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Biomedical subjects

I Wong

Publications and source records attributed to I Wong.

At least 37 records · Page 2Linked to original sources
PubMed1993

Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II).

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.

Adenosine Triphosphatases↗
PubMed1993

Acoustic vs. spirometric assessment of bronchial responsiveness to methacholine in children.

To study wheezing as an indicator of bronchial responsiveness during methacholine challenge (MC) in children, we used computer analysis of respiratory sounds and compared wheeze measurements to routine spirometry. MC was performed in 30 symptomatic subjects (sympt), age 11 +/- 3.1 years (mean +/- SD), with suspected asthma and in 12 controls (contr), age 10 +/- 3.4 years. Respiratory rate (RR), spirometry, arterial oxygen saturation (SaO2), and cough were registered until the concentration provoking a > or = 20% fall in forced expiratory flow in 1 second (FEV1;PC20), or the end point (8 mg/mL) was reached. For 1 min after each inhalation, sounds over the trachea and posterior right lower lobe were recorded together with calibrated airflow. Computer analysis of respiratory sounds was used for objective wheeze quantification. Wheezing was measured as its duration relative to inspiration (Tw/Ti) and expiration (Tw/Te). Seventeen of the sympt group developed wheezing (sympt/W) with > or = 5% Tw/Ti or > or = 5% Tw/Te. Thirteen of the sympt did not wheeze (sympt/no W). Three contr developed wheeze (contr/W) while 9 did not (contr/no W). In sympt/W, RR increased from 20 +/- 6.2 per min at baseline to 25 +/- 9.2 (P < 0.05) at the MC concentration provoking wheeze (PCw), and SaO2 decreased from 97.4 +/- 1.2% to 95.3 +/- 2.4 (P < 0.05). In contr/W, RR did not change, but SaO2 decreased from 97.3 +/- 1.5% to 95.7% +/- 1.2% (P < 0.05). Wheezing occurred at both recording sites and was as common during inspiration as during expiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Haemolytic Escherichia coli strains isolated from stools of healthy cats produce cytotoxic necrotizing factor type 1 (CNF1).

A total of 159 Escherichia coli colonies isolated from the stools of 23 healthy cats were studied for production of alpha-haemolysin (Hly), enterohaemolysin (EntHly), cytotoxic necrotizing factors (CNF1 and CNF2), verotoxins (VT) and heat-labile enterotoxin (LT). Hly+CNF1+, Hly+CNF2+, Hly+VT+ and Hly+ E. coli colonies were isolated from 12 (48%), 1 (4%), 1 (4%) and 2 (8%) respectively of the cats sampled. None of the 159 E. coli colonies produced LT or EntHly. Nine of 12 Hly+CNF1+ strains from the cats belonged to serogroup O6 and eleven to serotypes (O4:K?:H5 or H-, O6:K13:H1, O6:K53:H-, O6:K53:H1, O6:K53:H7 and O6:K14:H31) found among Hly+CNF1+ E. coli that cause urinary tract infections and sepsis in humans. Furthermore, 10 Hly+CNF1+ strains from the cats expressed the mannose-resistant haemagglutination (MRHA) type III. By contrast, the majority of nontoxigenic E. coli strains were MRHA negative and belonged to different O groups. We conclude that cats are a important reservoir of Hly+CNF1+ E. coli strains that possess similar characteristics to strains that can cause extraintestinal infections in humans and that Hly+ E. coli from cats usually do not produce shiga-like toxins with cytotoxic activity on Vero cells.

Animals↗
PubMed1992

Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding.

The Escherichia coli Rep helicase unwinds duplex DNA during replication. The functional helicase appears to be a dimer that forms only on binding DNA. Both protomers of the dimer can bind either single-stranded or duplex DNA. Because binding and hydrolysis of adenosine triphosphate (ATP) are essential for helicase function, the energetics of DNA binding and DNA-induced Rep dimerization were studied quantitatively in the presence of the nucleotide cofactors adenosine diphosphate (ADP) and the nonhydrolyzable ATP analog AMPP(NH)P. Large allosteric effects of nucleotide cofactors on DNA binding to Rep were observed. Binding of ADP favored Rep dimers in which both protomers bound single-stranded DNA, whereas binding of AMPP(NH)P favored simultaneous binding of both single-stranded and duplex DNA to the Rep dimer. A rolling model for the active unwinding of duplex DNA by the dimeric Rep helicase is proposed that explains vectorial unwinding and predicts that helicase translocation along DNA is coupled to ATP binding, whereas ATP hydrolysis drives unwinding of multiple DNA base pairs for each catalytic event.

