Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.     

Effects of Enriched Environment on COX-2, Leptin and Eicosanoids in a Mouse Model of Breast Cancer

Cyclooxygenase-2 (COX-2) and adipokines have been implicated in breast cancer. This study investigated a possible link between COX-2 and adipokines in the development of mammary tumors. A model of environmental enrichment (EE), known to reduce tumor growth was used for a syngeneic murine model of mammary carcinoma. 3-week-old, female C57BL/6 mice were housed in standard environment (SE) or EE cages for 9 weeks and transplanted orthotopically with syngeneic EO771 adenocarcinoma cells into the right inguinal mammary fat pad. EE housing influenced mammary gland development with a decrease in COX-2 expressing cells and enhanced side-branching and advanced development of alveolar structures of the mammary gland. Tumor volume and weight were decreased in EE housed mice and were associated with a reduction in COX-2 and Ki67 levels, and an increase in caspase-3 levels. In tumors of SE mice, high COX-2 expression correlated with enhanced leptin detection. Non-tumor-bearing EE mice showed a significant increase in adiponectin levels but no change in those of leptin, F₂-isoprostanes, PGF_2α, IL-6, TNF-α, PAI-1, and MCP-1 levels. Both tumor-bearing groups (SE and EE housing) had increased resistin, IL-6, TNF-α, PAI-1 and MCP-1 levels irrespective of the different housing environment demonstrating higher inflammatory response due to the presence of the tumor. This study demonstrates that EE housing influenced normal mammary gland development and inhibited mammary tumor growth resulting in a marked decrease in intratumoral COX-2 activity and an increase in the plasma ratio of adiponectin/leptin levels.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.     

Cuticle-degrading proteases of entomopathogenic fungi: from biochemistry to biological performance

Abstract

Entomopathogenic fungi are among the most successful biocontrol agents for preventing economic loss from insects. The identification of virulent species or isolates, the development of formulation technology and the improvement of efficiency are avenues being pursuing by researchers in diverse scientific disciplines. A successful entomopathogenic fungus deploys a combination of mechanical and biochemical processes to overcome the first defensive barrier in insects, the integument. A precise understanding of the mechanisms underlying fungal pathogenicity, particularly the roles of enzymes such as proteases, is essential in order to highlight the potential of entomopathogenic fungi and increase their virulence via genetic modifications. Cuticle-degrading proteases are divided into subtilisin-like (Pr1) and trypsin-like (Pr2) proteases, which are secreted in the initial stages of penetration. The biochemical structure contains the catalytic triad Asp39, His69 and Ser224 in addition to Ca²⁺ binding sites. Studies have shown a molecular weight of almost 19–47?kDa, an optimal pH of 7–12 and an optimal temperature of 35–45?°C. Different species or isolates of entomopathogenic fungi exhibit differences in the secretion and activity of cuticle-degrading proteases, which may indicate their virulence capacity. Genetic engineering techniques have been developed to create isolates with protease overexpression. Such isolates have significantly higher virulence against the host because they not only ensure fungal penetration but also exhibit direct toxicity to insects.     

Intracerebroventricular administration of vasoactive intestinal peptide inhibits food intake

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide expressed throughout the peripheral and central nervous systems. VIP and the VIP receptor VPAC(2)R are expressed in hypothalamic nuclei involved in the regulation of energy homeostasis. VIP has been shown to be involved in the regulation of energy balance in a number of non-mammalian vertebrates. We therefore examined the effects of intracerebroventricular (ICV) administration of VIP on food intake, energy expenditure and activity in adult male Wistar rats. VIP administration caused a potent short lived decrease in food intake and an increase in activity and energy expenditure. The pathways potentially involved in the anorexigenic effects of VIP were investigated by measuring the release of neuropeptides involved in the regulation of food intake from hypothalamic explants treated with VIP. VIP significantly stimulated the release of the anorexigenic peptide alpha-melanocyte stimulating hormone (αMSH). These studies suggest that VIP may have an endogenous role in the hypothalamic control of energy homeostasis.     

Assessment of IL-10, IL-1ß and TNF-α gene polymorphisms in patients with peri-implantitis and healthy controls

Molecular Biology Reports - Peri-implantitis (PI) is a multifactorial condition caused by the interactions of pathogens and the host immune response. Previous studies have demonstrated a...     

Frisking the whiskers: patterned sensory input in the rat vibrissa system

How are two prominent environmental features, surface texture and object location, transduced and encoded as rats whisk? Recent papers show that textures may excite intrinsic mechanical vibrations of the vibrissae. Although these vibrations are too rapid to be directly followed by cortical neurons, there is evidence that their speed is encoded by contact-dependent sensory signals. In addition to contact, sensory signals exist that report the angular position of the vibrissae. The combination of contact and reference signals may be used to decode spatial variations in the environment, particularly the location of objects in head-centered coordinates.