Differential activation of MAP kinase signaling pathways and nuclear factor-kappaB in bronchoalveolar cells of smokers and nonsmokers.

BACKGROUND: Prolonged exposure of alveolar macrophages (AM) to components of tobacco smoke, including nicotine and aromatic hydrocarbons, may lead to alterations in activation of cellular signaling pathways. In this study, we compared the spontaneous and LPS-stimulated activation of MAP kinases and NF-kappaB in bronchoalveolar cells (BAC) from smokers and nonsmokers. MATERIAL AND METHODS: BAC, which were predominantly comprised of AM, were obtained by bronchoalveolar lavage of healthy volunteering adult smokers and nonsmokers. Nuclear and cytoplasmic extracts were prepared from cell lysates. Activation of NF-kappaB was assessed by electrophoretic mobility shift assay. Degradation of the inhibitor of NF-kappaB (IkappaB) and total MAP kinases were assessed by Western blot analysis. Activation of MAP kinases, ERK, SAPK/JNK, and p38 were assessed by immunoprecipitation of cell lysates and kinase assays. RESULTS: LPS induced the activation of NF-kappaB in a dose-dependent manner, but BAC from smokers were approximately 10 times more sensitive, and showed faster kinetics of activation of NF-kappaB than BAC from nonsmokers. All three classes of MAP kinase-ERK, SAPK, and p38-were simultaneously activated by LPS in BAC from smokers and nonsmokers. However, the individual MAP kinases exhibited differential kinetics of activation. Activation of p38 was more rapid in BAC from smokers, whereas the activation of ERK and SAPK was similar in both groups. CONCLUSION: The differences in activation of NF-kappaB and MAP kinases in BAC from smokers and nonsmokers may relate to the differences in their microenvironment in situ as affected by chronic exposure to cigarette smoke. These differences may contribute to the increased susceptibility of smokers to infections, including infection with HIV-1, and lung disease.

Macrophage-activating cytokines in human immununodeficiency virus type 1-infected and -uninfected patients with pulmonary tuberculosis.

Tuberculosis (TB) is the most common opportunistic infection in human immunodeficiency virus type 1 (HIV-1)-infected patients globally and occurs throughout the course of HIV-1 disease. Here the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by peripheral blood mononuclear cells (PBMC) of HIV-1-infected versus -uninfected patients with newly diagnosed pulmonary TB (PTB) was compared. Findings were correlated with cytokine profiles, clinical presentation, and expression of inducible nitric oxide (iNOS). Most HIV-1/PTB patients with a CD4 cell count of 200-500 cells/microL had high IFN-gamma production and radiographic evidence of atypical PTB. Low IFN-gamma production and radiographic evidence of reactivated PTB characterized both HIV-1/PTB patients with a CD4 cell count >or=500 cells/microL and HIV-1-uninfected patients. TNF-alpha levels were similar in all HIV-1/PTB patients, regardless of CD4 cell count. Induction of iNOS in PBMC was low and was associated with low IFN-gamma production. These data underscore the potential pathogenic role of macrophage-activating cytokines in TB in HIV-1-infected patients.

Mechanisms of I(Ks) suppression in LQT1 mutants.

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K(+) channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K(+) current (I(Ks) )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I(Ks) currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I(Ks) current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Delta544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.

Endometriotic lesions synthesize and secrete a haptoglobin-like protein.

