Hypoxia-inducible factor 1: from molecular biology to cardiopulmonary physiology.
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Animals↗
Biomedical subjects
S Leung
Publications and source records attributed to S Leung.
Effect of anticardiolipin/beta2-glycoprotein I complexes on production of thromboxane A2 by platelets from patients with the antiphospholipid syndrome.
OBJECTIVE: Antiphospholipid antibodies (aPL) reactive with anionic phospholipids and beta2-glycoprotein I (beta2-GPI) are found in the sera of patients with autoimmune diseases. Clinically, aPL/beta2-GPI complexes are associated with arterial and venous thrombosis, fetal loss, and thrombocytopenia, i.e., the antiphospholipid syndrome (APS). The mechanism of thrombosis is not known. We hypothesized that aPL/beta2-GPI complexes could perturb the platelet membrane and increase production of thromboxane A2 (TXA2, a proaggregatory prostanoid). METHODS: We isolated an IgG fraction containing anticardiolipin antibody (aCL) and the plasma cofactor, beta2-GPI, from a patient with a high titer of aCL and thrombotic cerebrovascular disease. We then examined the effect of aCL, beta2-GPI, and the aCL/beta2-GPI complex on platelet TXB2 (a stable metabolite of TXA2) biosynthesis in vitro from 7 healthy controls. We also measured in vitro platelet TXB2 biosynthesis in 7 patients with APS and in 8 controls. RESULTS: We found: (1) significantly increased in vitro TXB2 production by platelets from controls after incubation with aCL/beta2-GPI complexes; (2) moderately increased TXB2 production by aCL alone; (3) no increase in TXB2 production by beta2-GPI alone; and (4) significantly increased 11-dehydro-TXB2, a metabolite of TXB2 production in vivo, in the urine of patients with APS compared with controls. CONCLUSION: These data suggest that aCL/beta2-GPI complexes play a role in activating platelets to produce TXA2, which could contribute to the prothrombotic state found in patients with APS.
Adult↗
Characterization of a teleost insulin-like growth factor II (IGF-II) gene: evidence for promoter CCAAT/enhancer-binding protein (C/EBP) sites, and the presence of hepatic C/EBP.
Insulin-like growth factor I (IGF-I) and IGF-II, acting under the regulatory control of growth hormone, are the principal mediators of vertebrate growth. We have previously demonstrated that like humans, but unlike rodents, rainbow trout maintain high hepatic IGF-II messenger RNA levels into adulthood. Here we describe a rainbow trout IGF-II gene with a proximal promoter that contains two CCAAT/enhancer-binding protein (C/EBP) binding sites (TCBS1 and TCBS2). Nuclear proteins corresponding in size to rat C/EBPalpha and C/EBPbeta were detected on Western immunoblots of growth-hormone-treated and mock-treated trout liver extracts. Electrophoretic mobility shift assay of these nuclear extracts further suggests the presence of C/EBPs in trout liver and confirms the ability of TCBS1 to form a complex with trout liver nuclear proteins that is identical in mobility and specificity to that formed by a mammalian consensus CBS construct. In both Western blot and mobility assay results, the growth-hormone-treated trout livers appeared to have a greater accumulation of C/EBP, suggesting a molecular mechanism by which growth hormone can influence the level of serum IGF-II.
Animals↗
Prognostic significance of chemotherapy dosage characteristics in children with osteogenic sarcoma.
High-dose methotrexate (HDMTX), adriamycin (ADR), and cisplatinum (CDDP) are effective agents in the treatment of osteogenic sarcoma (OS). Individual patient doses are determined by prior successful clinical trials but may be reduced due to ongoing toxicities. It is unknown whether individualized dose reductions of these drugs influence disease outcome. We retrospectively studied 27 consecutively enrolled children treated with HDMTX, ADR, and CDDP as adjuvant chemotherapy for OS, for correlations between disease outcome and several characteristics of drug dosings the cumulative MTX, CDDP, and ADR doses administered and the mean MTX blood levels for each patient. With a median follow-up of 59 months, the actuarial overall and disease-free survival rates were 70% and 59%, respectively. Factors which favorably influenced prognosis on univariate analysis were a cumulative ADR dose of > 300 mg/m2 (P = 0.0002) and a cumulative MTX dose > 114 gm/m2 (P = 0.0048). By multivariate analysis only the cumulative ADR dose > 300 mg/m2 retained prognostic value. We conclude that adjuvant chemotherapy dosages may need to be adjusted for therapeutic efficacy in addition to adjustments made for toxicity. The effect of different cumulative HDMTX and ADR dosages on prognosis in osteosarcoma patients needs to be evaluated in a prospective trial.
