Prevention of analytical false-positive increases of cardiac troponin I on the Stratus II analyzer.
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Autoanalysis↗
Biomedical subjects
B K De
Publications and source records attributed to B K De.
Primary pulmonary hypertension in non-cirrhotic portal fibrosis.
BACKGROUND: Primary pulmonary hypertension (PPH) has been reported in association with cirrhosis and extrahepatic portal venous obstruction; reports of PPH in noncirrhotic portal fibrosis (NCPF) are few. AIM: To evaluate pulmonary arterial pressure in patients with NCPF. METHODS: Twenty two patients with NCPF underwent hemodynamic studies for pulmonary arterial pressure after excluding secondary causes of pulmonary hypertension. Hemodynamic studies were carried out through the femoral route using 7F Swan-Ganz catheter. Splenoportal venography was done by percutaneous splenic puncture. RESULTS: The mean pulmonary arterial pressure was 12.9 +/- 3.1 mmHg with pulmonary capillary wedge pressure of 8.3 +/- 2.1 mmHg in 20 of 22 cases; in the remaining two cases, the corresponding pressures were 30 mmHg and 28 mmHg and 13 mmHg and 12 mmHg, respectively. CONCLUSION: Two of 22 patients with NCPF had PPH. PPH can thus develop without hepatocellular failure or recurrent embolization from portal axis thrombosis as has been described in cirrhosis.
Adult↗
Atmospheric transport of mosquitoes in northeast India.
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Animals↗
Haemochromatosis in a case of thalassaemia haemoglobin E disease presenting with cirrhosis of liver.
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Hemochromatosis↗
Problems and practical considerations in assessing accuracy with NIST SRM 909a: report of defective vials.
During an experimental period of 12 months in 1992-1993, while we were comparing the effectiveness of monthly vs quarterly use of the National Institute for Standards and Technology Standard Reference Material (NIST SRM) 909a as an accuracy material for the projected 30-year Fernald Medical Monitoring Program, we encountered three random defective vials with a glucose recovery of less than 30% of the NIST-assigned value. Analysis with five different multichannel instruments confirmed the original finding. Concomitant glucose recovery from adjacent vials was 97%-104%, as determined by using the same instruments, reagents, calibrators, and quality-control criteria on the same days. Recoveries of uric acid and cholesterol were also low (53-75% and 75-80%, respectively) in the three defective vials. Other analytes were unaffected. Studies to identify the cause of the defective vials were carried out with microbiological, electron microscopic, and biochemical techniques. When used for accuracy studies, each vial of NIST SRM 909a should have a concomitant check for glucose recovery to detect whether the vial is defective.
Chemistry, Clinical↗
Identification of a 100-kDa phosphoprotein in developing murine embryos as elongation factor 2.
Protein phosphorylation is a key regulatory mechanism for several functions. Although the complex control of organogenesis and growth most likely includes such mechanisms, few reports have examined protein phosphorylation in the developing mammal. The identification and characterization of mammalian embryonic phosphoproteins will allow a greater understanding of the regulation and mechanisms of developmental processes. Phosphorylation of the endogenous mouse proteins during development revealed a 100-kDa protein, located in the cytosolic fraction, to be the major substrate. The Ca(2+)-calmodulin kinase inhibitors, trifluorperazine and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, inhibited this phosphorylation. Inhibitors of protein kinase C (H-7)- and cAMP-dependent protein kinase, as well as the tyrosine kinase inhibitor, genistein, had no effect. One- and two-dimensional phosphoamino acid analysis indicated that phosphothreonine was the major phosphorylated amino acid. To determine the identity of this protein, the 100-kDa band was isolated and submitted for amino acid analysis and N-terminal sequencing. The N-terminal sequence Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, was identical to the N-terminal sequence of human, hamster and rat elongation factor 2 (EF-2). Western blotting analysis confirmed that the 100 kDa protein was EF-2. Our results of phosphorylated EF-2 in the developing mouse are in agreement with those reported in the avian embryo. However, our results differ in that phosphotyrosine detected in avian embryos could not be detected in murine embryos. This is the first report to demonstrate EF-2 in the developing mammalian embryo and its specific phosphorylation pattern. Our data suggest that the functional phosphoregulation of elongation factor 2 during protein synthesis in mammals is conserved from the developing embryo to the adult and thus emphasizes the importance of EF-2 in normal development and survival.
Amino Acid Sequence↗
Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system.
The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with infectious DNA from Autographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.
Amino Acid Sequence↗
Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease.
To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.
Antigens, Viral↗
Rapid and sensitive genotyping of Epstein-Barr virus using single-strand conformation polymorphism analysis of polymerase chain reaction products.
