Biomedical subjects

M Tan

Publications and source records attributed to M Tan.

At least 19 recordsLinked to original sources

PubMed2002

The effect of octreotide on kainate-induced wet dog shakes and seizure activity in male and female rats.

Systemic kainic acid (KA) administration to rats triggers wet dog shakes (WDS) followed by epileptic seizures. Although WDS are often associated with the occurrence of seizures, we have recently shown that following nitric oxide (NO) synthesis inhibition, the number of WDS decreased; subsequently the onset of seizure activity was shortened, and the number of convulsions was increased. Somatostatin (SS), whose release appears to be controlled by NO, inhibits seizure activity. There are sex differences in seizure susceptibility as well as in SS and NO activities in brain. The present study was undertaken to assess the effect of octreotide (OC), a stable SS analogue, on KA-induced WDS and seizures in rats, with emphasis on possible sex differences. WDS and seizures were induced by KA in male and female (proestrus) Sprague Dawley rats; OC or saline was injected 30 min before KA and the behavior was monitored for 120 min after KA. Octreotide increased the number of WDS and decreased the number of convulsions; this effect was more pronounced in males. Onset of KA-induced seizure activity was earlier in females than males; however, there was no effect of OC on seizure latency. Seizure activity started after the termination of WDS. These results show OC has opposite effects on WDS and convulsions, in that it stimulates the former and inhibits the latter. These results support our previous findings that WDS and seizure activity involve separate mechanisms and suggest that WDS may have an inhibitory effect on limbic seizures.

Analysis of Variance↗

PubMed2001

Persistent expression of cyclin D1 disrupts normal photoreceptor differentiation and retina development.

The differentiation of neuronal cells in the developing mammalian retina is closely coupled to cell cycle arrest and proceeds in a highly organized manner. Cyclin D1, which regulates cell proliferation in many cells, also drives the proliferation of photoreceptor progenitors. In the mouse retina, cyclin D1 protein normally decreases as photoreceptors mature. To study the importance of the down-regulation of cyclin D1 during photoreceptor development, we generated a transgenic mouse in which cyclin D1 was persistently expressed in developing photoreceptor cells. We observed numerous abnormalities in both photoreceptors and other nonphotoreceptor cells in the retina of these transgenic mice. In particular, we observed delayed opsin expression in developing photoreceptors and alterations in their number and morphology in the mature retina. These alterations were accompanied by disorganization of the inner nuclear and plexiform layers. The expression of cyclin D1 caused excess photoreceptor cell proliferation and apoptosis. Loss of the p53 tumor suppressor gene decreased cyclin D1-induced apoptosis and led to microscopic hyperplasia in the retina. These findings are distinct from other mouse models in which the retinoblastoma gene pathway is disrupted and suggest that the IRBP-cyclin D1 mouse model may recapitulate an early step in the development of retinoblastoma.

Animals↗

PubMed2001

Pseudoaneurysm of the superior mesenteric artery after pancreas transplantation treated by endovascular stenting.

Pseudoaneurysm after pancreas transplantation can have serious consequences, including rupture, hemorrhage, and graft loss. We describe a 38-year-old patient who presented with a pseudoaneurysm of the donor superior mesenteric artery 1 month after pancreas transplantation. Selective arteriography was performed and the lesion was repaired with endovascular placement of a 28-mm covered stent. Laparotomy was avoided. The pancreatic graft was continuing to function well 9 months later. As far as we know, this minimally invasive approach was not previously reported. According to published series, pseudoaneurysms often occur secondary to infection and require operative intervention necessitating graft pancreatectomy. Patients can present with serious symptoms including hypotension and shock. Therefore, it is important to detect pseudoaneurysm in a timely manner. Computed tomography and Doppler ultrasound are important diagnostic tools in this regard. We demonstrated the utility of endovascular stenting in the treatment of pseudoaneurysm after pancreas transplantation. When used in a timely manner in well selected patients, endovascular stenting can abrogate the need for operative intervention and its attendant morbidity.

Adult↗

PubMed2001

Measuring the diaspora for virus-specific CD8+ T cells.

The CD8(+) T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of D(b)NP(366)- and D(b)PA(224)-specific CD8(+) T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8(+) tetramer(+) populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the "whole mouse" virus-specific CD8(+) T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8(+)D(b)NP(366)+ and CD8(+)D(b)PA(224)+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 "activation marker" were detected consistently on virus-specific CD8(+) T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69(hi) T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of "resting" CD8(+) memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

Animals↗

PubMed2001

Circulating concentrations of IGF-I and IGFBP-3 are not predictive of incidence or clinical behavior of pediatric osteosarcoma.