Adolescent↗
Structural and functional analysis of hypoxia-inducible factor 1.
Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including those encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1. Hypoxia response elements from these genes consist of a HIF-1 binding site (that contains the core sequence 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding site. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1, whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HIF-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is mediated by the basic domain, and that HIF-1 alpha contains a carboxyl-terminal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-type hypoxia response element resulted in increased transcription in non-hypoxic cells and a superinduction of transcription in hypoxic cells, whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF-1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding activity increased exponentially as cellular oxygen tension decreased, with maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels were induced in vivo when animals were subjected to anemia or hypoxia. The HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chromosome 12.
Animals↗
Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2.
Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineered with CH1 domain-appended carbohydrates (CHOs) were generated to facilitate site-specific conjugation of radionuclides and anti-cancer drugs to antibodies. Such site-specific conjugation may minimize the incidence of immunoreactivity perturbation as is often observed with random conjugation. Since the compositions and structures of CHOs are important in determining the chemistry, efficiency, and extent of conjugation, the sequences of the CH1-appended CHOs were determined by exoglycosidase digestions and fluorophore-assisted CHO electrophoresis (FACE). The CHO species attached at HCN1 and HCN5 sites in hLL2HCN1 and hLL2HCN5, respectively, were distinct from each other, heterogeneous, and extensively processed. All of these CHOs were core-fucosylated complex-type oligosaccharides and contained Gal (galactose) and GlcNAc (N-acetylglucosamine) residues in the outer branches. Some of the outer branches were composed of Gal alpha1-3Galbeta1-4GlcNAc structure, also known as alpha-galactosyl epitope. Most of the CHOs were sialylated. While all HCN1-CHOs were biantennary, the majority of HCN5-CHOs (>60%) were triantennary. The CH1-appended CHOs have favorable structural characteristics suitable for site-specific conjugation. For efficient conjugation of large drug complexes, hLL2HCN5 is preferable to hLL2HCN1 because the attached CHO is larger in size and more remotely positioned from the V region. The effects of the alpha-galactosyl epitope found in these CHOs on the immunological properties of the immunoconjugates as efficient cancer diagnostics and therapeutics are being studied.
Antibodies, Monoclonal↗
Functional subdomains of STAT2 required for preassociation with the alpha interferon receptor and for signaling.
Two members of the STAT signal transducer and activator of transcription family, STAT1 and STAT2, are rapidly phosphorylated on tyrosine in response to alpha interferon (IFN-alpha). Previous work showed that in the mutant human cell line U6A, which lacks STAT2 and is completely defective in IFN-alpha signaling, the phosphorylation of STAT1 is very weak, revealing that activation of STAT1 depends on STAT2. We now find that STAT2 binds to the cytoplasmic domain of the IFNAR2c (also known as IFNAR2-2) subunit of the IFN-alpha receptor in extracts of untreated cells. STAT1 also binds but only when STAT2 is present. The activities of chimeric STAT2-STAT1 proteins were assayed in U6A cells to define regions required for IFN-alpha signaling. Previous work showed that a point mutation in the Src homology 2 (SH2) domain prevents STAT2 from binding to phosphotyrosine 466 of the IFNAR1 subunit of the activated receptor. However, we now find that the entire SH2 domain of STAT2 can be replaced by that of STAT1 without loss of function, revealing that other regions of STAT2 are required for its specific interaction with the receptor. A chimeric protein, in which the N-terminal third of STAT2 has replaced the corresponding region of STAT1, did preassociate with the IFNAR2c subunit of the receptor, became phosphorylated when IFN-alpha was added, and supported the phosphorylation of endogenous STAT1. These results are consistent with a model in which STAT2 and STAT1 are prebound to the IFNAR2c subunit of the resting receptor. Upon activation, the IFNAR1 subunit is phosphorylated on Tyr-466, allowing the SH2 domain of STAT2 to bind to it; this is followed by the sequential phosphorylation of STAT2 and STAT1.
Amino Acid Sequence↗
An assessment of DNA contamination risks in New York City Medical Examiner facilities.
