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Biomedical subjects

P Datta

Publications and source records attributed to P Datta.

At least 19 recordsLinked to original sources
PubMed1996

Effect of antibody specificity on results of selected digoxin immunoassays among various clinical groups.

We examined the specificity of three automated digoxin immunoassays (Abbott TDxFLx Digoxin II assay, Baxter-Dade Stratus II Digoxin assay, and Ciba Corning ACS Digoxin assay) applied without modification to (a) sera from 229 digoxin-free patients in 12 cohorts associated with nonspecific or endogenous digoxin-like immunoreactive factor (DLIF) interference, and (b) drug-free serum supplemented with the major metabolites and analogs of digoxin. We observed three patterns of apparent digoxin results among the DLIF samples: one common to kidney and liver failure patients, where TDx and Stratus assays showed significant positive results; one common to newborns and cord blood, where only the TDx assay had significant interference; and one from cardiac surgery patients, where the Stratus assay alone showed interference. Of the three assays, the ACS had the least interference from DLIF. The assays also behaved differently with respect to cross-reactivity with digoxin metabolites, digitoxin, and digitoxin metabolites. The ACS assay again had the least analog or metabolite cross-reactivity. The three methods agreed well on digoxin-positive specimens, with a mean bias of <0.15 microgram/L digoxin for each and discrepancies (defined as >3 SD between the assay pairs compared) of only 3-5%.

Antibody Specificity↗
PubMed1995

Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12.

TdcB activity expressed from the chromosomal gene and LacZ expression from single-copy tdc-lacZ transcriptional and translational fusions were measured in Escherichia coli strains harboring mutations in the genes encoding DNA gyrase, topoisomerase I and the HU protein. The pattern of tdc operon expression in these mutants suggests that relaxation of supercoiled DNA enhances tdc transcription in vivo.

DNA, Bacterial↗
PubMed1994

Mother-to-child transmission of human immunodeficiency virus type 1: report from the Nairobi Study.

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) is a significant problem in countries with endemic HIV-1 infection. Between 1986 and 1991, 365 children of HIV-1-infected mothers and 363 control children were studied in Kenya. The overall risk of transmission from mother to child, determined by serologic evidence of infection by age > or = 12 months and excess mortality in the HIV-1-exposed group, was 42.8% (range, 27.6%-62.2%). Marriage was the only maternal characteristic associated with transmission (odds ratio, 2.2; 95% confidence interval, 1.2-4.2; P < .05). Children who experienced growth failure were more likely to be infected. In 44% of children ultimately infected, the pattern of antibody response implied intrapartum or postnatal exposure to HIV-1. Of potential postnatal exposures examined, duration of breast-feeding beyond age 15 months and the mother being married were independently associated with increased risk of infection and seroconversion of children. The percentage of HIV infection attributable to breast-feeding > or = 15 months was 32%. The frequency of mother-to-child transmission of HIV-1 was high; a substantial proportion of infection occurred postnatally, possibly through breast-feeding.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Human immunodeficiency virus type 1 IgA antibody in breast milk and serum.

Breast-feeding plays a potentially significant role in mother to child transmission of human immunodeficiency virus type 1 (HIV-1). The additional transmission risk attributable to breast-feeding and the factors that enhance or inhibit transmission are presently unknown. One mechanism by which breast milk might inhibit HIV-1 transmission is the presence of specific antibodies directed against HIV-1 in breast milk of seropositive mothers. In this study serum and breast milk samples from women in Nairobi, Kenya, were tested to determine the prevalence of HIV-1 IgA antibodies. A Western blot test developed in our laboratory was used to detect anti-HIV-1 immunoglobulin A in serum and anti-HIV-1 secretory IgA (sIgA) in breast milk. Ninety-four percent of 63 HIV-1 seropositive women had anti-HIV-1 IgA in serum and 59% had anti-HIV-1 sIgA in their breast milk. No significant associations with maternal characteristics or serum anti-HIV-1 IgA or IgG banding patterns and the presence of anti-HIV-1 sIgA in breast milk were found. No protective effect of anti-HIV-1 sIgA was seen regarding mother to child transmission; however, further studies are necessary to determine the effect of these antibodies in maternal sera or in breast milk on the efficacy of HIV-1 transmission.