BACKGROUND: Preclinical studies suggest a role of insulin-like growth factor-1 (IGF-1) in the proliferation of osteosarcoma cells in vivo. The purpose of this study is to address the relationship between serum levels of IGF-1 and its binding protein (IGFBP-3), and the clinical behavior and outcome of osteosarcoma in children, and to compare those levels present in osteosarcoma patients with a normal population. PROCEDURE: Serum IGF-1 and IGFBP-3 levels were determined by ELISA in 37 patients with osteosarcoma treated on the same treatment regimen (OS-91 protocol), and who had available serum samples from diagnosis. IGF-1 and IGFBP-3 levels were compared with those previously established in the normal population, matched for age and gender, and were correlated with the presence of metastatic disease, histologic response to preoperative chemotherapy, and event-free survival. RESULTS: In osteosarcoma patients the median IGF-1 level was 275 ng/ml (range, 105-613) and the median IGFBP-3 level was 3.4 mg/L (range, 2.3-5.1). IGF-1 levels differed from those in the normal population (P = 0.029); although we anticipated higher IGF-1 levels than normal children, 68% of observed standardized scores were less than 0. Furthermore, IGF-1 or IGFBP-3 levels failed to correlate with the presence of metastatic disease (P = 0.12 and P = 0.12, respectively), histologic response (Rosen-Huvos grades 3/4 vs. grades 1/2) (P = 0.95 and P = 0.71, respectively), or event-free survival (P = 0.52 and P = 0.41, respectively). There was a strong association observed between IGF-1 and IGFBP-3 levels (P < 0.001). CONCLUSIONS: In this retrospective study of 37 patients, we found that circulating levels of IGF-1 and IGFBP-3 are not predictive of the development or clinical characteristics of pediatric osteosarcoma. However, further studies on a larger patient population should be performed in order to investigate this relationship.

Adolescent↗

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

Advantages of a posterior fourchette incision in anal sphincter repair.

PURPOSE: Delayed repair of obstetric-related anal sphincter injury remains problematic, and perineal wound breakdown is common. The aim of this study was to assess the outcome after overlap anal sphincter repair and to determine the advantages, if any, of a posterior fourchette incision (n = 18) compared with a conventional perineal incision (n = 32). METHODS: Fifty females of mean parity 2.8 (standard deviation, 1.6) underwent repair in a five-year period. The mean follow-up was 23 months. Assessment was by anal vector manometry, endoanal ultrasound, and continence scoring. RESULTS: Functional outcomes were similar in the two groups. Repair increased squeeze-pressure increment and improved continence scores in both groups. Postoperative wound complications were fewer when a posterior fourchette incision was used compared with a perineal incision (11 vs. 44 percent, respectively; P < 0.05). CONCLUSIONS: Delayed anal sphincter repair improves continence. A posterior fourchette approach is associated with fewer postoperative wound complications without compromising the quality of repair and the functional outcome.

Adult↗

PubMed2001

Thallium bone imaging as an indicator of response and outcome in nonmetastatic primary extremity osteosarcoma.

PURPOSE: We investigated one 201Tl bone scintigraphy method as a predictor of histologic response and event-free survival (EFS) of nonmetastatic extremity osteosarcoma. MATERIALS AND METHODS: We evaluated images of the primary tumor to determine whether they exhibited a donut of avidity for 40 patients enrolled on a single institutional protocol. Participants underwent three serial 201Tl bone scintigraphy studies during preoperative neoadjuvant chemotherapy. Intra- and interobserver variability of the method was assessed, and the presence of the donut of avidity was examined as a predictor of EFS and histologic response. RESULTS: Fifty-three percent of patients were female and 75% were Caucasian; the median age at diagnosis was 13.5 years. Intraobserver agreement was moderate to very good, ranging from 0.595 to 0.865. Interobserver agreement was moderate to good for all time points, ranging from 0.576 to 0.708. There was a significant difference in EFS among patients with and without the donut-shape at any of the three time points (P = 0.049); patients whose tumors displayed a donutshape had inferior EFS. CONCLUSION: The pattern of donut avidity in extremity OS is a predictor of lower EFS, but does not correlate with histologic response to therapy.

Adolescent↗

PubMed2001

Colonic J-pouch function at six months versus straight coloanal anastomosis at two years: randomized controlled trial.