DNA evidence holds an important position in criminal investigations and proceedings. The polymerase chain reaction (PCR) is often utilized to amplify polymorphic regions of DNA which are subsequently typed to produce distinct genotypes. The sensitivity of PCR-based techniques provides a major advantage over other DNA or conventional serological typing systems. Samples containing quantities of DNA in the picogram range are often typed. However, the unprecedented sensitivity of PCR is often cited as a criticism. One concern is that the interpretation of PCR typing can be affected by DNA contaminants from foreign sources. In this report, the level of DNA contamination in New York City Medical Examiner facilities and its potential affects on HLA-DQA1 typing were assessed. Two related studies conducted over a five week period measured and typed HLA-DQA1 from accumulated DNA on autopsy room and Forensic DNA Laboratory structures. The potential for DNA contamination from airborne sources was also evaluated in the autopsy suites. This study demonstrated the presence of small amounts of DNA on structural surfaces, but little evidence of airborne DNA contamination.
Air Pollutants↗
Comparison of analytical performance and biological variability of three bone resorption assays.
We have compared the analytical performance and biological variability of three commercially available bone resorption assays: Pyrilinks-D, Osteomark, and CrossLaps, for the measurement of urinary free deoxypyridinoline (Dpd), cross-linked N-telopeptides of type I collagen (NTx), and linear C-telopeptides of type I collagen (CTx), respectively. The intraassay and interassay CVs for precision of the Dpd and NTx assays were < 10% for analyte concentrations greater than the second calibrator (i.e., 3 nmol/L Dpd or 30 nmol bone collagen equivalents/L NTx). The CTx assay demonstrated poor precision for analyte concentration lower than the third calibrator (i.e., 200 micrograms/L). The NTx assay exhibited nonlinear recovery for sample dilutions prepared in buffer; however, this nonlinear recovery could be corrected for sample dilutions made in urine at a low analyte concentration. Supplement recoveries of each of the three assays were within 100% +/- 10% on average. All three analytes showed stability through five freeze-thaw cycles. The mean day-to-day variations were 16% for Dpd, and 23% for both NTx and CTx. Similar diurnal rhythm was observed for all three assays on average, with the peak in the early morning and the nadir in the afternoon. Mean amplitude of the diurnal variation was 37% for Dpd and NTx, and 57% for CTx. Variations within the reference intervals for a healthy premenopausal population were 28% for Dpd, 57% for NTx, and 56% for CTx. Pyrilinks-D has demonstrated analytical precision and accuracy equal or superior to Osteomark and CrossLaps in all areas. Dpd exhibits the least biological variability day-to-day, within individuals across the diurnal cycle, and within a healthy premenopausal population.
Adult↗
Characterization of beta-R1, a gene that is selectively induced by interferon beta (IFN-beta) compared with IFN-alpha.
We report preliminary characterization of a gene designated beta-R1, which is selectively expressed in response to interferon beta (IFN-beta) compared with IFN-alpha. In human astrocytoma cells, beta-R1 was induced to an equivalent extent by 10 IU/mL IFN-beta or 2500 IU/mL IFN-alpha2. To address the mechanism of this differential response, we analyzed induction of the beta-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. beta-R1 was readily induced by IFN-beta in the parental 2fTGH cell line, but not by recombinant IFN-alpha2, IFN-alpha Con1, or a mixture of IFN-alpha subtypes. IFN-alpha8 induced beta-R1 weakly. beta-R1 was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-alpha and -beta receptor, also failed to express beta-R1. U1A cells are partially responsive to IFN-beta and IFN-alpha8 but lacked beta-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of beta-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-beta compared with IFN-alpha.
Astrocytoma↗
STATs find that hanging together can be stimulating.
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Animals↗
Formation of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene transcription by interferon-alpha.
An upstream inverted repeat (IR) element mediates transcriptional activation of the interferon response factor-1 gene (IRF-1) by interferon (IFN)-alpha and IFN-gamma. IFN-alpha and IFN-gamma fail to induce IRF-1 in cells that lack signal transducer and activator of transcription 1 (STAT1), and STAT1 homodimers bind to IR elements in extracts of IFN-alpha-treated cells. We now report that STAT2 also plays an important role in the IFN-alpha-mediated transcriptional activation of the IRF-1 gene. A new factor, most likely a STAT1-STAT2 heterodimer, was detected with an IR probe in extracts of IFN-alpha-treated cells. STAT1 and STAT2 are already known to combine with p48, a DNA-binding protein, to form IFN-stimulated gene factor 3 (ISGF3), which binds to IFN-stimulated response elements (ISREs) distinct from the IR of the IRF-1 gene. In extracts of U2A cells, which lack p48, STAT1-STAT2 heterodimers were still formed, indicating that they do not contain p48. We manipulated the intracellular levels of STAT1-STAT2 heterodimers and STAT1 homodimers to examine their roles in the induction of IRF-1 by IFN-alpha. Although both dimers can induce IRF-1 transcription, the heterodimers are more potent and thus may be the major activators in vivo. Deletion analysis reveals that the C-terminal domain of STAT2 is important for transcriptional activation mediated by both STAT1-STAT2 heterodimers and ISGF3.
