PAI-1 gain-of-function genotype, factors increasing PAI-1 levels, and airway obstruction: The GALA II Cohort.

BACKGROUND: PAI-1 gain-of-function variants promote airway fibrosis and are associated with asthma and with worse lung function in subjects with asthma. OBJECTIVE: We sought to determine whether the association of a gain-of-function polymorphism in plasminogen activator inhibitor-1 (PAI-1) with airway obstruction is modified by asthma status, and whether any genotype effect persists after accounting for common exposures that increase PAI-1 level. METHODS: We studied 2070 Latino children (8-21y) with genotypic and pulmonary function data from the GALA II cohort. We estimated the relationship of the PAI-1 risk allele with FEV1/FVC by multivariate linear regression, stratified by asthma status. We examined the association of the polymorphism with asthma and airway obstruction within asthmatics via multivariate logistic regression. We replicated associations in the SAPPHIRE cohort of African Americans (n=1056). Secondary analysis included the effect of the at-risk polymorphism on postbronchodilator lung function. RESULTS: There was an interaction between asthma status and the PAI-1 polymorphism on FEV1 /FVC (P=.03). The gain-of-function variants, genotypes (AA/AG), were associated with lower FEV1 /FVC in subjects with asthma (ß=-1.25, CI: -2.14,-0.35, P=.006), but not in controls. Subjects with asthma and the AA/AG genotypes had a 5% decrease in FEV1 /FVC (P<.001). In asthmatics, the risk genotype (AA/AG) was associated with a 39% increase in risk of clinically relevant airway obstruction (OR=1.39, CI: 1.01, 1.92, P=.04). These associations persisted after exclusion of factors that increase PAI-1 including tobacco exposure and obesity. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in the FEV1 /FVC ratio associated with the risk genotype was modified by asthma status. The genotype increased the odds of airway obstruction by 75% within asthmatics only. As exposures known to increase PAI-1 levels did not mitigate this association, PAI-1 may contribute to airway obstruction in the context of chronic asthmatic airway inflammation.

Caveolin-1 serves as a negative effector in senescent human gingival fibroblasts during Fusobacterium nucleatum infection.

It is well established that aging is associated with increased susceptibility to infectious diseases. Fusobacterium nucleatum is a well-known bacterial species that plays a central bridging role between early and late colonizers in the human oral cavity. Further, the ability of F. nucleatum to invade gingival fibroblasts (GFs) is critical to the development of periodontal diseases. However, the mechanisms underlying the age-related infection of GFs by F. nucleatum remain unknown. We used young (fourth passage) and senescent (22nd passage) GFs to investigate the mechanisms of F. nucleatum infection in aged GFs and first observed increased invasion of F. nucleatum in senescent GFs. We also found that the co-localization of caveolin-1 (Cav-1), a protein marker of aging, with F. nucleatum and the knockdown of Cav-1 in GFs reduced F. nucleatum invasion. Additionally, F. nucleatum infection triggered the production of reactive oxygen species (ROS) through activation of NADPH oxidase in GFs, but senescent GFs exhibited significantly lower levels of NADPH oxidase activity and ROS production compared with young GFs in both the uninfected and infected conditions. Also, senescent GFs exhibited a decline in proinflammatory cytokine production and extracellular signal regulated kinase (ERK) phosphorylation following F. nucleatum infection. Interestingly, the knockdown of Cav-1 in senescent GFs increased NADPH oxidase activity and caused the upregulation of interleukin-6 and interleukin-8 and the phosphorylation of ERK. Collectively, the increased expression of Cav-1 might play a critical role in F. nucleatum invasion and could hinder the host response in senescent GFs.

Neuroprotection using gene therapy to induce vascular endothelial growth factor-A expression.

Engineered zinc-finger protein (ZFP) transcription factors induce the expression of endogenous genes and can be remotely delivered using adenoviral vectors. One such factor, Ad-32Ep65-Flag (Ad-p65), targets and induces expression of vascular endothelial growth factor (VEGF; also called VEGF-A) splice variants in their normal biological stoichiometry. We show that Ad-p65 transfection of primary motor neurons results in VEGF variant expression and a significant increase in axon outgrowth in these cells. Given the neuroprotective effects of VEGF and its ability to increase neurite outgrowth, we examined the efficacy of Ad-p65 to enhance motor neuron regeneration in vivo using rats that have undergone recurrent laryngeal nerve (RLN)-crush injury. Injection of Ad-p65 after RLN crush accelerated the return of vocal fold mobility and the percentage of nerve-endplate contacts in the thyroarytenoid muscle. Overall, adenoviral delivery of an engineered ZFP transcription factor inducing VEGF-A splice variant expression enhances nerve regeneration. ZFP transcription factor gene therapy to increase expression of the full complement of VEGF-A splice variants is a promising avenue for the treatment of nerve injury and neurodegeneration.

Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes.

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.

A case of localized persistent interstitial pulmonary emphysema.