The colonic J-pouch (pouch group) functions better than the straight coloanal anastomosis (straight group) immediately after ultra-low anterior resection, but there are few studies with long-term follow-up. This randomized controlled study compared functional outcome, anal manometry, and rectal barostat assessment of these two groups over a 2-year period. Forty-two consecutive patients were recruited, of which 19 of the straight group [17 men with a mean age of 62.1 +/- 2.3 (SEM) year] and 16 of the pouch group (11 men with a mean age of 61.3 +/- 3.2 year) completed the study. Four died from metastases and two emigrated; there was no surgical morbidity or local recurrence. At 6 months the Pouch patients had significantly less frequent stools (32.9 +/- 2.8 vs. 49 +/- 1.4/week; p < 0.05) and less soiling at passing flatus (38% vs. 73.7%; p < 0.05). At 2 years both groups had improved with no longer any differences in stool frequency (7.3 +/- 0.4 vs. 8 +/- 0.2/week) and soiling at passing flatus (38% vs. 53%). Defecation problems remained minimal in both groups. Anal squeeze pressures were significantly impaired in both groups up to 2 years (p < 0.05). The rectal maximum tolerable volume and compliance were not different between groups. Rectal sensory testing on the barostat phasic program showed impairment at 6 months and recovery at 2 years, suggesting that postoperative recovery of residual afferent sympathetic nerves may play a role in functional recovery. In conclusion, stool frequency and incontinence were less in the Pouch patients at 6 months; but after adaptation at 2 years the straight group patients yielded similar results. Nonetheless, this functional advantage can be given to patients with minimal added effort or complications by using the colonic J-pouch.

Adenocarcinoma↗

PubMed2001

The phase-shift mutation in the glucocorticoid receptor gene: potential etiologic significance of neuroendocrine mechanisms in lupus nephritis.

BACKGROUND: Glucocorticoid (GC), a mediator of the hypothalamo-pituitary-adrenal axis, has been found to play an important role in maintaining the stability of immune endo-environment of the body. The pathogenesis of lupus nephritis, an autoimmune disease, is thought to be related to the intrinsic hyposensitivity to GC secreted by adrenal gland, and impairs the regulation of the immuno-neuro-endocrine axis. METHODS: To test this hypothesis, we examined the response of 39 clinic patients with lupus nephritis to GC and analyzed the molecular structure and function of the GC receptor (GR) on peripheral blood mononuclear cells. RESULTS: There was no difference in the level of ACTH, GC and ligand affinity of GR between the patients and the controls. The GR number on mononuclear cells of lupus patients was lower than that of the controls. There was no difference in GR number between the patients with heterogeneous response, i.e. sensitive, dependent and resistance, to GC. The analysis of exon 9 of the GC receptor with PCR-amplified single strand conformation polymorphism (PCR-SSCP) method showed the polymorphism in exon 9 of GC receptor in 8 of the 39 lupus nephritis patients. DNA sequence analysis revealed an adenine insertion at the 2439 base pair of the GC receptor gene. This phase-shift mutation caused an additional 20 amino acids being translated into protein of GC receptor. CONCLUSION: The decreased number of GC receptor and the molecular variation of GR on mononuclear cells could explain the phenomenon of GC resistance, potentially to endogenous GC, which suggested an etiological significance of neuro-endocrine-immune mechanism in lupus nephritis. This may be useful in the design of lupus nephritis therapy.

Adolescent↗

PubMed2001

Successful outcome after transplantation of a donor liver with focal nodular hyperplasia.

Because of the increasing gap in the number of patients awaiting organ transplantation and the supply of organ donors, reevaluation of donor criteria is an important issue in clinical transplantation. It has become necessary to make maximal use of the currently available donor pool. We describe a case of successful orthotopic liver transplantation in a 57-year-old man with Laënnec's cirrhosis using a liver containing an 8-cm focal nodular hyperplasia (FNH) lesion involving segments II and III and the caudate lobe. The donor liver was procured from a 46-year-old woman declared brain dead after a subarachnoid hemorrhage. Definitive pathological diagnosis was made at laparotomy by obtaining a Tru-cut (Allegiance Health Care Inc, Toronto, Ontario, Canada) core biopsy specimen. The recipient operation was performed uneventfully except for bleeding from the biopsy site. The patient did well postoperatively and was discharged on tacrolimus, mofetil mycophenolate, and prednisone therapy. He continues to thrive 2(1/2) years posttransplantation with no change in the size of the lesion. In well-selected donors, FNH should not be a contraindication for use in transplantation. However, FNH must be differentiated from hepatocellular adenoma. Although FNH has a benign course with little propensity for bleeding and almost no malignant potential, hepatic adenoma is reported to have a 15% to 33% chance of bleeding and rupture with a well-documented potential for neoplastic degeneration, making the liver unsuitable for donation.