Base Sequence↗
The relationship between age and karyotypic abnormalities in myelodysplastic syndromes.
We performed a retrospective analysis of 143 consecutive patients with myelodysplastic syndrome to study the possible relationship between patient age and cytogenetic findings in this disorder. There were 96 men and 47 women, with a mean age of 63 years. Eighty-six patients were 63 years old and above, and 57 patients were younger than 63 years of age. The distributions of the five FAB subtypes were comparable in both groups of patients, except for a higher percentage of refractory anemia with ringed sideroblasts in older patients. The incidences of cytogenetic abnormalities were similar in the two groups. However, the younger patients tended to have a higher frequency of involvement of chromosomes 5 or 7 than the elderly patients (p < 0.05). The implications of our findings in relation to the biology of myelodysplastic syndrome are discussed.
Adult↗
Pituitary adenoma presenting as sinonasal tumor: pitfalls in diagnosis.
Pituitary adenomas may cause significant difficulties in histological diagnosis when presenting in unusual sites either as extension from an intrasellar lesion or as ectopic tumor. Three such cases, involving the sinonasal tract are described herein, and the differential diagnoses are discussed. Two of them were invasive intrasellar macroadenomas that presented as unilateral nasal polyp, and one was an ectopic pituitary adenoma involving the sphenoid sinus. There was notable cellular atypia in two cases, with nuclear pleomorphism, giant cells, chromatin clumping, and distinct nucleoli, leading to serious consideration of the possibility of poorly differentiated carcinoma. In none of the cases was the diagnosis of pituitary adenoma suspected clinically. The clues to diagnosis were an endocrine growth pattern comprising tumor cells arranged in packets, ribbons, or rosettes, with prominent delicate vascularized stroma; a high index of suspicion; and immunohistochemical showing of neuroendocrine markers and pituitary hormones in the tumor cells. A correct diagnosis is important because in contrast to neuroendocrine carcinoma as a whole or to poorly differentiated carcinoma, pituitary neoplasms have a much more favorable prognosis and a low metastatic potential.
Adenoma↗
Isolation and purification of anticardiolipin antibody from plasma of a patient with antiphospholipid syndrome: induced generation of platelet thromboxane A2 synthesis.
Antiphospholipid antibodies, particularly anticardiolipin antibodies (aCL) are autoantibodies frequently detected in the serum of patients with systemic lupus erythematosus (SLE) and the primary antiphospholipid antibody syndrome (PAPS). These patients commonly suffer from thrombosis, recurrent fetal loss and thrombocytopenia. Since platelet aggregation is pivotal in the genesis of thrombosis, we tested the hypothesis that perturbation of platelet membrane by aCL/beta 2-glycoprotein (aCL/beta 2GP) complex could trigger the biosynthesis of TXA2, a proaggregatory metabolite of AA. The preincubation of 14C-arachidonic acid (14C-AA)-labeled platelet pellets (14C-PP) from normal individuals with aCL alone followed by incubation with thrombin, resulted in a moderate increase in platelet thromboxane B2 (14C-TXB2) biosynthesis when compared to controls (without aCL). Similar incubations with beta 2GP-I alone resulted in negligible 14C-TXB2 biosynthesis. In contrast, the preincubations of normal 14C-PP with aCL/beta 2GP-I complex resulted in marked thrombin-induced TXB2 biosynthesis, underscoring the requirement of beta 2GP-I in aCL-induced platelet TXB2 biosynthesis. Taken together, these results are consistent with the view that aCL/beta 2GP-I platelet interactions do play a role, at least in part, in platelet hyperactivity and thrombosis in antiphospholipid antibody syndrome.
Adult↗
Primary and recurrent colorectal cancer masquerading as gynaecological malignancy.