Adenine Nucleotides↗
PubMed1992

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1991

Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.

The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: [formula: see text] where E, D, N, and P represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPi equal to 18 nM (koff/kon), 18 microM (k-1/k1), and 2 mM (k5/k-5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a conformational change, E.Dn.N----E'.Dn.N, which occurs at a rate of 300 s-1 (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate-limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s-1 for dTTP (alpha S) incorporation suggest that the chemical step (k3) occurs at a fast rate, greater than or equal to 9000 s-1. Following chemistry, the resulting ternary complex, E'.Dn+1.P, undergoes a second conformational change at a rate of 1200 s-1 (k4), leading to release of PPi and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 s-1 (k-2), greater than or equal to 18,000 s-1 (k-3) and 18 s-1 (k-4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1.0 x 10(4) for elongation of E.25/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1991

An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.

An exonuclease-deficient mutant of T7 DNA polymerase was constructed and utilized in a series of kinetic studies on misincorporation and next correct dNTP incorporation. By using a synthetic oligonucleotide template-primer system for which the kinetic pathway for correct incorporation has been solved [Patel, S.S., Wong, I., & Johnson, K. A. (1991) Biochemistry (first of three papers in this issue)], the kinetic parameters for the incorporation of the incorrect triphosphates dATP, dCTP, and dGTP were determined, giving, respectively, kcat/Km values of 91, 23, and 4.3 M-1 s-1 and a discrimination in the polymerization step of 10(5)-10(6). The rates of misincorporation in all cases were linearly dependent on substrate concentration up to 4 mM, beyond which severe inhibition was observed. Competition of correct incorporation versus dCTP revealed an estimated Ki of approximately 6-8 mM, suggesting a corresponding kcat of 0.14s-1. Moderate elemental effects of 19-, 17-, and 34-fold reduction in rates were measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP, respectively, indicating that the chemistry step is partially rate-limiting. The absence of a burst of incorporation during the first turnover places the rate-limiting step at a triphosphate binding induced conformational change before chemistry. In contrast, the incorporation of the next correct triphosphate, dCTP, on a mismatched DNA substrate was saturable with a Km of 87 microM for dCTP, 4-fold higher than the Kd for the correct incorporation on duplex DNA, and a kcat of 0.025 s-1. A larger elemental effect of 60, however, suggests a rate-limiting chemistry step. The rate of pyrophosphorolysis on a mismatched 3'-end is undetectable, indicating that pyrophosphorolysis does not play a proofreading role in replication. These results show convincingly that the T7 DNA polymerase discriminates against the incorrect triphosphate by an induced-fit conformational change and that, following misincorporation, the enzyme then selects against the resultant mismatched DNA by a slow, rate-limiting chemistry step, thereby allowing sufficient time for the release of the mismatched DNA from the polymerase active site to be followed by exonucleolytic error correction.

DNA Replication↗
PubMed1991

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells.

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.

Arginine↗
PubMed1989

Replication of measles virus in cultured human thymic epithelial cells.

Measles virus can replicate in cultures of both infantile and fetal human thymic epithelial cells. Virus-induced cytopathology including syncytium formation was first evident around 24 hr after viral inoculation of these cultures. At the same time, the cultures began to lose their characteristic thymus-like organizational structure. Viral antigens were detected in infected cells by indirect immunofluorescence, and the presence of progeny virions was demonstrated in culture fluids.

Animals↗
PubMed1989

Replication of cytomegalovirus in human thymic epithelial cells.