Focal Nodular Hyperplasia↗

PubMed2001

EXAFS study on the local atomic structures around iron in glycosylated haemoglobin.

Samples with 5.1%, 9.8% and 15.3% HbA1c were extracted from normal subjects and patients with slight and serious diabetes respectively. Extended x-ray absorption fine structure spectra of Fe K absorption were collected at the EXAFS experimental station of the Beijing Synchrotron Radiation Facility. The step-by-step fluorescent mode was employed with a count time of 10 s per point. Several independent scans were averaged to eliminate the statistical noise. Reference backscattering amplitudes and phaseshifts were calculated using the curve wave theory (FEFF code) of EXAFS. Apart from the nitrogen neighbours around the central iron atom, oxygen neighbours are also found. The Fe-N bond length increases by about 0.02 A for the sample with 15.3% HbA1c compared with the others, but the Fe-O bond length is almost unchanged. With increasing of HbA1c concentration, the content of Hb increases and the content of HbO2 decreases. This demonstrates that the glycosylation of haemoglobin will decrease its ability to carry oxygen.

Absorptiometry, Photon↗

PubMed2001

SAG/ROC/Rbx/Hrt, a zinc RING finger gene family: molecular cloning, biochemical properties, and biological functions.

The RING (really interesting new gene) finger proteins containing a characteristic C3HC4 or C3H2C3 motif appear to act as E3 ubiquitin ligase and play important roles in many processes, including cell-cycle progression, oncogenesis, signal transduction, and development. This review is focused on SAG/ROC/Rbx/Hrt (sensitive to apoptosis gene/regulator of cullins/RING box protein), an evolutionarily conserved RING finger family of proteins that were cloned recently by several independent laboratories through differential display, yeast two-hybrid screening, or biochemical purification. SAG/ROC2/Rbx2/Hrt2 is expressed in multiple mouse adult tissues, as well as early embryos. In humans, both SAG and ROC1 are ubiquitously expressed at a very high level in heart, skeletal muscle, and testis. Expression of both SAG and ROC1 is induced by mitogenic stimulation. SAG is also induced by a redox agent in cultured cells, as well as in in vivo mouse brain upon ischemia/reperfusion. Structurally, SAG consists of four exons and three introns with at least one splicing variant and two pseudogenes. The SAG gene promoter is enriched with multiple transcription factor binding sites. Biochemically, SAG binds to RNA, has metal-ion binding/free radical scavenging activity, and is redox-sensitive. Most importantly, like ROC1, SAG/ROC2 binds to cullins and acts as an essential component of E3 ubiquitin ligase. Biologically, SAG is a growth-essential gene in yeast. In mammalian cells, SAG protects apoptosis mainly through inhibition of cytochrome c release/caspase activation, and promotes growth under serum deprivation at least in part by inhibiting p27 accumulation. Blocking SAG expression via antisense transfection inhibits tumor cell growth. Thus, SAG appears to be a valid drug target for anticancer therapy.

Amino Acid Sequence↗

PubMed2001

p53CP is p51/p63, the third member of the p53 gene family: partial purification and characterization.

The p53 tumor suppressor is a transcription factor that upon activation by DNA-damaging agents induces growth arrest or apoptosis mainly through transactivation and transrepression of its downstream target genes. Two additional p53 family members, p73 and p51/p63, were recently identified and characterized. Although the three family members share some similarities in transcription activation and apoptosis induction, each of them appears to play a distinct role in development and tumor suppression. We have previously identified a nuclear protein, p53CP (p53 competing protein), that is not p53 but binds to the p53 consensus sequence. Here we report the partial purification of p53CP from HeLa cells by ammonium sulfate precipitation, followed by a series of chromatography steps through heparin-agarose, Mono S ion exchange and DNA affinity columns, coupled with a gel shift assay. Although p53CP activity is readily detectable in HeLa cells by gel shift assay, only a trace amount of p53CP protein was partially purified, which was not sufficient for direct protein sequencing. Using a monoclonal antibody (4A4) specific for all p51/p63 isoforms or a polyclonal antibody (N-18) recognizing the N-terminus-containing p51/p63 isoforms we detected a significant enrichment of p51/p63 protein in p53CP-containing fractions following each step of purification. Significantly, p51/p63 was detected only in the DNA affinity column fractions that contain p53CP activity. Thus, p53CP appears to be p51/p63, the third member of the p53 gene family.

Blotting, Western↗

PubMed2001

Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein.