To explore the identity and possible function of endometriosis protein-I (ENDO-I), which is an acidic glycoprotein synthesized and secreted by endometriotic lesions, partial amino acid sequence and cDNA sequence were determined. Partially purified, de novo-synthesized rat endometriosis glycoproteins were separated by two-dimensional SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with Coomassie blue. Protein corresponding to the size and pI of ENDO-I was cut from the membranes and analyzed by automated Edman degradation. ENDO-I amino acid sequence analysis identified 15 residues that shared significant homology with the beta-chain of rat, mouse, and human haptoglobin (Hp) and human Hp-related protein. Western blot analyses using anti-Hp antibody demonstrated cross-reactivity with de novo-synthesized ENDO-I protein in endometriosis culture media. For nucleotide sequence analysis, poly A-enriched mRNA was isolated from rat endometriotic tissues. A gene-specific oligonucleotide primer was designed and used for 3' rapid amplification of cDNA ends (RACE). Automated sequencing of RACE cDNA fragments identified 859 base pairs, of which 858 were identical to rat Hp. Reverse transcription-polymerase chain reaction was used to demonstrate that ENDO-I transcripts are differentially expressed by endometriosis but not by uterine tissues. In the human, distinct subtypes of Hp as well as proteins sharing epitopes with Hp have been used to diagnose a variety of diseases; therefore, Hp-like ENDO-I may prove to be a nonsurgical diagnostic tool to assess endometriosis. Hepatic Hp, induced by acute-phase stimuli, modulates macrophage function and angiogenic activity. If ENDO-I possesses similar activities, it may be involved with anomalies of the immune system or the etiology and pathophysiology of endometriosis.

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida.

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature.

Partial purification and amino acid sequence analysis of endometriosis protein-II (ENDO-II) reveals homology with tissue inhibitor of metalloproteinases-1 (TIMP-1).

De novo synthesized endometriosis protein-II (ENDO-II; M(r) 28,000 to 32,000; pI 7.0 to 9.0) was partially purified from rat endometriotic tissue culture media using affinity chromatography and two-dimensional SDS-PAGE. The protein was electrophoretically transferred to polyvinyl difluoride membranes which were stained with Coomassie blue R-250. The stained protein corresponding to ENDO-II (M(r) 28,000 to 32,000; pI 7.0 to 9.0) was cut from the membranes for amino acid sequencing. Partial amino acid sequence was determined by automated Edman degradation using a gas phase sequencer and phenylthiohydantoin analyzer. Sequence analysis of ENDO-II yielded 25 residues: C S C A P T H P Q T A F C N S D L V I R K F M G. Comparison to sequence databanks demonstrated significant homology with rat (100%) and human (84%) tissue inhibitor of metalloproteinases-1 (TIMP-1). Western blot analysis using a TIMP-1 antibody confirmed amino acid sequence analysis. In conclusion, ENDO-II shares sequence homology with TIMP-1 and cross-reactivity with TIMP-1 antibody and subsequently identifies production of TIMP-1 by endometriotic tissues. The synthesis and secretion of TIMP-1 by endometriosis may derange the normal proteolytic milieu of the peritoneal cavity and contribute to the etiology and underlying physiological sequelae associated with the disease endometriosis.

Interaction of Shigella toxin with globotriaosyl ceramide receptor-containing membranes: a fluorescence study.

The interaction of the B-subunit of Shigella toxin with a globotriaosyl ceramide receptor incorporated into phosphatidylcholine vesicles was studied by fluorescence spectroscopy. From the position of the maximum in the emission spectrum and the accessibility to acrylamide quenching, it is concluded that a single tryptophan of a free B-chain is located in a highly polar environment, most likely on the surface of the folded polypeptide chain. Binding of B-subunits to the membrane-associated globotriaosyl ceramide results in a strong enhancement of fluorescence intensity and a small blue-shift of the emission maximum; these effects suggest a conformational change in the protein which provides a new environment to a tryptophan residue. However, the polarity of this new environment is still relatively high--as indicated by the position of the emission maximum at 344 nm--and suggests that the receptor-bound B-chain remains largely on the membrane surface, without penetrating the hydrophobic interior of a lipid bilayer. On the other hand, the A-chains are shown to interact directly with the receptor-free lipid bilayers; this nonspecific interaction may play a role in the mechanism by which A-subunit traverses the membrane of a target cell.

The metabolic heterogeneity of pathogenic and nonpathogenic coagulase negative staphylococci.

The coagulase negative staphylococci are a metabolically heterogenous group of strains. The investigated pathogenic and nonpathogenic strains differed markedly in respiration activity and character of endogenous metabolism.