Overexpression,Purification and Functional Characterisation of Wild-Type HIV-1 Subtype C Protease and Two Variants Using a Thioredoxin and His-Tag Protein Fusion System

In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression of large quantities is prevented due to the enzyme’s autolytic nature and its inherent cytotoxicity in Escherichia coli cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin–hexahistidine fusion system for the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wild-type protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage of the thioredoxin–hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after cleavage of the tag. The novel thioredoxin–hexahistidine fusion system, therefore, enables the successful overexpression and purification of catalytically active HIV-1 proteases.     

The Isomerization of Δ5-Androstene-3,17-dione by the Human Glutathione Transferase A3-3 Proceeds via a Conjugated Heteroannular Diene Intermediate

The seemingly simple proton abstraction reactions underpin many chemical transformations, including isomerization reactions, and are thus of immense biological significance. Despite the energetic cost, enzyme-catalyzed proton abstraction reactions show remarkable rate enhancements. The pathways leading to these accelerated rates are numerous and on occasion partly enigmatic. The isomerization of the steroid Δ⁵-androstene-3,17-dione by the glutathione transferase A3-3 in mammals was investigated to gain insight into the mechanism. Particular emphasis was placed on the nature of the transition state, the intermediate suspected of aiding this process, and the hydrogen bonds postulated to be the stabilizing forces of these transient species. The UV-visible detection of the intermediate places this species in the catalytic pathway, whereas fluorescence spectroscopy is used to obtain the binding constant of the analog intermediate, equilenin. Solvent isotope exchange reveals that proton abstraction from the substrate to form the intermediate is rate-limiting. Analysis of the data in terms of the Marcus formalism indicates that the human glutathione transferase A3-3 lowers the intrinsic kinetic barrier by 3 kcal/mol. The results lead to the conclusion that this reaction proceeds through an enforced concerted mechanism in which the barrier to product formation is kinetically insignificant.     

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay.     

Bridelia ferruginea Inhibit Rat Heart and Liver Mitochondrial Membrane Permeability Transition Pore Opening Following Myocardial Infarction

International Journal of Peptide Research and Therapeutics - Despite advances in therapy, myocardial infarction (MI) remains a leading cause of death worldwide. Recently, the mitochondrion has been...     

Antifungal activity of microbial secondary metabolites

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.     

Climber species composition, abundance and relationship with trees in a Nigerian secondary forest

Climbers play different roles in forest biology and ecology and are the first to be eliminated during forest clearing but little is known about the species composition, distribution and relationship with tree species of this group of plants of tropical forest. This study thus investigated the species composition, abundance and tree relationship of climbers along altitudinal gradient in four 0.06 ha plots in a secondary forest at Ile‐Ife, Nigeria. All trees ≥10 cm g.b.h were examined for the presence of climbers in the plots. There were 49 climber species consisting of 35 liana and fourteen vine species distributed over 41 genera and 28 families in the forest. Lianas contributed 34% and vines 13.7% of the plant species in the forest. Climber basal area, density, number of species, genera and families increased with altitude. Forty‐two per cent (42%) of the trees in the forest carried climbers. There was significant positive correlation (P ≤ 0.05) between girth sizes of host trees of 31–50 cm with the girths of climbers on them indicating that trees of these girth sizes are highly susceptible to climber infestation. Tree species host density and size are important factors in determining the presence of climbers on a tree.     

Analysis of perillic acid in plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO₃. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C₁₈ column and the effluent monitored for UV absorption at the analytes' respective UV_max. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 μg/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.     

Cryptotrichosporon anacardii gen. nov., sp. nov., a new trichosporonoid capsulate basidiomycetous yeast from Nigeria that is able to form melanin on niger seed agar

Five yeast isolates obtained from cashew tree flowers in Nigeria resembled Cryptococcus neoformans phenotypically by producing brown pigmented colonies on niger seed agar, expressing a capsule, and being able to grow at 37 degrees C. However, rRNA gene sequences, including the 18S rRNA gene, the D1/D2 domains of the 26S rRNA gene and the ITS1+2 regions, suggested that these yeasts form a basal lineage within the Trichosporonales (Tremellomycetidae, Hymenomycetes, Basidiomycota, Fungi). Since the isolates could not be identified with any known genus and species within the Trichosporonales, we describe them as Cryptotrichosporon anacardii gen. et sp. nov. with CBS 9551(T) (=NRRL Y-27671) as the type strain. The taxonomic conflict between phenetic and molecular classification schemes within this group of fungi is discussed, and is resolved in favor of the latter.     

T1alpha/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.