Production and characterisation of monoclonal antibodies to ovine interleukin-5

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.     

Non-intertwined binary patterns of hydrophobic/nonhydrophobic amino acids are considerably better markers of regular secondary structures than nonconstrained patterns

Patterns of hydrophobic and hydrophilic residues (binary patterns) play an important role in protein architecture and can be roughly categorized into two classes regarding their preferential participation in α‐helices or β‐strands. However, a single binary pattern can be embedded into different longer patterns carrying opposite structural information and thus cannot be as much informative as expected. Here, we consider conditional binary patterns, or hydrophobic clusters, whose existence is conditioned by the presence of a minimum number of nonhydrophobic residues, called the connectivity distance, that separate two hydrophobic amino acids assumed to belong to two distinct patterns. Conditional binary patterns are distinct from simple ones in that they are not intertwined, i.e., they can not include or be included in other conditional patterns and therefore carry a much more differentiated information, in particular being dramatically better correlated with regular secondary structures (especially β ones). The distribution of these nonintertwined binary patterns in natural proteins was assessed relative to randomness, evidencing the structural bricks that are favored and disfavored by evolutionary selection. Several connectivity distances as well as several hydrophobic alphabets were tested, evidencing the clear superiority of a connectivity distance of 4, which mimics the minimum current length of loops in globular domains, and of the VILFMYW alphabet, selected from structural data (secondary structure propension and Voronoï tesselation), in highlighting fundamental properties of protein folds. Proteins 2003;51:236–244. © 2003 Wiley‐Liss, Inc.     

A new protein folding algorithm based on hydrophobic compactness: Rigid Unconnected Secondary Structure Iterative Assembly (RUSSIA). II: Applications

The RUSSIA procedure (Rigid Unconnected Secondary Structure Iterative Assembly) produces structural models of cores of small- and medium-sized proteins. Loops are omitted from this treatment and regular secondary structures are reduced to points, the centers of their hydrophobic faces. This methodology relies on the maximum compactness of the hydrophobic residues, as described in detail in Part I. Starting data are the sequence and the predicted limits and natures of regular secondary structures (alpha or beta). Helices are treated as rigid cylinders, whereas beta-strands are collectively taken into account within beta-sheets modeled by helicoid surfaces. Strands are allowed to shift along their mean axis to allow some flexibility and the alpha-helices can be placed on either side of beta-sheets. Numerous initial conformations are produced by discrete rotations of the helices and sheets around the direction going from the center of their hydrophobic face to the global center of the protein. Selection of proposed models is based upon a criterion lying on the minimization of distances separating hydrophobic residues belonging to different regular secondary structures. The procedure is rapid and appears to be robust relative to the quality of starting data (nature and length of regular secondary structures). This dependence of the quality of the model on secondary structure prediction and in particular the beta-sheet topology, is one of the limits of the present algorithm. We present here some results for a set of 12 proteins (alpha, beta and alpha/beta classes) of lengths 40-166 amino acids. The r.m.s. deviations for core models with respect to the native proteins are in the range 1.4-3.7 A.     

Myc-Induced Liver Tumors in Transgenic Zebrafish Can Regress in tp53 Null Mutation

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53^-/- mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53^-/- mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53.     

Relationship between Body Mass Index and Percent Body Fat in Vietnamese: Implications for the Diagnosis of Obesity

BackgroundThe burden of obesity in Vietnam has not been well defined because there is a lack of reference data for percent body fat (PBF) in Asians. This study sought to define the relationship between PBF and body mass index (BMI) in the Vietnamese population.MethodsThe study was designed as a comparative cross-sectional investigation that involved 1217 individuals of Vietnamese background (862 women) aged 20 years and older (average age 47 yr) who were randomly selected from the general population in Ho Chi Minh City. Lean mass (LM) and fat mass (FM) were measured by DXA (Hologic QDR 4500). PBF was derived as FM over body weight.ResultsBased on BMI ≥30, the prevalence of obesity was 1.1% and 1.3% for men and women, respectively. The prevalence of overweight and obesity combined (BMI ≥25) was ~24% and ~19% in men and women, respectively. Based on the quadratic relationship between BMI and PBF, the approximate PBF corresponding to the BMI threshold of 30 (obese) was 30.5 in men and 41 in women. Using the criteria of PBF >30 in men and PBF >40 in women, approximately 15% of men and women were considered obese.ConclusionThese data suggest that body mass index underestimates the prevalence of obesity. We suggest that a PBF >30 in men or PBF >40 in women is used as criteria for the diagnosis of obesity in Vietnamese adults. Using these criteria, 15% of Vietnamese adults in Ho Chi Minh City was considered obese.     

