Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.     

Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells.

The mutagenic potential of furfural was evaluated by means of the chromosome loss test in germ cells and the wing spot test in somatic cells of Drosophila melanogaster. The chromosome loss test was carried out employing repair-proficient as well as repair-deficient females. Males carried the compound Y chromosome, BSYy+. Two routes of administration were used: injection and feeding of adult males. Genetic damage was demonstrable after matings of treated males with females carrying the excision repair-deficient mutant mei-9a. The somatic mutation and recombination test was carried out treating 72-h transheterozygous mwh+/+flr3 larvae. Acute treatment of larvae was chosen as the method of exposure. Evidence indicates that furfural induces somatic damage as measured in the wing spot test.     

Biochemical changes in 24-month-old Wistar rats

Biogenic amines in pons and striatum have been dosed in twenty-four months old Wistar male rats. Choline acetyltransferase (CAT) and acetylcholine esterase (ACHesterase) activities, [3H] choline uptake by a synaptosomal preparation, [3H] quinuclidinyl benzylate (QNB) binding to muscarinic cholinergic receptors have been determined in hippocampus. Malondialdehyde (MDA) levels in plasma, in liver and in lungs and the characteristics of the skin collagen have been evaluated. Important differences are shown in comparison with three months old of the same strain animals.     

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy

Production of recombinant proteins, e.g. antibodies, requires constant real‐time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers. To date, this monitoring of mammalian cell culture‐based processes is done using laborious and time consuming enzyme‐linked immunosorbent assays (ELISA), two‐dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis, and chromatography‐based systems. Measurements are usually performed off‐line, requiring regular sample withdrawal associated with increased contamination risk. As information is obtained retrospectively, the reaction time to adapt to process changes is too long, leading to lower yield and higher costs. To address the resulting demand for continuous online‐monitoring systems, we present a feasibility study using attenuated total reflection spectroscopy (ATR) to monitor mAb and HCP levels of NS0 cell culture in situ, taking matrix effects into account. Fifty‐six NS0 cell culture samples were treated with polyelectrolytes for semi‐selective protein precipitation. Additionally, part of the samples was subjected to filtration prior to analysis, to change the background matrix and evaluate effects on chemometric quantification models. General models to quantify HCP and mAb in both filtered and unfiltered matrix showed lower prediction accuracy compared to models designed for a specific matrix. HCP quantification in the range of 2,000–55,000 ng mL^?1 using specific models was accurate for most samples, with results within the accepted limit of an ELISA assay. In contrast, mAb prediction was less accurate, predicting mAb in the range of 0.2–1.7 g L^?1. As some samples deviated substantially from reference values, further investigations elucidating the suitability of ATR for monitoring are required. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Microbiome Composition and Function Drives Wound-Healing Impairment in the Female Genital Tract

The mechanism(s) by which bacterial communities impact susceptibility to infectious diseases, such as HIV, and maintain female genital tract (FGT) health are poorly understood. Evaluation of FGT bacteria has predominantly been limited to studies of species abundance, but not bacterial function. We therefore sought to examine the relationship of bacterial community composition and function with mucosal epithelial barrier health in the context of bacterial vaginosis (BV) using metaproteomic, metagenomic, and in vitro approaches. We found highly diverse bacterial communities dominated by Gardnerella vaginalis associated with host epithelial barrier disruption and enhanced immune activation, and low diversity communities dominated by Lactobacillus species that associated with lower Nugent scores, reduced pH, and expression of host mucosal proteins important for maintaining epithelial integrity. Importantly, proteomic signatures of disrupted epithelial integrity associated with G. vaginalis-dominated communities in the absence of clinical BV diagnosis. Because traditional clinical assessments did not capture this, it likely represents a larger underrepresented phenomenon in populations with high prevalence of G. vaginalis. We finally demonstrated that soluble products derived from G. vaginalis inhibited wound healing, while those derived from L. iners did not, providing insight into functional mechanisms by which FGT bacterial communities affect epithelial barrier integrity.     

Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis

The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.     

Biocatalysis in ionic liquids - advantages beyond green technology

In recent years researchers have started to explore a particular class of organic solvents called room temperature ionic liquids - or simply ionic liquids - to identify their unique advantages for biocatalysis. Because they lack vapour pressure, ionic liquids hold potential as green solvents. Furthermore, unlike organic solvents of comparable polarity, they often do not inactivate enzymes, which simplifies reactions involving polar substrates such as sugars. Biocatalytic reactions in ionic liquids have also shown higher selectivity, faster rates and greater enzyme stability; however, these solvents present other challenges, among them difficulties in purifying ionic liquids and controlling water activity and pH, higher viscosity and problems with product isolation.