Expression and functional characterization of the cancer-related serine protease, human tissue kallikrein 14

Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.     

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of beta-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse beta-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and beta-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow-derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that beta-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: beta-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.     

Prey‐mediated changes in the selectivity of the predator Macrolophus pygmaeus (Heteroptera: Miridae)

When foraging in communities with mixed prey, generalist predators may be confronted with prey species that differ in quality, size and mobility and interact with one another. To examine prey selection, predation by Macrolophus pygmaeus (Heteroptera: Miridae) was recorded by providing a diet of either one or two prey species of Myzus persicae (third‐instar nymphs), Aphis gossypii (fourth‐instar nymphs), Trialeurodes vaporariorum (third‐instar nymphs) and Ephestia kuehniella (eggs). In the experiments, prey mobility, prey quality and prey biomass were considered. The biomass consumed by the predator was dependent on the combination of prey species and the quantity of biomass offered. In choice experiments with diets mixed of two prey species at equal densities, the predation to A. gossypii was significantly reduced in the presence of E. kuehniella but the rate of consumption of M. persicae, T. vaporariorum and E.kuehniella was not significantly affected by the coexistence of any other species in the mixed prey diet. When equal amounts of biomass from two prey species were provided in combination, the total consumed biomass was significantly reduced in the mixed prey diets composed of E. kuehniella eggs and aphid nymphs. Thus, under the mixed‐prey situation, prey selection by predators may be affected by interactions among prey species differing in traits such as quality, mobility and size.     

Expression of Activated PIK3CA in Ovarian Surface Epithelium Results in Hyperplasia but Not Tumor Formation

Background

The Phosphatidylinositol 3′-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear.

Methodology/Principal Findings

Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras.

Conclusions/Significance

While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.     

A method for laboratory studies on the polyphagous predator Macrolophus pygmaeus (Hemiptera: Miridae)

A method for studying the life history parameters of Macrolophus pygmaeus Rambur was developed and described. Fecundity of the predator was studied by placing one female and one male in a plastic cylinder in which a stem piece was used as an ovipositional substrate. Smaller cylinders were used to study the percentage of egg hatch of M. pygmaeus. Results demonstrated that this method is suitable for studying the fecundity and egg eclosion of M. pygmaeus. Allowing for easy modification, the method offers suitable conditions for maintenance of both insect and plant material and can be inexpensively modified for different experiments and the parts are easily constructed. The proposed method could also be employed for similar studies on other predatory hemipterans such as mirids and anthocorids and very likely on other phytophagous insect species that lay their eggs in or on plant stems.