Interstitial pulmonary emphysema is a well-documented complication of assisted mechanical ventilation in premature infants with respiratory distress syndrome. Localized persistent interstitial pulmonary emphysema (LPIPE) confined to a single lobe was incidentally presented in a 4-day-old female infant. This patient was a normal full-term baby with no respiratory distress symptom and no experience of assisted mechanical ventilation. Chest radiograph showed radiolucent area in right lower lobe zone, which needed differential diagnosis from other congenital lesions such as congenital cystic adenomatoid malformation and congenital lobar emphysema. CT scan showed irregular-shaped air cystic spaces and pathologically, cystic walls primarily consisted of compressed lung parenchyma and loose connective tissue intermittently lined by multinucleated foreign body giant cells.

Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte skeleton.

A distinctive pathological feature of Plasmodium falciparum malaria is the endothelial attachment of erythrocytes infected with mature asexual-stage parasites in microvessels of the major organs. Electron-dense protrusions described as knobs are displayed on the surface of parasitized erythrocytes and act as attachment points in cytoadherence. Parasite-encoded knob-associated histidine-rich protein (KAHRP) is a major component of knobs found on the cytoplasmic side of the host cell membrane. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of parasite-encoded cytoadherence receptors localized to knobs on the surface of parasitized erythrocytes. Despite its high antigenic diversity, PfEMP1 has a remarkably conserved cytoplasmic domain. We demonstrate in this study that the cytoplasmic domain of PfEMP1 (VAR(CD)) binds to host spectrin and actin and to full-length KAHRP in vitro. Apparent dissociation constants determined for VAR(CD)/F-actin and VAR(CD)/KAHRP interactions are 44.9+/-6.4 and 10. 7+/-2.2 nM, respectively. Further, we provide evidence that KAHRP polypeptides self-associate in solution to form structures similar to knobs and show binding of self-associated KAHRP clusters to spectrin-actin-protein 4.1 complexes. Findings in this study suggest that PfEMP1 is localized to the knob in P. falciparum-infected erythrocytes by binding to the host spectrin-actin junction and to self-associated KAHRP through its conserved cytoplasmic domain.

Human homologue of the Drosophila discs large tumor suppressor binds to p56lck tyrosine kinase and Shaker type Kv1.3 potassium channel in T lymphocytes.

Human homologue of the Drosophila discs large tumor suppressor protein (hDlg) belongs to a newly discovered family of proteins termed MAGUKs that appear to have structural as well as signaling functions. Consistent with the multi-domain organization of MAGUKs, hDlg consists of three copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate kinase-like domain. In addition, the hDlg contains an amino-terminal proline-rich domain that is absent in other MAGUKs. To explore the role of hDlg in cell signaling pathways, we used human T lymphocytes as a model system to investigate interaction of hDlg with known tyrosine kinases. In human T lymphocyte cell lines, binding properties of hDlg were studied by immunoprecipitation, immunoblotting, and immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg. Immunoblotting experiments revealed that the immunoprecipitates of hDlg contain p56lck, a member of the Src family of tyrosine kinases. The specificity of the interaction is demonstrated by the lack of p59fyn tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunoprecipitates. Direct interaction between hDlg and p56lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56lck expressed in the baculovirus-infected Sf9 cells. The p56lck binding site was localized within the amino-terminal segment of hDlg containing proline-rich domain. In addition, we show in vivo association of hDlg with Kv1.3 channel, which was expressed in T lymphocytes as an epitope-tagged protein using a vaccinia virus expression system. Taken together, these results provide the first evidence of a direct interaction between hDlg and p56lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.

Effects of somatostatin and somatotropin on the in vitro testicular steroidogenesis in hamster.

Adult hamsters were exposed to short-photoperiod, and injected with either somatotropin (GH), somatostatin (GHRIH), or saline for eight weeks. Hamster testis fragments of similar size were incubated with or without hCG. No significant differences in the basal media testosterone and estradiol levels were observed among groups. Treatment with GH potentiated the hCG-dependent increase in media testosterone. Contrary to what was expected, treatment with GHRIH did not only not reduce the hCG-related elevation in media testosterone, but even produced a numerical increase of it. Treatment with GHRIH potentiated the hCG-dependent increase in media estradiol, whereas treatment with GH produced only a numerical increase of the response. Furthermore, the combined exposure to GHRIH and hCG appeared to cause an increase in the efficiency of testicular aromatase. Since previous data indicated that the combined deficiency of lactotropic and somatotropic actions severely impairs testicular steroidogenesis, treatment with GHRIH should have caused further steroidogenic impairment in hamsters exposed to short-photoperiod. Since this does not appear to be the case, it could be postulated that GHRIH has a direct stimulatory or at least a protective effect on testicular steroidogenesis.

Mechanism of human placental aromatase: a new active site model.

Based on Akhtar's ferric peroxide mechanism and on recent studies in our own laboratory, we present a detailed proposal for aromatase action. This picture can account for the known stereochemical consequences at C-19 observed by others using isotopes of hydrogen and oxygen. The postulated process involves anchoring of the 19-hydroxymethyl and 19-oxo groups at the active site by a glutamate residue, which also serves to activate the 19-oxo group for attack by ferric peroxy species in the third oxidative step.