A new rhodamine‐based fluorescent chemodosimeter for mercuric ions in water media

A new rhodamine–ethylenediamine–nitrothiourea conjugate (RT) was synthesized and its sensing property as a fluorescent chemodosimeter toward metal ions was explored in water media. Analytical results from absorption and fluorescence spectra revealed that the addition of Hg²⁺ ions to the aqueous solution of the chemodosimeter RT caused a distinct fluorescence OFF–ON response with a remarkable visual color change from colorless to pink; however, no clear spectral and color changes were observed from other metal ions including: Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Ag⁺, Fe²⁺, Cr³⁺, Co³⁺, Ni²⁺, Ca²⁺, Mg²⁺, K⁺ and Na⁺. The sensing results and the molecular structure suggested that a Hg²⁺‐induced a desulfurization reaction and cyclic guanylation of the thiourea moiety followed by ring‐opening of the rhodamine spirolactam in RT are responsible for a distinct fluorescence turn‐on signal, indicating that RT is a remarkably sensitive and selective chemodosimeter for Hg²⁺ ions in aqueous solution. Hg²⁺ within a concentration range from 0.1 to 25 μM can be determined using RT as a chemodosimeter and a detection limit of 0.04 μM is achieved. Copyright © 2014 John Wiley & Sons, Ltd.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

New stilbenoid with inhibitory activity on viral neuraminidases from Erythrina addisoniae

Influenza occurs with seasonal variations and reaches the peak prevalence in winter causing the death of many people worldwide. A few inhibitors of viral neuraminidase, including amantadine, rimantadine, zanamivir, and oseltamivir, have been used as influenza therapy. However, as drug-resistant influenza viruses are generated rapidly, there is a need to identify new agents for chemotherapy against influenza. Therefore, research on more effective drugs has been given high priority. During the course of an anti-influenza screening program on natural products, two new compounds (1 and 2) along with seven known flavonoid derivatives (3-9) were isolated as active principles from an EtOAc-soluble extract of the root bark of Erythrina addisoniae. The stilbenoid (2) and chalcone (3, 4, and 6) compounds of the isolates exhibited stronger activity than the isoflavone ones. Compound 2, which is a formylated stilbenoid derivative, exhibited strong inhibition of both influenza H1N1 and H9N2 neuraminidases with IC(50) values of 8.80±0.34 μg/mL and 7.19±0.40 μg/mL, respectively.     

Therapeutic potential of the immunomodulatory activities of adult mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) include a select population of resident cells within adult tissues, which retain the ability to differentiate along several tissue‐specific lineages under defined media conditions and have finite expansion potential in vitro. These adult progenitor populations have been identified in various tissues, but it remains unclear exactly what role both transplanted and native MSCs play in processes of disease and regeneration. Interestingly, increasing evidence reveals a unique antiinflammatory immunomodulatory phenotype shared among this population, lending support to the idea that MSCs play a central role in early tissue remodeling responses where a controlled inflammatory response is required. However, additional evidence suggests that MSCs may not retain infinite immune privilege and that the context with which these cells are introduced in vivo may influence their immune phenotype. Therefore, understanding this dynamic microenvironment in which MSCs participate in complex feedback loops acting upon and being influenced by a plethora of secreted cytokines, extracellular matrix molecules, and fragments will be critical to elucidating the role of MSCs in the intertwined processes of immunomodulation and tissue repair. Birth Defects Research (Part C) 90:67–74, 2010. © 2010 Wiley‐Liss, Inc.