Biomedical subjects

A Shih

Publications and source records attributed to A Shih.

At least 19 recordsLinked to original sources

PubMed2007

DNA-PK phosphorylation sites on Oct-1 promote cell survival following DNA damage.

Octamer transcription factor-1 (Oct-1) has recently been shown to function as a stress sensor that promotes cell survival subsequent to DNA damage. Here, we show that the survival signal imparted by Oct-1 following exposure to ionizing radiation (IR) is dependent upon DNA-dependent protein kinase (DNA-PK)-dependent phosphorylation of a cluster of 13 specific ser/thr residues within the N-terminal transcriptional regulatory domain of Oct-1. Although IR treatment did not affect the recruitment of Oct-1 to the histone H2B promoter, the recruitment of RNA polymerase II, TATA-binding protein and histone H4 acetylation were strongly reduced, consistent with a decrease in Oct-1 transcriptional regulatory potential following IR exposure. Ser/Thr-Ala substitution of 13 sites present in Oct-1 transcriptional regulatory domain eliminated Oct-1 phosphorylation subsequent to IR exposure. Further, these substitutions prevented Oct-1 from rescuing the survival of IR-treated Oct-1-/- murine embryonic fibroblasts, providing a direct link between DNA-PK-dependent phosphorylation and the contribution of Oct-1 to cell survival. These results implicate Oct-1 as a primary effector in a DNA-PK-dependent cell survival pathway that is activated by double-stranded DNA breaks.

3T3 Cells↗

PubMed2001

Thyroid hormone promotes serine phosphorylation of p53 by mitogen-activated protein kinase.

L-Thyroxine (T(4)) nongenomically promotes association of mitogen-activated protein kinase (MAPK) and thyroid hormone receptor TRbeta1 (TR) in the cell nucleus, leading to serine phosphorylation of the receptor. The oncogene suppressor protein, p53, is serine phosphorylated by several kinases and is known to interact with TRbeta1. We studied whether association of p53 and TR is modulated by T(4) and involves serine phosphorylation of p53 by MAPK. TR-replete 293T human kidney cells were incubated with a physiological concentration of T(4) for 10-90 min. Nuclear fractions were immunoprecipitated and the resulting proteins separated and immunoblotted for co-immunoprecipitated proteins. Activated MAPK immunoprecipitates of nuclei from T(4)-treated cells accumulated p53 in a time-dependent manner; T(4) and T(4)-agarose were more effective than T(3). T(4)-induced nuclear complexing of p53 and MAPK was inhibited by PD 98059 (PD) and U0126, two MAPK kinase (MEK) inhibitors, and was absent in cells treated with MEK antisense oligonucleotide and in dominant negative Ras cells. T(4) also caused nuclear co-immunoprecipitation of TRbeta1 and p53, an effect also inhibited by PD. Nuclear complexing of p53 and MAPK also occurred in HeLa cells, which lack functional TR. Constitutively activated MAPK caused phosphorylation of a recombinant p53-GST fusion protein in vitro; thus, p53 is a substrate for MAPK. An indicator of p53 transcriptional activity, accumulation of the immediate-early gene product, c-Jun, was inhibited by T(4). This T(4) effect was reversed by PD, indicating that the transcriptional activity of p53 was altered by T(4)-directed MAPK-p53 interaction.

3T3 Cells↗

PubMed2001

Effect of stent on radiation dosimetry in an in-stent restenosis model.

