Biomedical subjects

S Zhou

Publications and source records attributed to S Zhou.

At least 91 records · Page 5Linked to original sources

PubMed2001

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human beta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice.

Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human beta-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human beta-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW(41/41) anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human beta-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human beta-globin gene sequences were present at approximately 1 copy per cell. The efficiency of the human beta-globin gene expression was determined to be up to 35% compared with the murine endogenous beta-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human beta-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human beta-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of beta-thalassemia and sickle-cell disease.

Anemia, Sickle Cell↗

PubMed2001

Novel gelatin-adriamycin sustained drug release system for intravesical therapy of bladder cancer.

To reduce recurrence in the patients with bladder cancer after tumor removal through open surgery or transurethral resection, a form of gelatin-adriamycin sustained drug release system was developed and its release kinetics both in vitro and in vivo, its efficacy in inhibiting BIU-87 bladder tumor cell growth in vitro and its safety in vivo were studied. The results showed that this system controlled adriamycin release over a period of 21 days in vitro and significantly inhibited BIU-87 cell growth. When this system was injected into rabbit bladder, it sustained adriamycin release for 12 days and the released drug could diffuse 1 cm around the injection point. No major complications were observed except minor acute nonspecific cystitis that could be tolerated well by the animals. This study suggests the possibility of applying this system locally in treating bladder cancer.

Administration, Intravesical↗

PubMed2001

In vitro and in vivo kinetic interactions of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid with thalidomide and diclofenac.

BACKGROUND: Previous studies have demonstrated that coadministration of L-thalidomide with the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) results in an increased area under the plasma concentration-time curve (AUC) of DMXAA, suggesting an explanation for the observed increase in the antitumour activity. The aims of this study were to investigate the effects of L-thalidomide on the in vitro metabolism of DMXAA in mouse and human liver microsomes using diclofenac as positive control, to examine the effects of L-thalidomide and diclofenac on the plasma protein binding of DMXAA in vitro, and to investigate whether the in vivo interactions can be predicted from in vitro data, particularly in humans. METHODS: Mouse and human liver microsomes were used to investigate the effects of L-thalidomide and diclofenac on DMXAA metabolism. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA, which were then compared with the results of in vivo mouse pharmacokinetic interaction studies. The protein binding of DMXAA in mouse and human plasma was determined using ultrafiltration followed by HPLC. RESULTS: Diclofenac at 100 microM caused significant inhibition of glucuronidation (> 70%) and 6-methylhydroxylation (> 54%) of DMXAA in mouse and human liver microsomes. In vivo diclofenac (100 mg/kg i.p.) resulted in a 24% and 31% increase in the plasma DMXAA AUC, and a threefold increase in T1/2 (P < 0.05) in male and female mice, respectively. In contrast, L-thalidomide at 100 microM had no inhibitory effect on DMXAA metabolism in vitro in either species, except for a decrease of about 25% in 6-methylhydroxylation in mice. L-Thalidomide at 500 microM resulted in further significant decreases in 6-methylhydroxylation in mice (30-60%) and human (30%) microsomes. Coadministration of L-thalidomide in male mice resulted in a 23% increase in DMXAA AUC and a twofold increase in T1/2 (P < 0.05). Neither L-thalidomide nor diclofenac at 50 or 500 microM had any significant effect on the in vitro plasma protein binding of DMXAA (500 microM) in mouse or human plasma. Based on our in vitro inhibition studies, we predicted a 20% increase in DMXAA AUC in mice with concomitant diclofenac, but little or no effect (< 5%) with L-thalidomide. CONCLUSION: Both L-thalidomide and diclofenac increased the plasma DMXAA AUC in mice. In the case of diclofenac, this appeared to be due to direct competitive inhibition of DMXAA metabolism, but this mechanism does not appear to be appropriate for L-thalidomide. From the in vitro human inhibition studies, it appears unlikely that concurrent diclofenac will cause an increase in the plasma AUC of DMXAA in patients. However, the effect of L-thalidomide on DMXAA could not be readily predicted from the in vitro data. Our study demonstrated that a predictive model based on direct inhibition of metabolism is appropriate for diclofenac-DMXAA interactions, but is inappropriate for the prediction of L-thalidomide-DMXAA interactions in mice and humans in vivo.

