Utilization of endoscopic inoculation in a mouse model of intrauterine infection-induced preterm birth: role of interleukin 1beta

A novel murine model of intrauterine infection/inflammation-induced preterm birth based on direct endoscopic intracervical inoculation is described. Using this model, we investigated infection-induced premature pregnancy loss in normal and interleukin (IL) 1beta-deficient mice. Seventy-four CD-1, HS, C57BL/6J wild type (IL-1beta+/+), and C57BL/6J IL-1beta-deficient (IL-1beta-/-) mice were inoculated intracervically using a micro-endoscope, at a time corresponding to 70% of average gestation. Intracervical injection of lipopolysaccharide (LPS) or Escherichia coli reliably induced premature birth: 100% of mice intracervically injected with LPS and 92% of mice with a positive endometrial E. coli culture delivered prematurely within 36 h after inoculation. No losses were observed in mice inoculated with saline. Pregnancy loss was associated with increased uterine tissue cyclooxygenase-2 gene expression and uterine content of IL-1beta, tumor necrosis factor alpha, macrophage inflammatory protein-1alpha, and IL-6, as well as elevation of nuclear factor-kappaB activity in uterine tissues. Although IL-1beta-/- mice exhibited decreased uterine cytokine production in response to bacteria and LPS, IL-1beta deficiency did not affect the rate of pregnancy loss. This model using direct intracervical bacterial or LPS inoculation is useful for studying preterm pregnancy loss in genetically altered mice in order to develop novel interventions for infection-associated preterm labor.     

Nature of the chromophore binding site of bacteriorhodopsin: the potential role of Arg82 as a principal counterion

The nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. We conclude that in the absence of divalent metal cations in close interaction with Asp85 and Asp212, a positively charged amino acid must be present in the same vicinity. We find that models in which Arg82 is pointed upward into the chromophore binding site and directly stabilizes Asp85 and Asp212 are successful in rationalizing the observed one-photon and two-photon properties. We conclude further that a water molecule is strongly hydrogen bonded to the chromophore imine proton. The chromophore "1Bu*+" and "1Ag*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying "1Bu*+" state is dominated by single excitations, whereas the second-excited "1Ag*-" state is dominated by double excitations. We can rule out the possibility of a negatively charged binding site, because such a site would produce a lowest-lying "1Ag*-" state, which is contrary to experimental observation. The possibility that Arg82 migrates toward the extracellular surface during the photocycle is examined.     

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.     

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.     

Structural changes and the storage of long-term memory in Aplysia

Long-term memory for sensitization of the gill-withdrawal reflex in Aplysia is associated with the growth of new synaptic connections between sensory and motor neurons. The duration of this structural change parallels the behavioral retention of the memory. Such changes can be reconstituted in dissociated cell culture by repeated presentations of the modulatory neurotransmitter serotonin (5HT) and are associated with an activity-dependent downregulation of NCAM-related cell adhesion molecules thought to contribute to cell recognition and axonal outgrowth during development. Thus, aspects of the mechanisms utilized for learning-related synaptic growth initiated by experience in the adult may eventually be understood in the context of the molecular logic that shapes synaptic circuitry during the later stages of neuronal development.     

Apolipoprotein J polymorphisms and serum HDL cholesterol levels in African blacks

Apolipoprotein J (apoJ, protein; APOJ, gene) is found in serum associated with high-density lipoprotein (HDL) subfractions, which also contain apolipoprotein A-I (apoA1) and cholesteryl ester transfer protein. ApoJ has been shown to be involved in a variety of physiological functions, including lipid transport. In earlier studies we reported the existence of a common genetic polymorphism (APOJ*1 and APOJ*2 alleles) using isoelectric focusing (IEF) and immunoblotting. In this study we determined the molecular basis of this polymorphism and together with another polymorphism at codon 328 (G-->A) evaluated its relationship with serum HDL cholesterol and apoA1 levels in 767 African blacks stratified by staff level: junior (less affluent, n = 450) and senior (more affluent, n = 317). The molecular analysis of the cathodally shifted APOJ*2 allele on IEF gels revealed an amino acid substitution of asparagine by histidine resulting from a missense mutation (A-->C) at codon 317 in exon 7. The frequency of the APOJ*2 (C) allele of codon 317 in the total sample was 0.267, whereas that of the less common allele A of codon 328 was 0.04. Despite their close proximity, no linkage disequilibrium was observed between the 2 polymorphisms. The impact of the codon 317 polymorphic variation was significant on serum HDL cholesterol (p = 0.003) and HDL3 cholesterol (p = 0.001) in junior staff. The adjusted mean values of these traits were higher in the codon 317 APOJ*2/*2 genotype than in the *1/*1 and *1/*2 genotypes. Overall, the APOJ codon 317 polymorphism explained 10.2% and 8.3% of the phenotypic variation in HDL cholesterol and HDL3 cholesterol, respectively, in junior staff. The codon 328 polymorphism showed a significant effect on HDL2 cholesterol (p = 0.039) and apoA1 (p = 0.007) only in junior women and accounted for 2.5% and 4.2% of the phenotypic variation in HDL2 cholesterol and apoA1, respectively. We also analyzed the combined effects of these genotypes at the 2 polymorphic sites. Significant effects on HDL cholesterol (p = 0.004) and HDL3 cholesterol (p = 0.008) in junior men and on HDL2 cholesterol (p = 0.003) in junior women were observed in the combined genotype data. The 2-locus genotypes explained 6.0% and 5.3% of the residual phenotypic variation of HDL cholesterol and HDL3 cholesterol in junior men and 10.4% of HDL2 cholesterol in junior women. These data indicate that the effect of the APOJ polymorphism on HDL cholesterol levels is modulated by socioeconomic status, as measured by staff level. Given the association of HDL and its subfractions with cardiovascular disease, these polymorphisms may lead to a better understanding of interracial differences in the risk of cardiovascular disease.     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.