integrated HIV-1 genome and enhances binding to core histones.

The HIV-1 Tat protein is required for viral replication and is a potent stimulator of viral transcription. Although Tat has been extensively studied in various reductive paradigms, to date there is little information as to how this activator mediates transcription from natural nucleosomally packaged long terminal repeats. Here we show that CREB-binding protein (CBP)/p300 interacts with the HIV-1 Tat protein and serves as a coactivator of Tat-dependent HIV-1 gene expression on an integrated HIV-1 provirus. The site of acetylation of Tat was mapped to the double-lysine motif in a highly conserved region, (49)RKKRRQ(54), of the basic RNA-binding motif of Tat. Using HLM1 cells (HIV-1(+)/Tat(-)), which contain a single copy of full-length HIV-1 provirus with a triple termination codon at the first AUG of the Tat gene, we find that only wild type, and not K50A, K51A, or K50A/K51A alone or in combination of ectopic CBP/p300, is able to produce full-length infectious virions, as measured by p24 gag ELISAs. Tat binds CBP/p300 in the minimal histone acetyltransferase domain (1253-1710) and the binding is stable up to 0.85 M salt wash conditions. Interestingly, wild-type peptide 41-54, and not other Tat peptides, changes the conformation of the CBP/p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB, indicating that Tat may influence the transcription machinery by helping CBP/p300 to recruit new partners into the transcription machinery. Finally, using biotinylated wild-type or acetylated peptides, we find that acetylation decreases Tat's ability to bind the TAR RNA element, as well as to bind basal factors such as TBP, CBP, Core-Pol II, or cyclin T. However, the acetylated Tat peptide is able to bind to core histones on a nucleosome assembled HIV-1 proviral DNA.

Acetylation↗

PubMed2000

Effect of mobile phase composition on the separation of propranolol enantiomers using a perphenylcarbamate beta-cyclodextrin bonded chiral stationary phase.

An analytical column packed with a novel perphenylcarbamate beta-cyclodextrin bonded chiral stationary phase was used to separate propranolol enantiomers. Good separation results were obtained using triethylammonium acetate (TEAA) buffer and methanol mixtures as the mobile phase. Effects of the methanol concentration, buffer concentration and pH value on the retention and the enantioselectivity of propranolol enantiomers were investigated on this column. The retention times and the separation factor decrease with increase of the methanol concentration as expected in reversed-phase HPLC. At trace TEAA amount, the solute eluted out with anti-Langmuirian band profiles, their retention times decreased quickly with increase of TEAA concentration and attained a minimum at a TEAA concentration of 20 ppm. Above 20 ppm, solute band profiles changed to a Langmuirian shape, the retention times of enantiomers increased with increasing buffer concentration, and eventually, they attained asymptotes at ca. 1% TEAA. A simulation considering the different interactions between the solute and the additive at above and below 20 ppm TEAA concentration as well as system peaks interference can successfully explain the anti-Langmuirian band profiles and the retention time variation trend.

Carbamates↗

PubMed2000

Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice.

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.

Acyl-CoA Dehydrogenase↗

PubMed2000

Interdialytic weight gain, compliance with dialysis regimen, and age are independent predictors of blood pressure in hemodialysis patients.

Hypertension is a common problem in patients undergoing chronic hemodialysis. The purpose of this study is to identify the clinical and demographic factors independently associated with blood pressure in this population. Data collected for the Dialysis Morbidity and Mortality Study Wave 1 by the US Renal Data System were analyzed. The mean predialysis blood pressure for this cohort of 5,369 patients was 149/79 mm Hg. Sixty-three percent of the patients were hypertensive; 27%, 25%, and 11% had stages 1, 2, and 3 hypertension, respectively. Young age, black race, male sex, diabetes as cause of end-stage renal disease, erythropoietin therapy, and smoking were associated with higher blood pressure in the univariate analysis. Patients skipping or shortening one or more dialysis treatments had higher blood pressure. The presence of congestive heart failure and coronary heart disease was associated with lower blood pressure. On multivariate analysis, high interdialytic weight gain, noncompliance with dialysis regimen, and younger age were independent predictors of higher blood pressure. In summary, hypertension is common and poorly controlled in patients undergoing chronic hemodialysis. Greater interdialytic weight gain and noncompliance with dialysis regimen are independently associated with higher blood pressure, and advancing age is associated with lower blood pressure levels in this population. Therapeutic regimens emphasizing reduction of interdialytic weight gain and improved compliance with the dialysis regimen need to be evaluated for improving the management of hypertension. The effect of age and other comorbid conditions, particularly cardiovascular disease, must be considered while studying the relationship between blood pressure and mortality in patients undergoing chronic hemodialysis.

Age Factors↗

PubMed2000

Inhibition of ethanol-induced liver disease in the intragastric feeding rat model by chlormethiazole.

The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

Animals↗

PubMed2000

Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G(1)/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G(1)/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G(1)/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G(1)/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G(1)/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.

CDC2-CDC28 Kinases↗

PubMed2000

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

Animals↗