Animals↗

PubMed2001

A difference between the rat and mouse in the pharmacokinetic interaction of 5,6-dimethylxanthenone-4-acetic acid with thalidomide.

PURPOSE: Coadministration of thalidomide, cyproheptadine or diclofenac has been shown to increase the area under the plasma concentration-time curve (AUC) of the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice. The aim of this study was to further investigate these pharmacokinetic DMXAA-drug interactions in the rat model. METHODS: The effects of coadministration of L-thalidomide, cyproheptadine or diclofenac on the pharmacokinetics of DMXAA were investigated in male Wistar Kyoto rats. The effects of L-thalidomide, cyproheptadine and diclofenac on microsomal metabolism and plasma protein binding of DMXAA were also investigated. RESULTS: No significant alteration in the plasma concentration profile for DMXAA was observed following L-thalidomide pretreatment in rats. In contrast, when combined with diclofenac or cyproheptadine, the plasma AUC of DMXAA was significantly (P<0.05) increased by 48% and 88% and the T1/2 by 36% and 107%, respectively, compared to controls. Both diclofenac and cyproheptadine at 500 microM caused a significant inhibition of DMXAA metabolism in rat liver microsomes. In contrast, L-thalidomide had no or little inhibitory effect on DMXAA metabolism in rat liver microsomes except for causing a 32% decrease in 6methylhydroxylation at 500 microM. None of the drugs had a significant effect on the plasma protein binding of DMXAA in the rat. CONCLUSION: These studies showed that coadministration of L-thalidomide did not alter the plasma DMXAA AUC in rats, in contrast to previous studies in mice, whereas diclofenac and cyproheptadine significantly reduced the plasma clearance of DMXAA in rats in a similar manner to their effect in mice. The cause of the species difference in the pharmacokinetic response to thalidomide by DMXAA is unknown, and indicates difficulties in predicting the outcome of such a combination in patients.

Animals↗

PubMed2001

Nuclear localization of Dpc4 (Madh4, Smad4) in colorectal carcinomas and relation to mismatch repair/transforming growth factor-beta receptor defects.

The tumor-suppressor protein Dpc4 (Smad4, Madh4) regulates gene expression. On binding of an extracellular ligand of the extensive transforming growth factor (TGF) superfamily to its cognate receptor complex, latent cytoplasmic Dpc4 is activated and translocated into the nucleus to function as part of various DNA-binding transcriptional activator complexes. The most relevant ligand/receptor pair to control the tumor suppressive function of Dpc4 remains uncertain, but is usually assumed to be TGF-beta and its heteromeric receptor. We exploited a fortuitous experiment of nature to directly test this hypothesis: the TGF-beta type II receptor gene is inactivated by mutation in nearly all colorectal carcinomas having microsatellite instability, as seen in hereditary nonpolyposis colorectal cancer (HNPCC) and in sporadic medullary colorectal cancers. Using a specific and sensitive immunohistochemical label for Dpc4, we examined nuclear localization of Dpc4 in 13 HNPCC, six medullary, and 41 sporadic nonmedullary colorectal carcinomas. In agreement with published rates, two (5%) of 41 sporadic tumors showed complete loss of Dpc4 protein, indicative of genetic inactivation. All 13 HNPCC and six medullary tumors had intact cytoplasmic and nuclear Dpc4 localization. The TGFBR2 gene was sequenced in three of the cancers from patients with HNPCC, and all of these harbored inactivating mutations. The specificity of the immunohistochemical assay was demonstrated in xenograft tumors of syngeneic cell lines that differed in DPC4 genetic status because of an engineered gene knockout. Thus, nuclear localization of Dpc4 can be maintained in cells with inactivated TGF-beta type II receptors, suggesting the persistence of tumor-suppressive action of an upstream signaling input, most likely a ligand/receptor complex distinct from TGF-beta. Identification of the relevant input would be expected to have implications for the understanding of tumorigenesis and the design of rational biological therapy.