AIDS Serodiagnosis↗
PubMed1994

Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR.

The efficient expression of the tdc operon of Escherichia coli requires the products of two regulatory genes, tdcR and tdcA. We have identified the transcription site of tdcR by primer extension mapping and established the translation start site of TdcR by mutational analysis of its reading frame. In a tdcR tdcABC deletion strain, tdcR+ promoted high-level LacZ expression from a lambda tdcAB-lacZ lysogen and mutations introduced in tdcR resulted in a greater than sixfold decrease in LacZ level. In-frame deletions of tdcA also reduced LacZ expression, and chromosomal and plasmid-borne tdcA+ increased the LacZ level in tdcA mutant lysogens. Interestingly, multicopy tdcA+ plasmids introduced into tdcR mutant strains completely restored tdc expression. In separate experiments we found that mutations in the tdc promoter DNA around positions -70, -140, and -175 greatly reduced tdc expression relative to that for the wild-type promoter and the tdcP mutation around -175 prevented multicopy tdcA+ from rescuing tdcR mutants. Furthermore, competition experiments revealed that a wild-type promoter fragment encompassing the -175 region cloned into a plasmid reduced tdc expression by titrating TdcA in vivo, and this effect was reversed with excess TdcA. These results suggest that in tdcR+ cells TdcR interacts with tdcP and/or TdcA to enhance tdc transcription whereas in tdcR mutant cells a new tdcP-TdcA complex around -175 in the native promoter bypasses the requirement for TdcR. On the basis of the accumulated data summarized here and elsewhere we propose that multiple transcription factors enhance tdc operon expression by bending and looping of the promoter DNA to form an active transcription complex.

Bacterial Proteins↗
PubMed1993

TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins.

The tdcB and tdcC genes of the tdcABC operon of Escherichia coli encode threonine dehydratase and a threonine-serine permease, respectively. These proteins are involved in transport and metabolism of threonine and serine during anaerobic growth. In this study, we functionally characterized tdcA, which encodes a 35 kDa polypeptide consisting of 312 amino acid residues. Non-polar and partially polar mutations introduced into tdcA drastically reduced the expression of the genes down-stream from tdcA. Complementation studies using single-copy chromosomal integrants of a tdcB-lacZ fusion harboring an in-frame deletion of tdcA with chromosomal or plasmid-borne tdcA+ in trans showed complete restoration of tdc operon expression in vivo. The amino acid sequence at the amino-terminal end of TdcA revealed a significant homology to the helix-turn-helix motifs of typical DNA binding proteins. Sequence alignment of TdcA with LysR also showed considerable sequence similarity throughout their entire lengths. Our results suggest that TdcA is related to the LysR family of proteins by common ancestry and, based on its functional role in tdc expression, belongs to the LysR family of transcriptional activators.

Amino Acid Sequence↗
PubMed1992

Increased risk of early measles in infants of human immunodeficiency virus type 1-seropositive mothers.

An increase in illness due to measles is one of the potential consequences of the human immunodeficiency virus (HIV) epidemic in Africa. During a study of perinatal HIV transmission conducted in Kenya, the risk of acquiring measles before vaccination (9 months of age) was found to be 3.8 times higher in infants born to HIV-seropositive mothers than in control infants (10 [9%] of 109 vs. 5 [3%] of 194 infants; P = .02; odds ratio, 3.8; 95% confidence interval, 1.2-13.2). The majority of infants who developed measles in this study had significant sequelae related to their measles infection. The increased risk of measles appeared to be related to relatively lower anti-measles antibody titers detected in cord blood samples of affected infants born to HIV-seropositive mothers. However, 94% of all infants were susceptible to measles on the basis of ELISA testing at age 6 months regardless of maternal HIV serology. These observations highlight the need for improved measles vaccination strategies in Africa and for studies to delineate the effects of HIV infection on the incidence, presentation, and sequelae of childhood infectious illnesses.