Cytomegalovirus (CMV) has often been cited as a cause of immune suppression in children, yet little is known of the mechanisms through which this agent might affect immune function. We have succeeded in using CMV to productively infect cultured human fetal and infantile thymic epithelial (TE) cells. Morphological changes were apparent by 2-4 days after viral inoculation. CMV-related early antigen (EA) and late antigen (LA) were detected by immunofluorescence after 8 days, and progeny infectious CMV was recovered from culture media after 12-17 days. TE cells that reacted with monoclonal antibodies specific for keratin and for GQ ganglioside were predominant throughout the culture period. In contrast, infection by CMV resulted in a significant decrease in numbers of cells reactive with monoclonal antibodies specific for mesoderm-derived components. Inoculation of TE cells with CMV also caused a diminution in levels of detectable interleukin-1 (IL-1)-related antigen by 17 days after infection.

Animals↗
PubMed1989

Infection of cultured human thymic epithelial cells by human immunodeficiency virus.

We have succeeded in growing the HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) in cultured human thymic epithelial (TE) cells. Expression of the HIV-1 proteins p17 and p24 was detected by immunofluorescence and reached a peak at 3 days after infection. Antigen capture and reverse transcriptase assays were used to detect HIV-1 in culture fluids, with positive results also being realized. The infection was cytolytic; cellular disarrangement, increased numbers of Hassall's corpuscles, and giant cells first appeared in monolayers of TE cells at 2 days after inoculation. By 4 days these changes were increased, and by 7 days, retraction and involution of TE cells were evident. The infection of TE cells by HIV-1 was blocked by preincubation with monoclonal OKT4A antibodies directed against CD4 target molecules.

Antibodies, Monoclonal↗
PubMed1989

Infection of cultured human fetal pancreatic islet cells by rubella virus.

A high incidence of insulin-dependent diabetes mellitus (IDDM) has been reported in children and young adults previously afflicted with congenital rubella syndrome (CRS). The authors have studied the effect of rubella virus infection on human pancreatic islet cells in tissue culture. These experiments were performed with the use of both monolayers and free-floating human fetal islets of Langerhans tissue. Levels of production of immunoreactive insulin by islet cells that had been infected by rubella virus were lower than those observed in control cultures, under conditions of high glucose concentration (11.1 mmol/L) in the medium. The presence of rubella viral antigens in human pancreatic beta and non-beta cells was demonstrated by double-label immunofluorescence. These results suggest that rubella virus can infect human pancreatic islet cells and that such infection may lead to significant reductions in levels of secreted insulin.

Antigens, Viral↗
PubMed1989

Effects of infection by HIV-1, cytomegalovirus, and human measles virus on cultured human thymic epithelial cells.

A tissue culture system for the growth of human fetal and infantile thymic epithelial (TE) cells has been established and characterized. We have investigated the effects of infection of these cells by human cytomegalovirus (CMV), measles virus, and human immunodeficiency virus type-1 (HIV-1). In the case of CMV, morphological changes were apparent by 2-4 days after viral inoculation of infantile TE cells. CMV-related antigens were detected by immunofluorescence after 12 days, and progeny infectious CMV was recovered from culture media after 18 days. Following infection by measles virus, distinctive, multinucleated giant TE cells appeared in both cultures of fetal and infantile TE cells. Measles virus-inoculated TE cells displayed an altered phenotype, as revealed by reaction with monoclonal antibodies with specificity for a variety of TE markers. Finally, infection of TE cells by HIV-1 resulted in cellular disarrangement, increased numbers of Hassall's corpuscles, and multinucleated giant cells. An increase in the number of cells reactive with monoclonal antibodies, specific for Hassall's corpuscles, was observed in the case of cells infected by either measles virus or HIV-1. These findings suggest that a variety of different viruses can successfully infect thymic epithelial tissue. Because of the important role of the thymus in development of the immune system, it is reasonable to conclude that viral infection of thymic tissue might play an important role in virus-mediated suppression of immune responsiveness.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Viral infection of human fetal islets of Langerhans. Replication of human cytomegalovirus in cultured human fetal pancreatic islets.

Tissue monolayer cultures of human fetal islets of Langerhans were infected with human cytomegalovirus (CMV). On the fourth day after inoculation, morphologic changes of monolayers were observed, including rounding up and floating of many cells. In addition, after this time CMV late antigen (LA) was detected in the inoculated cells. On the seventh day, CMV was recovered from the culture medium, and infected beta cells were identified by use of a double-label antibody technique. However, CMV apparently did not cause direct destruction of beta cells nor did infection by CMV lead to changes in production of insulin as measured by radioimmunoassay. Thus, despite the fact that CMV can successfully infect a variety of cells in fetal islets of Langerhans, the absence of a significant effect on beta cells does not support the notion that congenital CMV infection is involved in the origin of insulin-dependent diabetes mellitus.

Antigens, Viral↗
PubMed1988

Morphology, yield and functional integrity of islet-like cell clusters in tissue culture of human fetal pancreata obtained after different means of abortion.

Morphology, yield and function were studied in cultured islet-like cell clusters (ICC) from 140 human fetal pancreata obtained after abortions of different types performed at 11-23 weeks of gestation (12 by hysterotomy, 75 by mechanical dilation and extraction, and 53 induced with prostaglandin). After collagenase digestion and culture in medium supplemented with 10% human serum, up to 2000 free-floating ICC were formed from a single pancreas. Randomly scattered insulin- and glucagon-immunoreactive cells were found in the medullary part of the ICC. More than 100 ICC developed in 100% of the hysterotomies and 87% of the mechanical abortions, but in only 53% of the prostaglandin-induced abortions. Insulin and glucagon levels in the culture medium decreased rapidly during the first 7 days of culture, but then remained stable for at least 31 days. The hysterotomy-derived ICC responded to 10 mmol/l theophylline plus 20 mmol/l glucose by a 12.2 +/- 3.1 (SEM, N = 7) fold increase in insulin release, as compared with a 5.4 +/- 0.9 fold response of the prostaglandin ICC (N = 16; P less than 0.02). Despite the low proportion of B-cells, (pro)insulin biosynthesis accounted for 10% of the total protein biosynthesis in low (2 mmol/l) glucose. In conclusion, the yield and viability of the ICC were clearly better, if prostaglandin had not been used for the induction of the abortion.

Abortion, Induced↗
PubMed1988

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture.

The effects of recombinant human growth hormone (GH, 1 micrograms/ml) and insulin-like growth factor I (IGF-I, 200 ng/ml) on the production of insulin and glucagon by human fetal islet-like cell clusters (ICCs) were studied in tissue culture. ICCs were derived after collagenase digestion and culture of pancreases from 16 fetuses (mean gestational age 15.6 wk). The ICCs were cultured with or without GH or IGF-I for 7 or 31 days. Basal rates of insulin and glucagon production were not altered by GH during the first 17 days of culture, but the release of both hormones was increasingly augmented by GH during the last 2 wk of culture (131% increase in insulin and 85% in glucagon compared with controls). ICCs cultured for 7 days in the presence of GH secreted more insulin when incubated for 120 min in 20 mM than in 2 mM glucose (2.1-fold response, P less than .05), whereas ICCs maintained in basal medium did not respond to glucose. GH had no effect on DNA and insulin content or insulin biosynthesis. Exogenous IGF-I caused a 28% suppression of insulin release (P less than .05) between days 4 and 10 of culture but induced a 49% increase in the mean secretion rate during the last week (days 25-31, P less than .01). Glucagon release was not affected by exogenous IGF-I. In contrast to GH, exogenous IGF-I induced a twofold increase in the DNA content of the 7-day--cultured ICCs. However, insulin biosynthesis and release were markedly suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture Techniques↗
PubMed1988

Infection of human thymic epithelial cells by human cytomegalovirus and other viruses: effect on secretion of interleukin 1-like activity.

Cultures of human thymic epithelial (TE) cells are able to produce a interleukin 1 (IL-1) like activity. This IL-1 activity can be detected either using mouse thymocytes in a traditional IL-1 assay, or using thymic lymphocytes obtained from cases of pediatric cardio-vascular surgery. Production of IL-1 activity by TE cells was found to be maximal between 3 and 4 weeks after culture initiation. Human thymocytes worked best as targets in an IL-1 assay, when these cells were derived from donors younger than 1 year of age. Infection of human TE cells by any of human cytomegalovirus, herpes simplex virus type 2, adenovirus 7, Coxsackie B1, and respiratory syncytial virus led to marked reductions in the ability of these cells to secrete measurable IL-1 activity. In the case of TE cells infected by cytomegalovirus, respiratory syncytial virus, and Coxsackie B1, this abrogation of production of IL-1 activity occurred in the absence of any obvious virus-induced cytopathic effect.

Cells, Cultured↗