Carbonic anhydrase inhibitors. Benzenesulfonamides incorporating cyanoacrylamide moieties strongly inhibit Saccharomyces cerevisiae β-carbonic anhydrase

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogs) were assayed as inhibitors of the β-carbonic anhydrase (CA, EC 4.2.1.1) from Saccharomyces cerevisiae, ScCA. Some of these compounds were low nanomolar or subnanomolar ScCA inhibitors and showed selectivity ratios in the range of 4.91–69.86 for inhibiting the yeast enzyme over the offtarget human (h) isoforms hCA I and of 6.46–13.52 for inhibiting ScCA over hCA II. The model organism S. cerevisiae and this particular enzyme may be useful for detecting antifungals with a novel mechanism of action compared to the classical azole drugs to which significant drug resistance emerged. Indeed, some of these sulfonamides inhibited the growth of the yeast with CC₅₀-s in the range of 0.73–6.54 μM.     

An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells

Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric Env_IL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and Env_IL-21 may prove useful in improving antibody responses against HIV-1.     

Linkage and Association Mapping for Two Major Traits Used in the Maritime Pine Breeding Program: Height Growth and Stem Straightness

BackgroundIncreasing our understanding of the genetic architecture of complex traits, through analyses of genotype-phenotype associations and of the genes/polymorphisms accounting for trait variation, is crucial, to improve the integration of molecular markers into forest tree breeding. In this study, two full-sib families and one breeding population of maritime pine were used to identify quantitative trait loci (QTLs) for height growth and stem straightness, through linkage analysis (LA) and linkage disequilibrium (LD) mapping approaches.ResultsThe populations used for LA consisted of two unrelated three-generation full-sib families (n = 197 and n = 477). These populations were assessed for height growth or stem straightness and genotyped for 248 and 217 markers, respectively. The population used for LD mapping consisted of 661 founders of the first and second generations of the breeding program. This population was phenotyped for the same traits and genotyped for 2,498 single-nucleotide polymorphism (SNP) markers corresponding to 1,652 gene loci. The gene-based reference genetic map of maritime pine was used to localize and compare the QTLs detected by the two approaches, for both traits. LA identified three QTLs for stem straightness and two QTLs for height growth. The LD study yielded seven significant associations (P ≤ 0.001): four for stem straightness and three for height growth. No colocalisation was found between QTLs identified by LA and SNPs detected by LD mapping for the same trait.ConclusionsThis study provides the first comparison of LA and LD mapping approaches in maritime pine, highlighting the complementary nature of these two approaches for deciphering the genetic architecture of two mandatory traits of the breeding program.     

Simple and reliable detection of slime production of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">spp</Emphasis>. directly from blood culture bottles: Comparison of visual tube method and transmission electron microscopy

Early detection of slime production may be useful for clinical decision because of its suggestive property for potential pathogenic capacity of a Candida strain especially in patients with a prosthetic device. In this study we aimed to compare the visual tube method (VTM) with transmission electron microscopy (TEM) in order to confirm the reliability of the former method. In order to demonstrate the reproducibility of the tube method and to determine the correct timing for the test, Candida isolates directly obtained from blood culture (DBC) bottles and their two subsequent subcultures were used. The results of this study showed that VTM is a simple and reliable method which can be used in every clinical mycology laboratory, provided that the test is applied on DBC isolates; as the ability of slime production is decreased or lost even after the first subculturing. We suggest that this simple method can be used and may have some contributions to the ongoing studies on the controversial issue concerning removal of biomaterials in candidemic patients.     

Human genomics and microarrays: implications for the plastic surgeon

The Human Genome Project was launched in 1989 in an effort to sequence the entire span of human DNA. Although coding sequences are important in identifying mutations, the static order of DNA does not explain how a cell or organism may respond to normal and abnormal biological processes. By examining the mRNA content of a cell, researchers can determine which genes are being activated in response to a stimulus.Traditional methods in molecular biology generally work on a "one gene: one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain. To study each of the 60,000 to 80,000 genes in the human genome under each biological circumstance is not practical. Recently, microarrays (also known as gene or DNA chips) have emerged; these allow for the simultaneous determination of expression for thousands of genes and analysis of genome-wide mRNA expression.The purpose of this article is twofold: first, to provide the clinical plastic surgeon with a working knowledge and understanding of the fields of genomics, microarrays, and bioinformatics and second, to present a case to illustrate how these technologies can be applied in the study of wound healing.     

Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II

Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1–2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.     

The effect of aminoguanidine against cholestatic liver injury in rats

We investigated the protective role of aminoguanidine (AG) in rat liver injury induced by chronic biliary obstruction. Secondary biliary cirrhosis was induced by bile duct ligation for 14 days. Swiss albino rats were divided into three groups: Common bile duct ligated (CBDL) rats; Group A, CBDL rats treated with AG as Group B and simple laparotomy group known as the Sham group; Group C. Group B received 200 mg/kg of AG intraperitoneally daily throughout 14 days. The present data showed decreased gama glutamyl transferase (GGT), aspartate aminotransferase (AST), bilirubin and alanine aminotransferase (ALT) levels in the AG treated rats, when compared with CBDL rats (p < 0.05). In the AG treated rats, tissue levels of malondialdehyde (MDA) were significantly lower than that in CBDL rats (p < 0.001). Although the levels of glutathione (GSH) in AG treated rats were higher and myeloperoxidase (MPO) were lower than that in CBDL rats, the difference was not statistically significant (p > 0.05). The levels of interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were significantly lower and although the levels of interleukin-6 (IL-6) were lower in AG treated rats than that in CBDL rats, the difference was not statistically significant. Administration of AG in the rats with biliary obstruction resulted in inhibition of ductular proliferation and portal inflammation. The present study demonstrates that intraperitoneal administration of AG in CBDL rats maintains antioxidant defenses, reduces liver oxidative and cytokine damage and ductular proliferation and portal inflammation. This effect of AG may be useful in the preservation of liver injury in cholestasis.