Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

HU binds and folds single-stranded DNA

The nucleoid-associated protein HU plays an important role in bacterial nucleoid organization and is involved in numerous processes including transposition, recombination and DNA repair. We show here that HU binds specifically DNA containing mismatched region longer than 3 bp as well as DNA bulges. HU binds single-stranded DNA (ssDNA) in a binding mode that is reminiscent but different from earlier reported specific HU interactions with double-helical DNA lesions. An HU dimer requires 24 nt of ssDNA for initial binding, and 12 nt of ssDNA for each additional dimer binding. In the presence of equimolar amounts of HU dimer and DNA, the ssDNA molecule forms an U-loop (hairpin-like) around the protein, providing contacts with both sides of the HU body. This mode differs from the binding of the single-strand-binding protein (SSB) to ssDNA: in sharp contrast to SSB, HU binds ssDNA non-cooperatively and does not destabilize double-helical DNA. Furthermore HU has a strong preference for poly(dG), while binding to poly(dA) is the weakest. HU binding to ssDNA is probably important for its capacity to cover and protect bacterial DNA both intact and carrying lesions.     

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.     

Involvement of KATP and KvLQT1 K+ channels in EGF-stimulated alveolar epithelial cell repair processes

Several respiratory diseases are associated with extensive damage of lung epithelia, and the regulatory mechanisms involved in their regeneration are not clearly defined. Growth factors released by epithelial cells or fibroblasts from injured lungs are important regulators of alveolar repair by stimulating cell motility, proliferation, and differentiation. In addition, K(+) channels regulate cell proliferation/migration and are coupled with growth factor signaling in several tissues. We decided to explore the hypothesis, never investigated before, that K(+) could play a prominent role in alveolar repair. We employed a model of mechanical wounding of rat alveolar type II epithelia, in primary culture, to study their response to injury. Wound healing was suppressed by one-half upon epidermal growth factor (EGF) titration with EGF-antibody (Ab) or erbB1/erbB2 tyrosine-kinase inhibition with AG-1478/AG-825. The addition of exogenous EGF slightly stimulated the alveolar wound healing and enhanced, by up to five times, alveolar cell migration measured in a Boyden-type chamber. Conditioned medium collected from injured alveolar monolayers also stimulated cell migration; this effect was abolished in the presence of EGF-Ab. The impact of K(+) channel modulators was examined in basal and EGF-stimulated conditions. Wound healing was stimulated by pinacidil, an ATP-dependent K(+) channel (K(ATP)) activator, which also increased cell migration, by twofold, in basal conditions and potentiated the stimulatory effect of EGF. K(ATP) or KvLQT1 inhibitors (glibenclamide, clofilium) reduced EGF-stimulated wound healing, cell migration, and proliferation. Finally, EGF stimulated K(ATP) and KvLQT1 currents and channel expression. In summary, stimulation of K(+) channels through autocrine activation of EGF receptors could play a crucial role in lung epithelia repair processes.     

Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons

Hedgehog (Hh) signal transduction is directly required in zebrafish DRG precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursors.     

Glutamate 346 of human Na+-H+ exchanger NHE1 is crucial for modulating both the affinity for Na+ and the interaction with amiloride derivatives

A NHE1 variant that exhibits very high resistance to (3-methyl sulfonyl-4-piperidinobenzoyl) guanidine methane sulfonate (HOE694), a potent inhibitor of Na(+)-H(+) exchangers, was selected and characterized. Sequencing of the coding region corresponding to the N-terminal domain of this variant revealed the presence of only one mutation located within membrane-spanning segment 9 (M9). This base pair change replaces a glutamate (Glu) with an aspartate (Asp). We reproduced this amino acid change in wild-type NHE1 and found that this mutation alone is responsible for the huge decrease in sensitivity to the HOE694 compound and to ethylisopropylamiloride (EIPA). We found that the NHE1-Glu(346)Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHE1, with the size, rather than the charge, of the amino acid in position 346 having the greatest effect. Interestingly, its affinity for Na(+) was at least 4-fold lower than that of wild-type NHE1. Mutation of amino acids in the vicinity of Glu(346) did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu(346) or involvement of Glu(346) in a coordination site with NHE inhibitors and with Na(+).     

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli

Summary A strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30° C and 42° C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the (topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.Abbreviations Ap^r ampicillin resistance - Cm^r chloramphenicol resistance - Km^r Kanamycin resistance - St^r streptomycin resistance - Tc^r tetracycline resistance     

Improving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp. S38: structural comparison and mutational analysis

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M-1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site-directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant.