A modeled structure of an aptamer-gp120 complex provides insight into the mechanism of HIV-1 neutralization.

The HIV-1 envelope glycoprotein, gp120, is a key target for a class of drugs called entry inhibitors. Here we used molecular modeling to construct a three-dimensional model of an anti-gp120 RNA aptamer, B40t77, alone and in complex with gp120. An initial model of B40t77 was built from the predicted secondary structure and then subjected to a combination of energy minimization and molecular dynamics. To model the B40t77-gp120 complex, we docked the B40t77 predicted structure onto the CD4-induced epitope of the gp120 crystal structure. A series of gp120 point mutations in the predicted B40t77-gp120 interface were measured for their binding affinity for B40t77 by surface plasmon resonance. According to the model, of the 10 gp120 amino acids that showed a reduction in the level of binding when mutated to alanine, all of them are modeled as making direct contact with B40t77 as part of a hydrogen bonding network. Comparison by electron microscopy of the B40t77-gp120 complex with gp120 alone revealed that only the longest dimension of the complex significantly increased in length, in a manner consistent with the predicted model. Binding assays revealed that B40t77 can weaken the binding of gp120 to the monoclonal antibodies B6, B12, and 2G12, none of which have binding sites that overlap with B40t77, as well as strengthen the binding to the antibody 19b. Thus, B40t77 may induce distant conformational changes in gp120 that disrupt its association with host cells and may suggest a mechanism for aptamer neutralization of HIV-1.

A deletion and point mutation study of the human papillomavirus type 16 major capsid gene.

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A-->T mutation at residue 266 (A266T), and of a C-->G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428-483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2-9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428-465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.

A Gel Filtration-Based Method for the Purification of Infectious Rotavirus Particles for Environmental Research Applications.

This article describes a rapid method for purifying infectious rotavirus particles from cell culture for environmental research. The method is based on size-exclusion chromatography using TOSOH TSKgel® G5000PWXL-CP with a TSKgel® Size Exclusion G2500PWxl guard column, set up on an AKTA Explorer10. Four peaks were identified from the chromatogram and the corresponding fractions were collected and analysed by electron microscopy, 1-step quantitative reverse transcription polymerase chain reaction (RT-PCR) and qNano measurement. Infectivity potential of the recovered virus particles was determined using cell culture. Our analysis reveals that the first fraction contains majority of the intact triple-layered infectious virions while the other three fractions contain mixtures of empty capsids and intact infectious virions. Our results also indicate that there is a gross overestimation of rotaviruses in crude extracts due to encapsidated RNA in the order of 2.3 × 1011 particles and we note that estimates by qNano are similarly skewed (1.36 × 1013 particle) possibly due to empty capsids and cellular debris. In summary we present a method for purification (~12 h) of rotaviruses for a more robust and accurate quantification of virus size, surface charge and particle concentration in environmental contexts.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins.

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size ( approximately 55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb.

Investigation of the role of a beta(1-4) agarase produced by Pseudoalteromonas gracilis B9 in eliciting disease symptoms in the red alga Gracilaria gracilis.

Gracilaria species are an important source of agar. The South African Gracilaria industry has experienced a number of setbacks over the last decade in the form of complete or partial die-offs of the agarophyte growing in Saldanha Bay, which may be attributed to bacterial infection. Since a positive correlation was observed between the presence of agarolytic epiphytes and bacterial pathogenicity, we investigated the role of an agarase in the virulence mechanism employed by a bacterium that elicits disease in Gracilaria gracilis. The recombinant plasmid pDA1, isolated from a Pseudoalteromonas gracilis B9 genomic library, was responsible for the agarolytic activity exhibited by Escherichia coli transformants when grown on solid medium. A BLAST search of the GenBank database showed that an 873 bp ORF (aagA) located on pDA1 had 85 % identity to the beta-agarase (dagA) from Pseudoalteromonas atlantica ATCC 19262(T) (or IAM 12927(T)) at the amino acid level. AagA was purified from the extracellular medium of an E. coli transformant harbouring pDA1 by using a combination of gel filtration and ion-exchange chromatography. AagA has an M(r) of 30 000 on SDS-PAGE. TLC of the digestion products of AagA showed that the enzyme cleaves the beta-(1,4) linkages of agarose to yield predominately neoagarotetraose. Western hybridization confirmed that the cloned agarase was in fact the extracellular beta-agarase of P. gracilis B9. The observed relationship between disease symptoms of G. gracilis and the agarolytic phenotype of P. gracilis B9 was confirmed. Transmission electron microscope examination of cross sections of both healthy G. gracilis and G. gracilis infected with P. gracilis, revealed a weakening of the cell structure in the latter plants. Immunogold-labelled antibodies localized the agarase in situ to the cell walls of bleached G. gracilis. Thus, the weakening observed in the cell structure of G. gracilis infected with P. gracilis can be attributed to degradation of the mucilaginous component of the cell wall of the bleached thalli.

An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccation-tolerant and -sensitive tissues.

The ability of tissues to survive desiccation is common in seeds but rare in vegetative tissues. In this study the ultrastructure of hydrated and dehydrated tissues were examined at different stages of the life cycle of the resurrection grass, Eragrostis nindensis Ficalho & Hiern. Conventional fixation techniques are unsuitable for dry tissues as rehydration occurs during fixation in aqueous fixatives. Thus a cryofixation and freeze-substitution method was developed. As a result of the improved fixation methods, it was possible to identify the stage and nature of the damage in the desiccation-sensitive tissues. E. nindensis has desiccation-tolerant orthodox seeds, but the young seedlings are not tolerant to extreme water loss. However, like the seeds, most of the leaves of the adult plant are tolerant to desiccation (only the oldest outermost leaf on a tiller are not). Desiccation-induced damage in these outer leaves was observed in the later stage of dehydration, dominated by the appearance of abundant cell wall fractures (1 wall fracture per 50 µm2). Unlike the outer leaves, the leaves of seedlings appeared similar to those of the hydrated ones upon desiccation. Irreparable damage occurred on rehydration of these tissues possibly as a result of the absence of protection mechanisms observed during desiccation of the inner desiccation-tolerant leaves of the mature plants. The mesophyll tissues of these leaves become compact with extensive cell wall folding on drying. The bundle sheath cells maintained their shape with desiccation but became packed with small vacuoles.

Fibrinogen is degraded and internalized during incubation with neutrophils, and fibrinogen products localize to electron lucent vesicles.

A biologically relevant relationship exists between neutrophils and coagulation processes. Several studies have focused on the ability of neutrophil proteases (both intracellular and membrane-associated) to degrade fibrinogen. The present study investigates the events following the interaction of activated neutrophils with soluble fibrinogen. During incubation of PMA-stimulated neutrophils with fibrinogen at 37 degrees C, fibrinogenolysis occurred, and degraded fibrinogen became associated with the neutrophil. Immunoelectron microscopy identified these fibrinogen products to be located within electron lucent vesicles, and not on the surface of the cell, suggesting that they are internalized. Although a specific interaction between fibrinogen and the neutrophil membrane might assist uptake, in the presence of physiological concentrations of fibrinogen, internalization occurred largely via a non-specific pinocytic process. Studies at low temperature revealed that both intact and degraded forms of fibrinogen can associate with neutrophils. The fibrinogen products detected intracellularly in experiments performed at 37 degrees C might represent uptake of degraded as well as intact forms of fibrinogen, the latter being rapidly degraded intracellularly. This route of fibrinogenolysis contributes minimally to the overall extent of the degradation process, the majority occurring extracellularly. Neutrophils thus possess a proteolytic mechanism for preventing accumulation of surface ligand, perhaps allowing them to evade the immunomodulatory effects of such ligands during inflammation.

Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies.

BACKGROUND AND AIM: The methionine choline-deficient (MCD) diet leads to steatohepatitis in rodents. The aim of the present study was to investigate species, strain and sex differences in this nutritional model of non-alcoholic steatohepatitis (NASH). METHODS: Male and female Wistar, Long-Evans and Sprague-Dawley rats, and C57/BL6 mice (n = 6 per group) were fed a MCD diet for 4 weeks. Control groups received an identical diet supplemented with choline bitartrate (0.2% w/w) and methionine (0.3% w/w). Liver pathology (steatosis and inflammation) and ultrastructure, liver lipid profile (total lipids, triglycerides, lipid peroxidation products), liver : body mass ratios and serum alanine aminotransferase (ALT) levels were compared between these groups. RESULTS: The MCD diet-fed male rats developed greater steatosis (P < 0.001), had higher liver lipid content (P < 0.05) and had higher serum ALT levels (P < 0.005) than did female rats. Wistar rats (both sexes) had higher liver lipid levels (P < 0.05), serum ALT levels (P < 0.05), and liver mass : body mass ratios (P < 0.025) than did Long-Evans and Sprague-Dawley rats. In female groups, Wistar rats showed greater fatty change than did the other two strains (P < 0.05). All rats fed the MCD diet developed hepatic steatosis, but necrosis and inflammation were minor features and fibrosis was absent. Compared with Wistar rats, male C57/BL6 mice showed a marked increase in inflammatory foci (P < 0.001), end products of lipid peroxidation (free thiobarbituric acid reactive substances) (P < 0.005), and mitochondrial injury, while showing less steatosis (P < 0.005), lower hepatic triglyceride levels, (P < 0.005) and lower early lipid peroxidation products (conjugated dienes and lipid hydroperoxides; P < 0.005 and P < 0.01, respectively). CONCLUSIONS: The Wistar strain and the male sex are associated with the greatest degree of steatosis in rats subjected to the MCD diet. Of the groups studied, male C57/BL6 mice develop the most inflammation and necrosis, lipid peroxidation, and ultrastructural injury, and best approximate the histological features of NASH.