To make clinicians more aware of the phenomenon of primary and recurrent colorectal and anal carcinoma masquerading as primary gynaecological malignancy, we reviewed the records of 8 women referred to our gynaecological oncology unit with primary colorectal cancer (1), recurrent colorectal cancer (6) and primary anal cancer (1). Seven of these patients presented with abnormal vaginal bleeding or discharge. All patients had Papanicolaou smears performed; 7 were abnormal and 1 unsuitable for cytological assessment. None of the 6 patients with recurrent carcinoma had been previously treated with more than standard anterior or abdominoperineal resection; no radiotherapy had been given, and only 1 patient had received chemotherapy. These patients were treated in our gynaecological oncology unit for their recurrence by surgery and/or chemotherapy and/or irradiation. All 6 had further recurrences in the pelvis despite this aggressive therapy. Follow-up of colorectal cancer in women should involve gynaecological history, pelvirectal examination and Pap smear at each visit. Correct diagnosis of the colorectal origin of a genital tract tumour is made on careful history, examination and biopsy. An abnormal Pap smear may be the first indication of recurrent colorectal cancer in the cervix and vagina, although most patients ultimately present with abnormal vaginal bleeding. The presence of a tumour invading both cervix and posterior vaginal wall is suggestive of spread from a colorectal tumour compared to the more common lateral spread of a cervical primary.
Adult↗
Evaluation of a quality assurance program for quantitation of human immunodeficiency virus type 1 RNA in plasma by the AIDS Clinical Trials Group virology laboratories.
A number of quantitative assays have been developed by using amplification techniques to measure human immunodeficiency virus type 1 RNA in the plasma of infected individuals. The Virology Committee of the AIDS Clinical Trials Group in the Division of AIDS, National Institute of Allergy and Infectious Diseases, has established a quality assurance program (QAP) for quantitative assays of HIV-1 RNA levels in plasma. The primary objective of the QAP was to ascertain that a laboratory could maintain the precision required to have a 90% power to detect a fivefold difference in RNA copy number between two samples in the same batch. To achieve this goal, the QAP required an intra-assay standard deviation of no greater than 0.15 log10 RNA copies per ml. Panels for proficiency testing consisted of coded replicate samples and a common set of standards. To date, 41 laboratories have participated in the program and have used both commercial and in-house assays. We demonstrated that 65% of the laboratories were capable of attaining the necessary level of intra-assay precision. The fitted regressions indicated that the differences among laboratories that used the same kit were generally greater than the differences among population-average regressions for the kits themselves. The use of an external QAP and a common set of standards reduced differences both among laboratories that used the same kit and among laboratories that used different kits. Thus, use of a common set of standards across clinical trial protocols would allow for cross-protocol comparisons.
Clinical Protocols↗
Function of Stat2 protein in transcriptional activation by alpha interferon.
Alpha interferon (IFN-alpha)-induced transcriptional activation requires the induction of a complex of DNA-binding proteins, including tyrosine-phosphorylated Stat1 and Stat2, and of p48, a protein which is not phosphorylated on tyrosine and which comes from a separate family of DNA-binding proteins. The isolation and characterization of U6A cells, which lack Stat2, have allowed the introduction of normal and mutant forms of Stat2 so that various functions of the Stat2 protein can be examined. As reported earlier, Stat1, which is the second target of tyrosine phosphorylation in IFN-alpha-treated cells, is not phosphorylated in the absence of Stat2. We show that all mutations that block Stat2 phosphorylation also block Stat1 phosphorylation. These include not only the mutations of Y-690 and SH2 domain residues that are involved in tyrosine phosphorylation but also short deletions at the amino terminus of the protein. Two mutants of Stat2 that are not phosphorylated on tyrosine can act as dominant negative proteins in suppressing wild-type Stat2 phosphorylation, most likely by competition at the receptor-kinase interaction site(s). We also show that the COOH-terminal 50 amino acids are required for transcriptional activation in response to IFN-alpha. Mutants lacking these amino acids can be phosphorylated, form IFN-stimulated gene factor 3, and translocate to the nucleus but cannot stimulate IFN-alpha-dependent transcription. Seven acidic residues are present in the deleted COOH-terminal residues, but 24 acidic residues still remain in the 100 carboxy-terminal amino acids after deletion. Thus, transcriptional activation is unlikely to depend on acidic amino acids alone.
Amino Acid Sequence↗