Structural analysis of the N‐terminal fragment of the antiangiogenic protein endostatin: A molecular dynamics study

Endostatin is a potent antiangiogenic protein derived from the noncollagenous domain 1 (NC1) of collagen XVIII. The mechanism by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid N‐terminal fragment of murine endostatin has antitumor, antimigration, and antipermeability activities comparable to the full soluble protein. To understand how this peptide can exert such elaborate function, we performed structural analysis using molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here, we show that the N‐terminal peptide of murine endostatin is able to assume a well‐defined structure, folding into a zinc‐dependent β‐hairpin conformation. Analyzing the folding mechanism, we were able to understand why the N‐terminal peptide of human endostatin with the same length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and of a shorter form of the human peptide with 25 residues. Finally, we show that the β‐hairpin conformation assumed by the zinc‐bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis inhibitors: the integrin‐binding portion of the NC1 domain of collagen IV. Indeed, our docking simulations show that arresten, canstatin, and the endostatin peptide bind to the same spot of αVβ3 integrin, suggesting similar interactions via a common binding site on this receptor. Proteins 2011;. © 2011 Wiley‐Liss, Inc.     

High-density genetic mapping for coffee leaf rust resistance

Coffee leaf rust caused by the fungus Hemileia vastatrix causes considerable economic losses for coffee producers. Although agrochemical products can provide sufficient disease control, the use of resistant cultivars is a safer alternative. This resistance may be constrained by one or a few genetic factors, mainly those found in material originating from interspecific hybrids. In this study, the genetic analysis of an F ₂ population consisting of 224 plants derived from a crossing of Híbrido de Timor UFV 427-15 (resistant) with Catuaí Amarelo IAC 30 (susceptible) showed that a dominant gene confers the resistance of coffee to race II of H. vastatrix. From a genetic map saturated with 25 amplified fragment length polymorphism (AFLP) markers linked to the resistance gene, we developed a high-density genetic map with six sequence-characterized amplified region (SCAR) markers delimiting a chromosomal region of 9.45 cM and flanking the dominant gene at 0.7 and 0.9 cM. This is the first saturated and high-density genetic map obtained from this region containing the resistance gene. The results of this study are of great importance for the introduction of molecular markers for marker-assisted selection; they will also facilitate studies related to the cloning, structure, and function of race-specific genes involved in the resistance of coffee trees to H. vastatrix.     

Host suitability, respiration rate and the outcome of larval competition in strains of the cowpea weevil, Callosobruchus maculatus

Abstract.  Variation between strains of Callosobruchus maculatus in several life-history traits is well known. Differences in functional anatomy of egg pores and larval respiration rates have also been reported in strains from Brazil and Yemen. The response of five strains of C. maculatus to seeds of two host species, cowpea ( Vigna unguiculata ) and mung bean ( Vigna radiata ), was measured along with the larval respiration rates of the same strains on both hosts. There was significant variability of response to the two hosts. Strains with higher larval respiration rate (µL O₂/insect/day) showed higher seed consumption, which significantly affected adult emergence per seed on both hosts. This finding provides support for the hypothesis that differential feeding rate is an important mechanistic component of the larval competition outcome observed in strains of C. maculatus .     

Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.     

Development of a bioadhesive nanoformulation with Glycyrrhiza glabra L. extract against Candida albicans

Abstract

Glycyrrhiza glabra L. is considered an important source of bioactive compounds. This study aimed at the development of an efficient solution for the treatment of oral candidiasis. Several extracts of Glycyrrhiza glabra L. were prepared using different solvents and their potential in vitro antifungal activity was assessed. Ethanolic extracts showed the most promising results against C. albicans. This extract was incorporated into mucoadhesive nanoparticles (PLA, PLGA and alginate), which were further included in an oral gel, an oral film and a toothpaste, respectively. The results showed that nanoparticles were successfully produced, presenting a mean size among 100–900?nm with high encapsulation efficiency. In vitro studies showed that the most bioadhesive formulation was the oral film with extract-loaded PLGA nanoparticles, followed by the toothpaste with extract-loaded alginate nanoparticles and the oral gel with extract-loaded PLA nanoparticles.     

Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.