Racial/Ethnic Differences in Correlates of Spontaneous and Medically-Indicated Late Preterm Births among Adolescents.

STUDY OBJECTIVE: To investigate the racial/ethnic differences in the correlates of spontaneous and medically-indicated late preterm birth (LPTB), defined as deliveries between 34 0/7 and 36 6/7 weeks gestation, among US adolescents. DESIGN: Population-based, retrospective cohort study. SETTING: Births in the United States to adolescents in 2012. PARTICIPANTS: Adolescents (younger than 20 years; n = 171,573) who delivered nonanomalous singleton first births between 34 and 44 weeks of gestation. INTERVENTIONS AND MAIN OUTCOME MEASURES: Bivariate and multivariable logistic regression were used to evaluate the associations between maternal risk factors and spontaneous and medically-indicated LPTB, stratified according to maternal race/ethnicity. RESULTS: Risk factors for spontaneous LPTB included single marital status among Asian adolescents; no insurance coverage among whites, Asian, and Hispanic adolescents; inadequate prenatal care among all racial/ethnic groups except American Indian, and adequate plus prenatal care among all races/ethnicities; prenatal smoking among whites and black adolescents; insufficient gestational weight gain among all racial/ethnic groups except American Indian; and prepregnancy underweight among white, black, and Hispanic adolescents. Risk factors for medically-indicated LPTB included inadequate prenatal care among white, black, and Hispanic adolescents, and adequate plus prenatal care among all racial/ethnic groups except Asian; insufficient gestational weight gain among white, black, and Hispanic adolescents; and prepregnancy overweight and obesity among white, black, and Hispanic adolescents. CONCLUSION: Our results show racial/ethnic differences in the correlates of spontaneous and medically-indicated LPTB among US adolescents and support the need for risk-specific interventions among different racial/ethnic groups.

Occurrence and detection of AmpC beta lactamases among clinical isolates of E. coli and K. pneumoniae causing UTI.

Presence of Bush class C enzymes in uropathogenic strains of Klebsiella pneumoniae & E. coli resistant to extended spectrum cephalosporins is an emerging threat to clinical therapeutics. These resistant strains result in considerable treatment failure and cannot be detected by routine antibiotic sensitivity screening methods. An effort was therefore made to study AmpC beta lactamase production in E. coli and Klebsiella pneumoniae strains causing UTI. A total of 126 E. coli and 49 K. pneumnoniae strains isolated from urine samples were selected for study out of which AmpC beta lactamase production was seen in 23% E. coli (29 isolates) and 18% K. pneumoniae (49 isolates). The susceptibility of AmpC beta lactamase producers to Imipenem, Nitrofurantoin and Amikacin was found to be 100%, 92% and 80% respectively. Thereby the present study emphasizes the importance of monitoring and control of usage of extended spectrum cephalosporins.

Interactions of chaperone alpha-crystallin with the molten globule state of xylose reductase. Implications for reconstitution of the active enzyme.

alpha-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of alpha-crystallin we used xylose reductase (XR) from Neurospora crassa as the model system. Denaturation studies using the structure-perturbing agent guanidinium chloride indicated that XR folds through a partially folded state that resembles the molten globule. Fluorescence and delay experiments revealed that alpha-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of alpha-crystallin.XR-m interaction was revealed by temperature shift experiments implicating the involvement of hydrophobic interactions in the formation of the complex. Reconstitution of active XR was observed on cooling the alpha-crystallin.XR-m complex to 4 degrees C or on addition of ATP at 37 degrees C. ATP hydrolysis is not a prerequisite for XR release since the nonhydrolyzable analogue 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the alpha-crystallin.XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of alpha-crystallin in vivo is discussed.

Site and significance of cysteine residues in xylose reductase from Neurospora crassa as deduced by fluorescence studies.

Inactivation of xylose reductase (XR) by p-hydroxy-mercury benzoate (PHMB) was found to be biphasic with second-order rate constants of 80 and 6 M-1s-1 for the fast (kf) and slow (ks) phase respectively. Spectroscopic studies indicated that the inactivation was due to modification of one Cys residue per molecule of XR and not due to subsequent disruption of the quaternary structure. The binding of NADPH to XR (Kd 0.9 microM) was depressed on modification of the enzyme by PHMB (Kd 2.3 microM). The dependence of PHMB induced inactivation of XR in the presence of alcohols and varying temperature revealed that the Cys residue is situated in a hydrophobic microenvironment and is not involved in hydrogen bonding. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemo-affinity labeling and double inhibition studies indicates that Cys residues involved in the reaction with PHMB (SHI) and OPTA (SHII) are distinctly different. Experimental evidence presented here serves to implicate that SHI located in a hydrophobic microenvironment at the high affinity NADPH binding site of XR plays a role in the binding of the coenzyme to XR, whereas SHII serves to maintain the conformation of the active site essential for catalysis by interacting with the NH2 group of an essential lysine residue.

Conformation and microenvironment of the active site of xylose reductase inferred by fluorescent chemoaffinity labeling.

Conformation and microenvironment at the active site of xylose reductase (XR) from Neurospora crassa was probed with fluorescent chemoaffinity labeling (FCAL) using o-phthalaldehyde as a chemical initiator. Formation of a single isoindole derivative resulted in complete inactivation of the enzyme as judged by spectroscopic and fluorescence studies. Kinetic analysis of the 2,4,6-trinitrobenzenesulfonic-acid-modified XR implicated the presence of an essential lysine residue at the active site of XR. Modification of lysine in XR abolished the ability of the enzyme to form isoindole derivative, indicating that the lysine residue involved in the reaction with 2,4,6-trinitrobenzenesulfonic acid and o-phthalaldehyde is the same and that the probe o-phthalaldehyde is directed to the active site. Fluorescence studies revealed that inactivation of XR by Gdn/HCl precedes gross conformational change and the possibility of secondary-conformational change was eliminated by acrylamide quenching studies. The enzyme inactivated by low concentrations of Gdn/HCl retained its ability to form the fluorescent XR-isoindole derivative indicating that inactivation is not due to conformational changes at or near the active site of XR. Gdn/HCl also had no effect on the high-affinity and low-affinity NADPH-binding sites of XR. Energy-transfer experiments further revealed structural integrity at the active site of the Gdn/HCl-inactivated XR. Changes in the fluorescence emission maximum of 1-(beta-hydroxyethylthio)-2-beta hydroxyethyl isoindole (EA adduct) in solvents of varying polarity was studied, the data obtained were utilized to interpret the fluorescence behaviour of XR-isoindole derivative and assess the polarity at the active site. Experimental evidence presented here serves to suggest that the inactivation of XR by Gdn/HCl precedes conformational changes at the active site located in a microenvironment of low polarity.

Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa.

Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.