Two distinct wild-type Epstein-Barr virus (EBV) strains (A and B) that have significantly diverged at the two small RNA-encoding region (EBER) have been identified (Arrand et al., 1989). In order to test whether single-strand conformation polymorphism analysis (SSCP) would correlate with these sequence variations, we designed primer pairs specific for EBER-encoding regions for amplification of divergent sequences by polymerase chain reaction (PCR). The PCR-amplified products from six EBV-positive cell lines were analyzed by SSCP method, and the results were compared with the prototype strains B95-8 (type A) and AG876 (type B). Type-specific point mutations were detected as demonstrated by shifts in mobility due to conformational changes of DNA sequences. The locations of point mutations were identified by direct sequencing of the PCR amplified DNA. Of the three primer pairs designed, the pair that amplified a 190 bp fragment spanning six type-specific point mutations gave the best resolution in SSCP analysis. This pair is now preferred for initial genotyping of EBV-infected tumor biopsies. Thus, SSCP is a simple, fast and efficient technique for genotyping of EBV-associated diseases.
Antigens, Viral↗
Effect of ionophore A23187 on the membrane permeability in mouse fibroblasts.
Addition of the divalent cation ionophore A23187 to transformed mouse fibroblasts (3T6) resulted in an increase in the cell membrane permeability to normally impermeant solutes (e.g., nucleotides). The membrane permeability was assessed by following the efflux of prelabeled adenine nucleotides, the influx of p-nitrophenyl phosphate in cells attached to plastic dishes and reconstitution of intracellular protein synthesis in the presence of exogenously added normally impermeant factors required for macromolecular synthesis. The permeability change of 3T6 cells was found to be dependent on the specific presence of external calcium ion. The permeabilization was found to occur preferably in alkaline pH and specific to certain transformed cells. It is preceded by rapid efflux of K+, influx of Na+ and partial hydrolysis of cellular nucleotides in 3T6 cells. Similar ion fluxes were previously found to precede cell permeabilization by electrogenic ionophores for monovalent ions and by exogenous ATP. Our data suggest that a calcium dependent process caused the K+ release and excess Na+ entry, causing dissipation of the membrane potential and subsequent formation of aqueous channels.
4-Nitrophenylphosphatase↗
Comparative analysis of nucleotide sequences of the partial envelope gene (5' domain) among human T lymphotropic virus type I (HTLV-I) isolates.
Human T-cell lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The nucleotide sequences of 640 bp of the proviral genome (positions 5158-5797) derived from 11 HTLV-I-infected persons were analyzed using the polymerase chain reaction and M13-based sequencing techniques. Patterns of single nucleotide substitutions were characterized from the extracellular domain of the envelope gene (gp46). Compared with other retroviruses, the nucleotide sequences of the HTLV-I external envelope gene are highly conserved among the genotypes studied. We found no evidence of dual infections with HTLV-II among the seropositive asymptomatic persons or in patients with either ATL or HAM/TSP. No unique sequence differences were observed in the envelope gene of the HTLV-I isolates derived from patients coinfected with human immunodeficiency virus type 1 (HIV-1). However, comparative analysis of these data and other published HTLV-I envelope sequences indicated the presence of four subtypes of HTLV-I in relation to their geographic origin.
Base Sequence↗
Ontogeny of epidermal growth factor receptor tyrosine kinase in rat liver.
The binding of epidermal growth factor (EGF) to its receptor and the activity of the receptor intrinsic protein-tyrosine kinase were studied during the ontogeny of rat liver. The number of EGF receptors during pre- and postnatal development was first compared in crude liver plasma membranes using 1) specific binding of 125I-labeled EGF and 2) immunoblot analysis using any antireceptor polyclonal rabbit antibody. Both methods detected the expression of the EGF receptor in fetal rat liver on day 17 of gestation, but in an amount markedly less than the adult. Within 24 h, there was a more than twofold increase in EGF binding to plasma membranes as well as a marked increase in receptor immunoreactivity. However, after birth, there was a precipitous drop in receptor number to less than 20% of the adult level by the end of the first postnatal day (P less than 0.001). Next, the presence of EGF-stimulated tyrosine kinase activity (autophosphorylation) was determined during the same stages of development. Electrophoresis of membranes phosphorylated in the presence or absence of EGF followed by autoradiography demonstrated autokinase activity stimulated by EGF in day 18 and 19 fetal liver plasma membranes, but not in membranes on day 17 of gestation. Similar to the pattern observed with EGF binding, there was a decrease in autokinase activity in early neonatal plasma membranes followed by an increase to near adult levels by 7 days postnatally. Quantitation of the amount of 32P radioactivity associated with the EGF receptor bands in each age group, correlated with the degree of autophosphorylation assessed by autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Comparison of inactivated, live and recombinant DNA vaccines against influenza virus in a mouse model.