Purpose: Intravascular brachytherapy is the leading modality being evaluated for treatment of in-stent restenosis. Stent struts may have an effect on the dose distributions of various radiation sources. We evaluated dosimetry in a stented coronary artery model using a variety of beta and gamma sources and stent materials.Methods: We determined the dose distributions with and without stent in the in-stent restenosis model. Three beta-particle emitting radionuclides, 90Y (2.3 MeV), 144Pr (3.0 MeV), and 106Rh (3.5 MeV), and two gamma-ray emitters, 192Ir (380 keV) and 125I (30 keV), were studied. Stent materials included stainless steel, nitinol, and tantalum. Monte Carlo dose calculations were performed in a stent model of multiple stent struts placed at 1.5 mm from the source. Isodose curves were generated and the ratios of dose rates with and without stent, the stent factors, were evaluated. A stent factor of greater or less than unity represents dose enhancement or reduction in the presence of a stent.Results: For the three beta radionuclides, dose reduction was found on the adventitial side of the stent strut and dose enhancement was noted on the luminal side. On the luminal side, the maximum dose enhancement ranges from 7% to 29%, and the dose reduction on the adventitial side ranges from 13% to 43%. Both the reduction and enhancement effects were most pronounced for the high atomic number material, tantalum. For a given stent material, the dose reduction and enhancement are similar for the three beta radionuclides. For the gamma sources, the stent had no effect for the high-energy 192Ir, but for the low-energy 125I, drastic dose reduction on the adventitial side was observed (up to 86% for tantalum stent), and about 10% dose enhancement on the luminal side was also noted. The dose reduction with 125I was more pronounced than that seen with the beta sources.Conclusions: The presence of stent struts significantly affects dose distributions of 90Y, 106Rh, 144Pr, and 125I. The maximum dose reduction can be as much as 86%. 192Ir was unaffected. These factors need to be considered in choosing radionuclides and dose prescriptions in treating in-stent restenosis.

Journal Article↗

PubMed2001

Intravesical Escherichia coli lipopolysaccharide stimulates an increase in bladder nerve growth factor.

OBJECTIVE: To investigate the effects of the intravesical instillation of Escherichia coli lipopolysaccharide (LPS) on nerve growth factor (NGF, which may mediate the pain associated with inflammation) protein and mRNA in the bladders of mice. MATERIALS AND METHODS: E. coli LPS was instilled into the bladders of female mice; the whole-bladder NGF content was then determined by an enzyme-linked immunosorbent assay and the NGF mRNA content of the bladder determined by semiquantitative reverse transcription-polymerase chain reaction. Bladder NGF was also evaluated by immunohistochemistry in some of the mice. RESULTS: LPS stimulated a significant increase in bladder NGF 90 min after instillation, but bladder NGF content was significantly less than that in bladders of control mice 3 and 7 h after LPS instillation. Twenty-four hours after the intravesical infusion of saline or LPS, there was no difference in NGF content in bladders from saline or LPS-infused mice. Immunohistochemistry confirmed the presence of increased NGF in the mucosa of detrusor from bladders 90 min after LPS instillation. Bladder NGF mRNA increased more slowly in response to LPS, and 7 and 24 h after LPS instillation the relative abundance of NGF mRNA was 1.5 and 2.0 times greater in LPS-infused bladders, respectively. CONCLUSIONS: E. coli LPS can stimulate increased NGF message and protein in the bladder. The increase in NGF protein preceded the increase in mRNA, suggesting that this increase was not the result of gene transcription. It is possible that NGF participates in the pathogenesis of pain associated with bacterial cystitis.

Administration, Intravesical↗

PubMed2001

Severe anemia.

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Journal Article↗

PubMed2000

Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR.

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.

Cell Line↗

PubMed2000

Broad specificity of GDNF family receptors GFRalpha1 and GFRalpha2 for GDNF and NTN in neurons and transfected cells.

The glial cell line-derived neurotrophic factor (GDNF) family of ligands binds to lipid anchored proteins termed GDNF family receptor (GFR)alphas, and then activates the RET receptor tyrosine kinase, by ligand GFRalpha. The binding of soluble GFRalphas to transfected cells suggested that different GFRalphas were dedicated to particular ligands, with GDNF acting primarily or entirely through GFRalpha1, and neurturin (NTN), through GFRalpha2. More recent evidence has suggested the possibility of cross-talk between these ligands and the two receptors. We examined here whether crosstalk between the GDNF ligands and the GFRalphas is biologically relevant, using midbrain dopaminergic, and parasympathetic, submandibular gland neurons. By biochemical and genetic addition and/or deletion of GFRalpha1 and 2, we show that in both neuronal cell types, robust biological activities of GDNF or NTN can be mediated by either GFRalpha1 or GFRalpha2, although GDNF is slightly more potent in dopaminergic (DA) neurons which normally express GFRalpha1, and NTN in submandibular neurons which normally express GFRalpha2. Throughout the body, GDNF and NTN are likely to have important biological actions on both GFRalpha1- and GFRalpha2-expressing cells.