Animals↗

PubMed2001

Study of laser vas deferens occlusion.

The objective of this study was to determine threshold levels for high-power laser output Nd:YAG laser photocoagulation and to determine the possible reversibility of laser vas occlusion. The study included vas deferens of 220 rabbits and 20 samples of men in vitro, applying the Nd:YAG laser instrument, doing vasopuncture by a catheter needle, guiding the fiber into the vas, and performing the irradiation occlusion experiment to determine effective threshold value of photocoagulation. The effect and safety of occlusion were followed-up over a year, and reopening a experiment was done in 60 occluded nodes of rabbits. The postoperative sperm disappearance rate was 100%, and reopening rate was 72.7% without obvious complications. High-power lasers may be used as photocoagulation, and its merits are reliable, effective, and rapid recovery. The vas threshold lesion value of rabbits and men in vitro are 50 W/0.5 s, 45 W/1 s, respectively, and irradiation depth reached the middle-ring muscularis.

Animals↗

PubMed2001

Metabolic response of Platynota stultana pupae during and after extended exposure to elevated CO(2) and reduced O(2) atmospheres.

The metabolic response of Platynota stultana pupae to elevated CO(2) and reduced O(2) atmospheres was measured using microcalorimetry. Initial measurements at 20 degrees C immediately upon placement in controlled atmosphere indicated a decrease in metabolic heat rate (MHR) of 27, 45, 56, 56, and 72% in an atmosphere of 5, 10, 20, 40, and 79% CO(2), respectively, and a decrease of 20, 50, 66 and 100% under 6, 2, 1, and 0% O(2). With extended exposure to controlled atmospheres, MHR increased under 5, 10, and 20% CO(2) and 6 and 2% O(2); however, the increase was greater and occurred more rapidly with lower CO(2) and higher O(2) concentration. The MHR at 40 and 79% CO(2) remained at the initial reduced level for 8 and 6 days, respectively, then decreased with longer exposure. The MHR of pupae held under 1 and 0% O(2) remained at the initial reduced level for 22 days. Upon transfer to air, the MHR of pupae increased from the reduced levels and then decreased. When the MHR decreased by no more than 30%, as a result of controlled atmosphere treatment, the pupae still developed into adults. However, when the MHR decreased by more than 50%, the energy supply was insufficient and the pupae died. Pupa mortality was comparable between 5% CO(2) and 6% O(2), and 10% CO(2) and 2% O(2). The MHR was reduced less under 20% CO(2) than under 2 or 1% O(2); however, the pupae were more susceptible to 20% CO(2) than 2 or 1% O(2). These and other data indicate an increased toxicity of high CO(2) over low O(2) atmospheres that may be related to an increase in membrane permeability as a result of CO(2) treatment.

Journal Article↗

PubMed2001

A mechanistic study of electrospray mass spectrometry: charge gradients within electrospray droplets and their influence on ion response.

A model has been developed to account qualitatively for the effects of ion pairing, surface activity, and electrophoretic mobility in electrospray mass spectrometry. The model is tested with various salt and amino acid mixtures. The data suggest that the axial charge gradient arising from electrophoretic separation at droplet genesis may persist within the electrosprayed droplets at least until the first droplet fission, accounting for the field dependence of detected ion clustering of quaternary ammonium salts and for the relatively field-invariant charge distribution of horse heart myoglobin samples.

Journal Article↗

PubMed2001

The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.

Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Effects of anticancer drugs on the metabolism of the anticancer drug 5,6-dimethylxanthenone-4-acetic (DMXAA) by human liver microsomes.