The protective efficacy of influenza hemagglutinin expressed from recombinant vaccinia virus was compared with that induced by inactivated or infectious influenza vaccines. Intraperitoneal and intranasal routes of vaccination were compared. All the vaccines except the intranasally administered, inactivated vaccine induced detectable levels of neutralizing and hemagglutination-inhibiting antibodies in the serum of mice at 28 days postvaccination. Immunization with any of the intranasally administered vaccines reduced the amount of influenza virus nucleoprotein antigen in lungs after challenge with a homologous, mouse-adapted strain of influenza virus. Intraperitoneally administered vaccines failed to provide such protection. These results indicated that the route of vaccine administration may be the most critical factor for inducing protective immunity. The results also showed that in this mouse model a recombinant DNA-based vaccine could provide protection equivalent to that provided by conventional attenuated and inactivated influenza vaccines.
Animals↗
Isolation of human T-cell lymphotropic virus type 2 from Guaymi Indians in Panama.
Human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma and with a chronic degenerative myelopathy. However, another major type of HTLV, HTLV-II, has been isolated only sporadically, and little is known of disease associations, transmission routes, and risk factors for HTLV-II infection. Recent studies indicate that a high percentage of certain groups of i.v. drug users and blood donors are infected with HTLV-II. Seroepidemiologic studies have found an elevated rate of seroreactivity to HTLV among Guaymi Indians from Bocas del Toro Province, Panama. To identify the cause of seroreactivity among this unique population we used HTLV-II-specific polymerase chain reaction techniques to detect HTLV genetic sequences from blood leukocytes of three seropositive Guaymi Indians. The HTLV-II primer-amplified polymerase chain reaction products from two of these subjects were partially sequenced and matched published HTLV-II nucleotide sequences in both p24 gag (94% of 107 bases) and pol (98% of 112 bases) regions. A CD4+ T-lymphocyte line established from one of these same subjects produced HTLV-II-specific proteins when tested in antigen-capture and immunoblot assays, as well as mature HTLV particles. The demonstration of HTLV-II infection in this geographically and culturally isolated Central American Indian population without typical risk factors for HTLV infection suggests that HTLV-II infection is endemic in this population and provides an important clue to potential natural reservoir for this virus.
Base Sequence↗
Expression of influenza A and B virus nucleoprotein antigens in baculovirus.
Full-length cDNA clones of the nucleoprotein (NP) genes of influenza A/Ann Arbor/6/60 and B/Ann Arbor/1/86 viruses were constructed from virion RNA and subsequently expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector, Autographa californica nuclear polyhedrosis virus. Western blot analysis of lysates prepared from Sf9 cells infected with the recombinant viruses confirmed that the baculovirus-expressed NP antigens were reactive with monoclonal antibodies specific for either type A or B NP and with anti-NP antibodies in human serum samples. Electrophoretic analysis indicated that the expressed NP antigens comigrated with NP purified from influenza A or B virions and that the recombinant NP antigens represented greater than 10% of total protein in infected cells. Dilutions of clarified Sf9 cell lysates were used as antigens in a standard enzyme immunoassay to detect serum antibody specific for influenza A or B viruses. The results from assays using the baculovirus-expressed NP antigens showed good correlation with the results obtained using bacterially expressed NP antigen as well as complement fixation. Therefore, baculovirus-expressed NP antigens have the potential to be used to develop reproducible and routine assays for the serodiagnosis of influenza virus infections as an alternative to the complement fixation or haemagglutination inhibition tests.
Adult↗
Multiple primer pairs for the detection of HTLV-I by PCR.
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Biomarkers, Tumor↗
Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume.
Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.
Adenosine Triphosphate↗
Detection of human immunodeficiency virus (HIV) and human lymphotropic virus (HTLV) type I or II dual infections by polymerase chain reaction.
To type the specific human T-lymphotropic virus (HTLV) coinfection in acquired immunodeficiency syndrome (AIDS) patients, we have utilized the polymerase chain reaction technique. The primer pairs employed for HTLV-I and HTLV-II are derived from pol and env regions and gag region for human immunodeficiency virus (HIV). The data indicate that the selected primer pairs are specific for each virus and are able to detect HTLV-I and -II coinfection in lymphoid cells established from AIDS patients.
Acquired Immunodeficiency Syndrome↗