Antibodies, Viral↗
PubMed1992

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.

Amino Acid Sequence↗
PubMed1992

Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli.

Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP). Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid. Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88. The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence. In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together. These regulatory proteins also induced significant bending of the tdc promoter DNA. Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.

Anaerobiosis↗
PubMed1991

Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome.

The physical and genetic structure of 37 kilobases of DNA encompassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping. Re-examination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA.

Cloning, Molecular↗
PubMed1991

Adjuvant activity of non-ionic block copolymers. V. Modulation of antibody isotype by lipopolysaccharides, lipid A and precursors.

Non-ionic block copolymers and lipopolysaccharides are both effective immunological adjuvants which are thought to act via distinct mechanisms. We hypothesized that they might produce synergistic effects when used together. We prepared a series of lipopolysaccharide (LPS) preparations ranging from the smallest precursor, lipid X through complete LPS with O-polysaccharide chains. Three preparations with reduced toxicity, monophosphoryl lipid A, partially hydrolysed Ra-LPS and LPS of Rhodopseudomonas sphaeroides were also utilized. All LPS preparations except the smallest were effective adjuvants for inducing early antibody responses to trinitrophenyl-conjugated hen egg albumin (TNP-HEA) when injected in squalane-in-water emulsions with copolymer L141. Only the larger LPS preparations induced sustained antibody responses. By itself, emulsions of copolymer L141 induced a predominant IgG1 antibody isotype response with lesser amounts of IgG2a and IgG2b. Surprisingly, all of the LPS preparations tested increased the proportion of IgG2 isotypes even though some had little effect on overall titres. The detoxified Ra-LPS (Ra-detox) was the most effective preparation for both increasing antibody titres and inducing the desirable IgG2a and IgG2b isotypes. These results demonstrate that the combination of LPS and block polymer adjuvants can produce synergistic effects without unacceptable toxicities.

Adjuvants, Immunologic↗
PubMed1990

Impact of maternal HIV infection on obstetrical and early neonatal outcome.

In a case-control study of 177 HIV-seropositive and 326 seronegative women and their newborns in Nairobi, Kenya, maternal HIV infection at term was independently associated with travel to other African countries [odds ratio (OR) 4.9, P less than 0.0001], history of a blood transfusion since 1980 (OR 3.5, P = 0.01), history of more than one sexual partner in the previous 5 years (OR 1.8, P = 0.02) and unmarried status (OR 1.8, P = 0.02). Neonates of HIV-positive and HIV-negative women differed little with respect to occurrence of congenital malformations, stillbirths, in-hospital mortality, sex, APGAR score, or gestational age. However, the mean birth weight of singleton neonates of HIV-positive women was significantly lower than that of controls (3090 versus 3220 g, P = 0.005), and birth weight was less than 2500 g in 9% of cases and 3% of controls (OR 3.0, P = 0.007). Among neonates of HIV-seropositive women, birth weight was less than 2500 g in 17% if mothers were symptomatic and 6% if mothers were asymptomatic (OR 3.4, P = 0.08).

Adult↗
PubMed1990

A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli.

A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of a cDNA clone encoding the lima bean lectin.

The complete amino acid sequence of the lima bean (Phaseolus lunatus) lectin was deduced from the nucleotide sequence of a cDNA clone. The lectin appears to be synthesized as a prepeptide consisting of a signal sequence of 21 residues and a mature protein of 241 amino acids. Comparison of the lima bean lectin sequence to the sequences of other leguminous seed lectins indicates regions of extensive homology. Northern blot analysis showed absence of lectin mRNA in the leaves, roots, or stems of 16-day-old lima bean plants.

Amino Acid Sequence↗
PubMed1989

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain.

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