Cerebral ischemia resulting from a disruption of blood flow to the brain initiates a cascade of events that causes neuron death and leads to neurologic dysfunction. Reactive oxygen species are thought, at least in part, to mediate this disease process. The authors recently cloned and characterized an antioxidant protein, SAG (sensitive to apoptosis gene), that is redox inducible and protects cells from apoptosis induced by redox agents in a number of in vitro cell model systems. This study reports a neuroprotective role of SAG in ischemia/reperfusion-induced brain injury in an in vivo mouse model. SAG was expressed at a low level in brain tissue and was inducible after middle cerebral artery occlusion with peak expression at 6 to 12 hours. At the cellular level, SAG was mainly expressed in the cytoplasm of neurons and astrocytes, revealed by double immunofluorescence. An injection of recombinant adenoviral vector carrying human SAG into mouse brain produced an overexpression of SAG protein in the injected areas. Transduction of AdCMVSAG (wild-type), but not AdCMVmSAG (mutant), nor the AdCMVlacZ control, protected brain cells from ischemic brain injury, as evidenced by significant reduction of the infarct areas where SAG was highly expressed. The result suggests a rather specific protective role of SAG in the current in vivo model. Mechanistically, SAG overexpression decreased reactive oxygen species production and reduced the number of apoptotic cells in the ischemic areas. Thus, antioxidant SAG appears to protect against reactive oxygen species-induced brain damage in mice. Identification of SAG as a neuroprotective molecule could lead to potential stroke therapies.

Adenoviridae↗

PubMed2001

Use of reverse transcriptase polymerase chain reaction for diagnosis and staging of alveolar rhabdomyosarcoma, Ewing sarcoma family of tumors, and desmoplastic small round cell tumor.

PURPOSE: To compare the use of reverse transcriptase polymerase chain reaction (RT-PCR) with that of morphology-based methods for diagnosis, staging, and detection of metastatic disease in pediatric alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma family of tumors (ESFT), and desmoplastic small round cell tumors (DSRCT). MATERIALS AND METHODS: RT-PCR assays for the EWS-FLII, EWS-ERG, PAX3-FKHR, PAX7-FKHR, and EWS-WTI fusion transcripts were performed on RNA extracted from the primary tumor tissue, bone marrow, and body fluids obtained at initial presentation and relapse. Molecular findings were compared with original histologic diagnoses and results of staging procedures. RESULTS: Eighty-eight samples from 47 patients with ARMS (n = 13), ESFT (n = 31), or DSRCT (n = 3) were analyzed. The detection rate of metastatic disease was significantly higher with RT-PCR (95%) as compared with the morphologic methods (70%) for the three pediatric sarcomas studied. In primary tumors with characteristic fusion transcript, RT-PCR was positive in all cases with morphologic evidence of metastatic disease. Moreover, in six patients (3 with ARMS, 2 with DSRCT, and 1 with ESFT) with metastatic disease, micrometastases in bone marrow (4) and other sites (2) were detected by RT-PCR alone. Importantly, none of the patients with localized disease diagnosed had micrometastases detected by RT-PCR in bone marrow. CONCLUSIONS: The high sensitivity and specificity of RT-PCR for the characteristic fusion transcripts of pediatric sarcomas make it an ideal method to aid in the routine staging of these patients. In addition, the 100% sensitivity of RT-PCR in detection of micrometastasis makes it useful for follow-up and detection of minimal residual disease. However, the clinical significance of molecularly-detectable disease remains unknown. Further studies should aim to elucidate the therapeutic and prognostic implications of micrometastases detected by RT-PCR alone.

Biomarkers, Tumor↗

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

Volume of mastoid pneumatization: three-dimensional reconstruction with ultrahigh-resolution computed tomography.

The volume of the mastoid air cell system was measured in 69 patients with normal middle ears. All patients underwent axial ultrahigh-resolution computed tomography. Mastoid pneumatization was marked on each axial slice, and 3-dimensional reconstruction was performed. The volumes were measured with a volumetric algorithm. A polyethylene tubing phantom with a density similar to that of bone on computed tomography was devised. The polyethylene tubing was tied in a particular fashion so as to create interconnecting air spaces with a known volume. The phantom was scanned with the imaging parameters used for scanning the temporal bone. The air in the tubing was marked, and 3-dimensional reconstruction for the marked phantom air was performed. The volume of the interconnecting air spaces was measured and found to be identical to its known volume, thereby verifying the accuracy of the method used. The mean mastoid volume was 6.61 cm3. The smallest volume measured was 1.3 cm3, and the largest was 12.7 cm3. The importance of this technique lies in its high accuracy, ease of use, and ability to directly correlate mastoid size and clinical findings.

Adult↗