Animals↗

PubMed2000

CC-chemokine receptor 5 polymorphism and age of onset in familial multiple sclerosis. Multiple Sclerosis Genetics Group.

Multiple sclerosis (MS) is a common disease of the central nervous system characterized by myelin loss and progressive neurological dysfunction. An underlying genetic susceptibility plays a clear role in the etiology of MS, likely acting in concert with an undefined environmental exposure. Full-genome screenings in multiplex MS families have identified several susceptibility regions, supporting a polygenic model for MS. Among these regions, evidence for weak linkage was observed at 3p/3cen suggesting the presence of an MS gene(s) of modest effect. Encoded here are two chemokine receptors, CCR5 and CCR2B. We examined the chromosome 3p21-24 region in 125 MS families (322 total affecteds and 200 affected sib-pairs), and performed genetic analyses of CCR5 and CCR2B loci and two nearby markers (D3S1289 and D3S1300) using both linkage- and association-based tests. No evidence of linkage to MS was observed for any of the tested markers. Affected relative-pair (SimIBD) and sib-pair analyses (ASPEX), and association testing (sib-TDT) for each locus were also not significant. However, age of onset was approximately 3 years later in patients carrying the CCR5delta32 deletion (P=0.018 after controlling for gender effects). Thus, chemokine receptor expression may be associated with differential disease onset in a subset of patients, and may provide a therapeutic target to modulate inflammatory demyelination.

Age of Onset↗

PubMed2000

The function of PML in p53-dependent apoptosis.

The PML gene of acute promyelocytic leukaemia (APL) encodes a growth- and tumour-suppresor protein that is essential for several apoptotic signals. The mechanisms by which PML exerts its pro-apoptotic function are still unknown. Here we show that PML acts as a transcriptional co-activator with p53. PML physically interacts with p53 both in vitro and in vivo and co-localizes with p53 in the PML nuclear body (PML-NB). The co-activatory role of PML depends on its ability to localize in the PML-NB. p53-dependent, DNA-damage-induced apoptosis, transcriptional activation by p53, the DNA-binding ability of p53, and the induction of p53 target genes such as Bax and p21 upon gamma-irradiation are all impaired in PML-/- primary cells. These results define a new PML-dependent, p53-regulatory pathway for apoptosis and shed new light on the function of PML in tumour suppression.

Animals↗

PubMed1999

Regulation of learning by EphA receptors: a protein targeting study.

EphA family receptor tyrosine kinases and their ephrin-A ligands are involved in patterning axonal connections during brain development, but until now a role for these molecules in the mature brain had not been elucidated. Here, we show that both the EphA5 receptor and its ephrin-A ligands (2 and 5) are expressed in the adult mouse hippocampus, and the EphA5 protein is present in a phosphorylated form. Because there are no pharmacological agents available for EphA receptors, we designed recombinant immunoadhesins that specifically bind to the receptor binding site of the ephrin-A ligand (antagonist) or the ligand binding site of the EphA receptor (agonist) and thus target EphA function. We demonstrate that intrahippocampal infusion of an EphA antagonist immunoadhesin leads to impaired performance in two behavioral paradigms, T-maze spontaneous alternation and context-dependent fear conditioning, sensitive to hippocampal function, whereas activation of EphA by infusion of an agonist immunoadhesin results in enhanced performance on these tasks. Because the two behavioral tasks have different motivational, perceptual, and motor requirements, we infer the changes were not caused by these performance factors but rather to cognitive alterations. We also find bidirectional changes in gene expression and in electrophysiological measures of synaptic efficacy that correlate with the behavioral results. Thus, EphA receptors and their ligands are implicated as mediators of plasticity in the adult mammalian brain.

Animals↗

PubMed1999

Autoreactivity to myelin antigens: myelin/oligodendrocyte glycoprotein is a prevalent autoantigen.