AIMS: To investigate the effects of various anticancer drugs on the major metabolic pathways (glucuronidation and 6-methylhydroxylation) of DMXAA in human liver microsomes. METHODS: The effects of various anticancer drugs at 100 and 500 microM on the formation of DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) in human liver microsomes were determined by high performance liquid chromatography (h.p.l.c.). For those anticancer drugs showing significant inhibition of DMXAA metabolism, the inhibition constants (Ki) were determined. The resulting in vitro data were extrapolated to predict in vivo changes in DMXAA pharmacokinetics. RESULTS: Vinblastine, vincristine and amsacrine at 500 microM significantly (P < 0.05) inhibited DMXAA glucuronidation (Ki = 319, 350 and 230 microM, respectively), but not 6-methylhydroxylation in human liver microsomes. Daunorubicin and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) at 100 and 500 microM showed significant (P < 0.05) inhibition of DMXAA 6-methylhydroxylation (Ki = 131 and 0.59 microM, respectively), but not glucuronidation. Other drugs such as 5-fluoroucacil, paclitaxel, tirapazamine and methotrexate exhibited little or negligible inhibition of the metabolism of DMXAA. Pre-incubation of microsomes with the anticancer drugs (100 and 500 microM) did not enhance their inhibitory effects on DMXAA metabolism. Prediction of DMXAA-drug interactions in vivo based on these in vitro data indicated that all the anticancer drugs investigated except DACA appear unlikely to alter the pharmacokinetics of DMXAA, whereas DACA may increase the plasma AUC of DMXAA by 6%. CONCLUSIONS: These results indicate that alteration of the pharmacokinetics of DMXAA appears unlikely when used in combination with other common anticancer drugs. However, this does not rule out the possibility of pharmacokinetic interactions with other drugs used concurrently with this combination of anticancer drugs.

Antineoplastic Agents↗

PubMed2001

Modulation of QT interval by cardiac sympathetic nerve sprouting and the mechanisms of ventricular arrhythmia in a canine model of sudden cardiac death.

INTRODUCTION: We previously reported that there is a high incidence of sudden cardiac death (SCD) in dogs with myocardial infarction (MI), complete AV block (CAVB), and nerve growth factor (NGF) infusion to the left stellate ganglion (LSG). Whether or not QT interval prolongation underlines the mechanism of SCD was unclear. METHODS AND RESULTS: We analyzed QT intervals in three groups of dogs. All dogs had CAVB and MI. The LSG group (n = 9) and right stellate ganglion (RSG) group (n = 6) received NGF infusion via the osmotic pumps over a 5-week period to LSG and RSG, respectively. The control group (n = 6) received no NGF. The dogs either died suddenly or were sacrificed within 2 to 3 months after MI. Heart rhythm and QT and RR intervals were monitored using implantable cardioverter defibrillator ECG recordings. There was a time-dependent increase of QTc intervals in the LSG group and a time-dependent decrease of QTc intervals in the RSG group. At the end of NGF infusion, QTc intervals in the LSG group (408 +/- 41 msec) were significantly longer than those in the control (350 +/- 41 msec; P < 0.05) and RSG groups (294 +/- 23 msec; P < 0.01). In the LSG group, 4 of 9 dogs died of SCD. There was no SCD in either the RSG or control group. Immunocytochemical staining showed NGF infusion to LSG and RSG resulted in left and right ventricular sympathetic nerve sprouting and hyperinnervation, respectively. CONCLUSION: NGF infusion to the LSG in dogs with MI and CAVB resulted in increased QT interval and incidence of ventricular tachycardia, ventricular fibrillation, and SCD, whereas NGF infusion to the RSG shortened QT interval and reduced the incidence of ventricular tachycardia. These findings indicate that QT interval prolongation is causally related to the occurrence of ventricular arrhythmia in dogs with nerve sprouting, MI, and CAVB.

Animals↗

PubMed2001

The evaluation of a precordial ECG BELT: technologist satisfaction and accuracy of recording.

The standard method for performing electrocardiogram (ECG) recordings presents a challenge to technicians because of the need to correctly position the individual precordial electrodes according to 6 bony thoracic landmarks. A proposed new method using a 6-lead ECG BELT for precordial application was compared to the standard method to determine the level of agreement among automated interpretations. A comparison of automated interpretations from repeat standard recordings served as the control. Results indicate that BELT and standard automated interpretations disagreed significantly more frequently than repeat standard recording automated interpretations of the cardiac rhythm. The BELT's most obvious weakness was the inability to obtain a recording with a stable ECG baseline, triggering automated detection of "baseline artifact or wander," and requiring a repeat recording. These findings suggest that the ECG BELT is not adequate for clinical application in its current form.

