Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.     

Heterologous expression of an alginate lyase from Streptomyces sp. ALG-5 in Escherichia coli and its use for preparation of the magnetic nanoparticle-immobilized enzymes

The marine alginate lyase from Streptomyces sp. ALG-5, which specifically degrades poly-G block of alginate, was functionally expressed as a His-tagged form with an Escherichia coli expression system. The recombinant alginate lyase expressed with pColdI at 15 °C exhibited the highest alginate-degrading activity. The recombinant alginate lyase was efficiently immobilized onto two types of magnetic nanoparticles, superparamagnetic iron oxide nanoparticle, and hybrid magnetic silica nanoparticle, based on the affinity between His-tag and Ni²⁺ that displayed on the surfaces of nanoparticles. An alginate oligosaccharide mixture consisting of dimer and trimer was prepared by the immobilized alginate lyase. The immobilized enzymes were re-used repeatedly more than 10 times after magnetic separation.     

Oncogenic CagA promotes gastric cancer risk via activating ERK signaling pathways: a nested case-control study

Background

CagA cellular interaction via activation of the ERK signaling pathway may be a starting point in the development of gastric cancer. This study aimed to evaluate whether genes involved in ERK downstream signaling pathways activated by CagA are susceptible genetic markers for gastric cancer.

Methods

In the discovery phase, a total of 580 SNPs within +/−5 kbp of 30 candidate genes were genotyped to examine an association with gastric cancer risk in the Korean Multi-center Cancer Cohort (100 incident gastric cancer case-control sets). The most significant SNPs (raw or permutated p value<0.02) identified in the discovery analysis were re-evaluated in the extension phase using unconditional logistic regression model (400 gastric cancer case-control sets). Combined analyses including pooled- and meta-analysis were conducted to summarize all the results.

Results

24 SNPs in eight genes (ERK, Dock180, C3G, Rap1, Src, CrkL, Mek and Crk) were significantly associated with gastric cancer risk in the individual SNP analyses in the discovery phase (p<0.05). In the extension analyses, ERK rs5999749, Dock180 rs4635002 and C3G rs7853122 showed marginally significant gene-dose effects for gastric cancer. Consistently, final combined analysis presented the SNPs as significantly associated with gastric cancer risk (OR = 1.56, [95% CI: 1.19–2.06], OR = 0.61, [95% CI: 0.43–0.87], OR = 0.59, [95% CI: 0.54–0.76], respectively).

Conclusions

Our findings suggest that ERK rs5999749, Dock180 rs4635002 and C3G rs7853122 are genetic determinants in gastric carcinogenesis.     

The multifunctional LigB adhesin binds homeostatic proteins with potential roles in cutaneous infection by pathogenic Leptospira interrogans

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9-11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats.     

Functional interaction between herpes simplex virus type 2 gD and HVEM transiently dampens local chemokine production after murine mucosal infection

Herpes virus entry mediator (HVEM) is one of two principal receptors mediating herpes simplex virus (HSV) entry into murine and human cells. It functions naturally as an immune signaling co-receptor, and may participate in enhancing or repressing immune responses depending on the natural ligand used. To investigate whether engagement of HVEM by HSV affects the in vivo response to HSV infection, we generated recombinants of HSV-2(333) that expressed wild-type gD (HSV-2/gD) or mutant gD able to bind to nectin-1 (the other principal entry receptor) but not HVEM. Replication kinetics and yields of the recombinant strains on Vero cells were indistinguishable from those of wild-type HSV-2(333). After intravaginal inoculation with mutant or wild-type virus, adult female C57BL/6 mice developed vaginal lesions and mortality in similar proportions, and mucosal viral titers were similar or lower for mutant strains at different times. Relative to HSV-2/gD, percentages of HSV-specific CD8(+) T-cells were similar or only slightly reduced after infection with the mutant strain HSV-2/gD-Δ7-15, in all tissues up to 9 days after infection. Levels of HSV-specific CD4(+) T-cells five days after infection also did not differ after infection with either strain. Levels of the cytokine IL-6 and of the chemokines CXCL9, CXCL10, and CCL4 were significantly lower in vaginal washes one day after infection with HSV-2/gD compared with HSV-2/gD-Δ7-15. We conclude that the interaction of HSV gD with HVEM may alter early innate events in the murine immune response to infection, without significantly affecting acute mortality, morbidity, or initial T-cell responses after lethal challenge.     

Functional role of kallikrein 6 in regulating immune cell survival

Background

Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur.

Methodology/Principal Findings

Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice.

Conclusion/Significance

KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis.     

Levosulpiride, (S)-(-)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl) methyl]-2-methoxybenzamide, enhances the transduction efficiency of PEP-1-ribosomal protein S3 in vitro and in vivo

Many proteins with poor transduction efficiency were reported to be delivered to cells by fusion with protein transduction domains (PTDs). In this study, we investigated the effect of levosulpiride on the transduction of PEP-1 ribosomal protein S3 (PEP-1-rpS3), and examined its influence on the stimulation of the therapeutic properties of PEP-1-rpS3. PEP-1-rpS3 transduction into HaCaT human keratinocytes and mouse skin was stimulated by levosulpiride in a manner that did not directly affect the cell viability. Following 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced inflammation in mice, levosulpiride alone was ineffective in reducing TPA-induced edema and in inhibiting the elevated productions of inflammatory mediators and cytokines, such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor- α. Anti-inflammatory activity by PEP-1-rpS3 + levosulpiride was significantly more potent than by PEP-1-rpS3 alone. These results suggest that levosulpiride may be useful for enhancing the therapeutic effect of PEP-1-rpS3 against various inflammatory diseases. [BMB reports 2011; 44(5): 329-334].