Autoreactive T cells specific for myelin antigens are thought to play a role in the pathogenesis of multiple sclerosis (MS). We compared T cell proliferative responses in peripheral blood following challenge in vitro with myelin/oligodendrocyte glycoprotein (recombinant protein, rMOG), myelin basic protein (MBP) and proteolipid apoprotein (PLP) in 50 patients with MS and 40 healthy controls. T cell reactivity against rMOG (defined by a specific stimulation index of 2.5 or greater) was present in 13 (26%) MS patients and 12 (30%) healthy controls and was MHC-restricted, as anti-MHC class II antibodies abolished all proliferative responses. By contrast, reactivity against PLP was present in only one (2%) MS patient and six (15%) controls, and no reactivity against MBP was found in any subject. Thus, by the criteria of the present study, an increased reactivity of circulating T cells to MOG is present to a similar degree in healthy individuals and in patients with MS. This finding raises the possibility that additional factors contribute to the pathogenicity of these autoreactive T cell populations in demyelinating disorders of the central nervous system.

Adult↗

PubMed1999

Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells.

We have examined the effects of l-thyroxine (T4) on the activation of signal transducer and activator of transcription 3 (STAT3) and on the STAT3-dependent induction of c-Fos expression by epidermal growth factor (EGF). T4, at a physiological concentration of 100 nM, caused tyrosine phosphorylation and nuclear translocation (i.e. activation) of STAT3 in HeLa cells in as little as 10-20 min. Activation by T4 of STAT3 was maximal at 30 min (15+/-4-fold enhancement; mean+/-S.E.M.) in 18 experiments. This effect was reproduced by T4-agarose (100 nM) and blocked by CGP 41251, genistein, PD 98059 and geldanamycin, inhibitors of protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK) kinase and Raf-1 respectively. Tyrosine-phosphorylated MAPK also appeared in nuclear fractions within 10 min of treatment with T4. In the nuclear fraction of T4-treated cells, MAPK immunoprecipitate also contained STAT3. The actions of T4 were similar in HeLa and CV-1 cells, which lack thyroid hormone receptor (TR), and in TR-replete skin fibroblasts (BG-9). T4 also potentiated the EGF-induced nuclear translocation of activated STAT1alpha and STAT3 and enhanced the EGF-stimulated expression of c-Fos. Hormone potentiation of EGF-induced signal transduction and c-Fos expression was inhibited by CGP 41251, geldanamycin and PD 98059. Therefore the non-genomically induced activation by T4 of STAT3, and the potentiation of EGF by T4, require activities of PKC, PTK and an intact MAPK pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Expression and prognostic significance of proliferating cell nuclear antigen and Ki-67 in malignant ovarian germ cell tumors.

BACKGROUND: Both proliferating cell nuclear antigen (PCNA) and Ki-67 are proliferative markers known to correlate with the cell proliferative state. The aim of this study was to evaluate the usefulness of PCNA and Ki-67 immunoreactivity in the assessment of clinicopathologic features and prognosis in patients with malignant ovarian germ cell tumors. METHODS: Thirty-one patients with surgically resected malignant ovarian germ cell tumors were investigated. The clinicopathologic features and survival data of these patients were recorded. Immunohistochemical staining with monoclonal antibodies (PC 10 for PCNA, and MIB-1 for Ki-67) were performed on paraffin embedded tissue from each patient. The correlation of the immunoreactivity of these two markers with the clinicopathologic features and prognosis were studied. RESULTS: All of the tumors were positive for PCNA and Ki-67, but the intensity of expression varied widely. The immunoreactivity in each tumor was also heterogeneous. The scoring of PCNA and Ki-67 was determined by a semiquantitative method. Both advanced tumor stage (stages III and IV) and high PCNA score (scores 3 and 4) indicated a poorer prognosis for survival than did early stage (stages I and II) and a low PCNA score (scores 1 and 2) (p = 0.017 and p = 0.008, respectively). In addition, the proportion of tumor relapse and tumor-induced death was more accurately predicted by PCNA and Ki-67 scoring than by tumor staging (chi 2 = 0.3159, chi 2 = 0.7186 and chi 2 = 1.9689, respectively). CONCLUSIONS: PCNA and Ki-67 proliferative markers appear promising to differentiate patients into low- and high-risk groups. In the presence of a high PCNA or Ki-67 score, aggressive postoperative chemotherapy is warranted, even if the disease is in a very early stage.

Adolescent↗

PubMed1998

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.