Electrocardiography↗

PubMed2001

Identification of functionally variant MDR1 alleles among European Americans and African Americans.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.

Base Sequence↗

PubMed2001

Critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells.

Under conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8(+) T cells. In this work we studied the down-regulation of the virus-specific CD8(+)-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-alpha/beta]), type II (IFN-gamma), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8(+)-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8(+) T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.

Acute Disease↗

PubMed2001

Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593-1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835-9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused approximately 40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

3T3 Cells↗

PubMed2001

Nuclear localization of CBF1 is regulated by interactions with the SMRT corepressor complex.

The CSL family protein CBF1 is a nuclear mediator of Notch signaling and has been predicted to contain an N-terminal nuclear localization signal in exon 4. Surprisingly, we found that CBF1 carrying mutations at codon 233 or 249 within exon 7 was restricted to the cytoplasm. In mammalian and yeast two-hybrid assays, these mutations were also associated with a loss of CBF1-mediated transcriptional repression and a severely impaired interaction with the corepressors SMRT and CIR. Overexpression of SMRT rescued the ability of mutant CBF1 to target to the nucleus of transfected cells and similarly rescued nuclear targeting of enhanced green fluorescent protein (EGFP)-CBF1 exons 6 to 9 CBF1(6-9)carrying the codon 233 or 249 mutations. Carboxy-terminally truncated SMRT with amino acids (aa) 1291 to 1495 deleted was unable to rescue the nuclear targeting of mutant EGFP-CBF1(6-9). In yeast two-hybrid assays, the SMRT aa 1291 to 1495 domain interacted with SKIP and SMRT aa 1291 to 1495 colocalized with SKIP within the nuclei of cotransfected cells. Comparison of the intracellular localization of CBF1(6-9) with that of CBF1(5-9) further supported the suggestion that nuclear targeting of CBF1 is dependent on the formation of a CBF1-SMRT-SKIP corepressor complex. These observations suggest that nuclear targeting of CBF1 is itself a component of CBF1-mediated gene regulation and that in the absence of signaling, CBF1 enters the nucleus precommitted to a transcriptional repression function. The activators NotchIC (the intracellular domain of Notch) and Epstein-Barr virus EBNA2 also mediated nuclear targeting of mutant CBF1, consistent with the competition model for activator versus corepressor binding to CBF1.

Cell Nucleus↗

PubMed2001

The basic helix-loop-helix transcription factor Cph2 regulates hyphal development in Candida albicans partly via TEC1.

Candida albicans undergoes a morphogenetic switch from budding yeast to hyphal growth form in response to a variety of stimuli and growth conditions. Multiple signaling pathways, including a Cph1-mediated mitogen-activated protein kinase pathway and an Efg1-mediated cyclic AMP/protein kinase A pathway, regulate the transition. Here we report the identification of a basic helix-loop-helix transcription factor of the Myc subfamily (Cph2) by its ability to promote pseudohyphal growth in Saccharomyces cerevisiae. Like sterol response element binding protein 1, Cph2 has a Tyr instead of a conserved Arg in the basic DNA binding region. Cph2 regulates hyphal development in C. albicans, as cph2/cph2 mutant strains show medium-specific impairment in hyphal development and in the induction of hypha-specific genes. However, many hypha-specific genes do not have potential Cph2 binding sites in their upstream regions. Interestingly, upstream sequences of all known hypha-specific genes are found to contain potential binding sites for Tec1, a regulator of hyphal development. Northern analysis shows that TEC1 transcription is highest in the medium in which cph2/cph2 displays a defect in hyphal development, and Cph2 is necessary for this transcriptional induction of TEC1. In vitro gel mobility shift experiments show that Cph2 directly binds to the two sterol regulatory element 1-like elements upstream of TEC1. Furthermore, the ectopic expression of TEC1 suppresses the defect of cph2/cph2 in hyphal development. Therefore, the function of Cph2 in hyphal transcription is mediated, in part, through Tec1. We further show that this function of Cph2 is independent of the Cph1- and Efg1-mediated pathways.

Amino Acid Sequence↗