Mutational analysis of the yeast multidrug resistance ABC transporter Pdr5p with altered drug specificity

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.     

Loss of heterozygosity and mutational analyses of the ACTRII gene locus in human colorectal tumors

The activin type II receptorgene (ACTRII) is mutated in 58.1% of microsatellite-unstable (MSI-H) colorectal cancers and is a close relative of the TGFbeta-1 type II receptor, which is known to be involved in both MSI-H and non-MSI-H colorectal carcinogenesis. We therefore sought to determine whether ACTRII was involved in non-MSI-H colorectal cancers. We evaluated ACTRII inactivation by allelic deletion, loss of mRNA expression, or somatic mutation in 51 non-MSI-H colon cancers. Loss of heterozygosity (LOH) at the ACTRII locus (2q23.1) was found in 9 (17.6%) of 51 primary tumors. Loss of ACTRII mRNA expression was seen in one (14.3%) of the seven LOH-positive primary tumors from which total RNA was available. We also performed DNA sequencing analysis of tumors showing LOH. One LOH-positive primary tumor exhibited a novel germline missense sequence alteration (amino acid substitution, 117 Ile to Phe) that was not found in 23 additional normal individuals, implying that this alteration is not a frequent polymorphism. We conclude that ACTRII is probably involved in both non-MSI-H and MSI-H colorectal carcinogenesis, but more frequently in the latter subgroup.     

Collagen-Carboxymethylcellulose Biocomposite Wound-Dressings with Antimicrobial Activity

Microbial infections associated with skin diseases are frequently investigated since they impact on the progress of pathology and healing. The present work proposes the development of freeze-dried, glutaraldehyde cross-linked, and non-cross-linked biocomposite dressings with a porous structure, which may assist the reepithelization process through the presence of collagen and carboxymethylcellulose, along with a therapeutic antimicrobial effect, due to silver nanoparticles (AgNPs) addition. Phisyco-chemical characterization revealed the porous morphology of the obtained freeze-dried composites, the presence of high crystalline silver nanoparticles with truncated triangular and polyhedral morphologies, as well as the characteristic absorption bands of collagen, silver, and carboxymethylcellulose. In vitro tests also assessed the stability, functionality, and the degradability rate of the obtained wound-dressings. Antimicrobial assay performed on Gram-negative (Escherichia coli), Gram-positive (Staphyloccocus aureus) bacteria, and yeast (Candida albicans) models demonstrated that composite wound dressings based on collagen, carboxymethylcellulose, and AgNPs are suitable for skin lesions because they prevent the risk of infection and have prospective wound healing capacity. Moreover, the cell toxicity studies proved that the obtained materials can be used in long time treatments, with no cytotoxic effects.     

Ligand-independent activation of vascular endothelial growth factor receptor 2 by fluid shear stress regulates activation of endothelial nitric oxide synthase

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm2) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.     

Ghrelin does not mediate the somatotroph and corticotroph responses to the stimulatory effect of glucagon or insulin-induced hypoglycaemia in humans

OBJECTIVE: Acylated ghrelin, a gastric peptide, possesses a potent GH- but also significant ACTH/cortisol-releasing activity mediated by the activation of GH secretagogue receptors (GHS-R) at the hypothalamus-pituitary level. The physiological role of ghrelin in the control of somatotroph and corticotroph function is, however, largely unclear. Glucagon is known to induce a clear increase of GH, ACTH and cortisol levels in humans, at least after intramuscular administration. In fact, glucagon is considered to be a classical alternative to insulin-induced hypoglycaemia (ITT) for the combined evaluation of the function of GH and the hypothalamus-pituitary-adrenal (HPA) axis. We aimed to clarify whether ghrelin mediate the GH and corticotroph responses to intramuscular glucagon or ITT, which has recently been reported able to induce a surprising ghrelin decrease. SUBJECTS: To this aim we enrolled six normal young male subjects [age (mean +/- SD): 29.0 +/- 8.0 years, body mass index (BMI) 21.9 +/- 2.5 kg/m(2)]. DESIGN AND MEASUREMENTS: In all the subjects we studied ghrelin, GH, ACTH, cortisol and glucose levels after glucagon (GLU; 0.017 mg/kg intramuscularly), ITT (0.1 IU/kg insulin intravenously) or saline administration. RESULTS: Saline infusion was not followed by any significant variation in ghrelin, GH and glucose levels while ACTH and cortisol showed the expected spontaneous morning trend toward a decrease. GLU administration increased (P < 0.01) circulating GH, ACTH and cortisol as well as insulin and glucose levels. ITT induced an obvious increase (P < 0.01) of GH, ACTH and cortisol levels. The ITT-induced increases in GH and ACTH, but not cortisol, levels were higher (P < 0.01) than those after GLU. Circulating ghrelin levels were not modified by GLU. On the other hand, ghrelin levels underwent a transient reduction (P < 0.01) after insulin-induced hypoglycaemia. CONCLUSIONS: Ghrelin does not mediate the GH and ACTH responses to glucagon or to the ITT. In fact, ghrelin levels are not modified at all by glucagon and transiently decrease during the ITT. These findings support the assumption that ghrelin does not play a major role in the physiological control of somatotroph and corticotroph function.