Animals↗

PubMed1998

Experimental System for X-ray Cone-Beam Microtomography

: A laboratory test of X-ray tomography employing a diverging beam of X-rays rather than the usual parallel X-ray beam is described. We chose to test and demonstrate the advantages of divergent beam tomography by imaging an extracted juvenile human premolar using an ordinary dental X-ray source and a cooled CCD camera. Experiments with a three-piece cover-glass sample and with the human tooth demonstrated that three-dimensional reconstruction can be achieved at 34 µm per pixel resolution employing an X-ray tube spot 800 µm in its smallest direction without requiring close contact with the fluorescent screen. Reconstruction of a 256 x 256 pixel single-plane image from 100 projection images took only 45 sec on a personal computer with a Pentium 166 MHz processor. We have also demonstrated a volume reconstruction of 256 x 256 x 256 voxels from the data. Successful extension of this work to submicrometer projection X-ray microscopy is predicted. Improved resolution of medical tomography is another possible application.

Journal Article↗

PubMed1998

The ethics of tailoring the patient's chart.

Because psychiatrists cannot include everything they observe and everything the patient says in the medical chart, they must select and tailor what goes into the chart. They should tailor the chart to focus on what is significant for the diagnosis and treatment of the patient. However, sometimes they tailor the chart for other purposes: to ensure that managed care will cover continued hospitalization, to protect themselves against malpractice claims, to secure a civil commitment, or to obtain a long-term placement for the patient. The authors of this paper present and analyze four cases in which psychiatrists tailor charts for these purposes. They discuss whether each psychiatrist's actions are ethically justified and consider whether tailoring the chart is a deceptive practice. In each case, they present reasons for and against this practice and suggest truthful alternatives designed to improve patient care, preserve social trust in the profession of psychiatry, and challenge serious failings in the health care system.

Beneficence↗

PubMed1997

Mutational analysis of the BRCA1 tumor suppressor gene in endometrial carcinoma.

OBJECTIVE: To investigate whether alteration of BRCA1 tumor suppressor gene occurs in sporadic endometrial carcinomas. METHODS: Genomic DNAs were prepared from 33 consecutively collected endometrial carcinoma tissues for BRCA1 mutational analysis. To screen for BRCA1 mutation, polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) technique were used with 41 overlapping PCR primer pairs for the 23 coding exons of BRCA1. Tumors that demonstrated SSCP variants were further subjected to direct DNA sequencing in the appropriate exons to identify the DNA alteration. RESULTS: In addition to detecting a previously described polymorphism in exon 11, single strand conformation polymorphism analysis of the 33 endometrial cancers identified 3 tumors with mobility shifts. Two tumors shifted in exon 3 and showed the same pattern of band shift. The other tumor shifted in exon 9. DNA sequencing revealed sequence alterations in the 3 tumors; all appeared heterozygous. In the 2 tumors shifted in exon 3, the sequence alteration caused no amino acid change and was consistent with an infrequent silent polymorphism. In the third tumor, a missense alteration at codon 191 was detected and was recognized as germline in origin. CONCLUSIONS: Because a normal allele of BRCA1 was retained in the tumor where a germline missense alteration was detected, the heterozygous DNA alteration should not be cancer predisposing in terms of the two-hit model for inactivation of the tumor suppressor gene. We conclude that mutation of BRCA1 may not be involved in the development of sporadic endometrial cancer.

DNA Mutational Analysis↗

PubMed1997

NGF binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different stages of development.

To clarify the role of the common neurotrophin receptor p75 in modulating the survival response of sensory and sympathetic neurons to NGF at different stages of development, we compared the actions of wild-type NGF with a mutated NGF protein that binds normally to TrkA, the NGF receptor tyrosine kinase, but has greatly reduced binding to p75. At saturating concentrations, the NGF mutant promoted the survival of similar numbers of trigeminal sensory and sympathetic neurons as NGF. At subsaturating concentrations, the NGF mutant was less effective than wild-type NGF in promoting the survival of embryonic sensory neurons and postnatal sympathetic neurons but was equally effective as wild-type NGF in promoting the survival of embryonic sympathetic neurons. Whereas the levels of trkA and p75 were similar in embryonic sensory neurons and postnatal sympathetic neurons, the level of p75 was significantly lower than that of trkA in embryonic sympathetic neurons. These results indicate that binding of NGF to p75 enhances the sensitivity of NGF-dependent neurons to NGF at stages in their development when the levels of p75 and TrkA are similar.

Animals↗