Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.     

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.     

Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy

Brain areas damaged by stroke and seizures express high levels of the 72-kd heat shock protein (HSP72). Whether HSP72 represents merely a marker of stress or plays a role in improving neuron survival in these cases has been debated. Some induced tolerance experiments have provided correlative evidence for a neuroprotective effect, and others have documented neuroprotection in the absence of HSP72 synthesis. We report that gene transfer therapy with defective herpes simplex virus vectors overexpressing hsp72 improves neuron survival against focal cerebral ischemia and systemic kainic acid administration. HSP72 overexpression improved striatal neuron survival from 62.3 to 95.4% in rats subjected to 1 hour of middle cerebral artery occlusion, and improved survival of hippocampal dentate gyrus neurons after systemic kainic acid administration, from 21.9 to 64.4%. We conclude that HSP72 may participate in processes that enhance neuron survival during transient focal cerebral ischemia and excitotoxin-induced seizures.     

A comparison of two methods of evaluation of teat skin pathology

A split-plot design with repeated measures was used to test the relationship between visual teat skin condition score, the degree of transepidermal water loss from the skin, and the colonization by Staphylococcus aureus on experimentally chapped and inoculated teats of 20 lactating Holstein cows. Visual teat skin chapping score and the number of S. aureus colonies obtained from a teat skin swab were correlated (r = 0.53). Transepidermal water loss and S. aureus count were not correlated (r = 0.02). Results indicated that visual teat skin evaluation is superior to measurements of transepidermal water loss in the prediction of the susceptibility of teat skin to colonization by S. aureus.     

Inhibition of carbamyl phosphate synthetase-I and glutamine synthetase by hepatotoxic doses of acetaminophen in mice

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS-PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.     

Childhood chronic inflammatory demyelinating neuropathies: clinical course and long-term follow-up

Chronic inflammatory demyelinating neuropathy (CIDP) is a rare disease in childhood. We reviewed the clinical characteristics, response to therapy, and long-term prognosis in 13 children (1.5 to 16 years of age) diagnosed with CIDP at Washington University Medical Center, St. Louis, and the Royal Children's Hospital, Melbourne, Australia, between 1979 and 1994. The most common presenting symptom (in 11/13 [85%]) was lower extremity weakness associated with difficulty in walking. Preceding events within 1 months of onset, mostly intercurrent infections or vaccinations, occurred in seven children (54%). The disease was monophasic in three children (23%). One relapse occurred in four (30%) and multiple relapses in six (46%). All patients had at least short-term response to steroids. Three children (23%) recovered completely during the first year. Ten children (77%) had residual weakness after an average follow-up of 6 years. There seems to be two populations of children with CIDP. One subgroup, with a favorable prognosis, progressed to peak disability over less than 3 months; these children often have a monophasic course with complete resolution of symptoms and signs and withdrawal from all medications by 1 year after onset. A second subgroup progressed for 3 months or longer; these children all required substantial does of prednisone for prolonged periods and had considerable long-term morbidity with persistent weakness.     

Attenuation of acetaminophen hepatotoxicity in mice as evidence for the bioavailability of the cysteine in D-glucose-L-cysteine in vivo

A substantial fraction of the cysteine added to total parenteral nutrition (TPN) solutions is converted to the corresponding thiazolidine derivative, while in solution with relatively large concentrations of glucose typical of TPN (700 mM and higher). It was recently reported (Roberts et al. (1987) J. Med. Chem. 30, 1891-1896) that this thiazolidine, D-glucose-L-cysteine (DGC), offered no significant protection against the hepatic injury caused by 5 mmol/kg of acetaminophen in mice, suggesting that the cysteine present as DGC is poorly bioavailable in vivo. In the present study, fasted male ICR mice given 1.6 or 2.6 mmol/kg of acetaminophen sustained hepatic injury, estimated by elevations in plasma alanine aminotransferase (ALT) activities. Administration of 2.5 mmol/kg of N-acetylcysteine (NAC) 1 h before acetaminophen given i.p. prevented the rise in plasma ALT activities, apparently through support of glutathione (GSH) synthesis. Administration of 2.5 mmol/kg of DGC prior to acetaminophen resulted in slightly lower mean plasma ALT activities than were observed in animals given saline before acetaminophen, but the effect was not statistically significant. When DGC was given 1 h before p.o. administration of 1.6 or 2.6 mmol/kg of acetaminophen, the protective effects of DGC were statistically significant (P < 0.01, 0.025, respectively), although NAC afforded significantly greater protection than did DGC at the higher dose of acetaminophen. Given 4 h before acetaminophen, DGC attenuated acetaminophen-induced increases in plasma ALT activities significantly, whereas NAC was without effect. These results indicate that the cysteine in DGC is at least partially bioavailable in vivo and, further, that DGC may function as a slow release formulation of cysteine.     

Characterisation of the unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines of the SuSa testicular teratoma cell line

Three etoposide-selected resistant sublines of the SuSa testicular teratoma cell line expressing 9-, 21- and 33-fold levels of resistance, proved increasingly cross resistant to cisplatin with levels approximating to 3-, 4- and 6-fold in sublines VPC2, VPC3 and VPC4, respectively. Cisplatin resistance was not associated with any significant modifications in levels of total glutathione or associated enzyme activities. Decreased platinum (Pt) accumulation was detected, although this did not correlate either with total platination levels judged immunochemically or with peak induction of interstrand crosslinks (ISC) determined by alkaline elution. Following exposure to cisplatin in the least resistant subline, VPC2, total platination levels were markedly decreased (3-fold) relative to those of the parental cells, whilst peak ISC levels were markedly increased (4-fold). In the most highly resistant subline, VPC4, peak levels of ISCs were even higher (9-fold), although total platination levels remained comparable with those in parental cells. Both VPC2 and VPC4 cells appeared highly proficient in removing ISCs, unlike the parental cells. However, whilst VPC2 cells appeared to share deficient removal of the intrastrand platinated lesions with parental cells, VPC4 cells proved proficient in removing specific adducts in the sequence pApG. This unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines derived from an inherently repair deficient parental cell line, SuSa, therefore appears to be associated with enhanced removal of the specific intrastrand crosslinks in the sequence pApG and/or of DNA-DNA ISCs. Similar mechanisms have been implicated in two other cisplatin resistant SuSa sublines selected following in vitro exposure to the drug itself or to fractionated X-irradiation.     

Peroxidase-catalyzed effects of indole-3-acetic acid and analogues on lipid membranes, DNA, and mammalian cells in vitro

This study aimed to explore the mechanisms and molecular parameters which control the cytotoxicity of derivatives of indole-3-acetic acid (IAA) when oxidatively activated by horseradish peroxidase (HRP). Lipid peroxidation was measured in liposomes, damage to supercoiled plasmid DNA assessed by gel electrophoresis, free radical intermediates detected by EPR following spin trapping, binding of IAA-derived products demonstrated by 3H labelling, stable products measured by HPLC, and cytotoxicity in hamster fibroblasts measured by clonogenic survival. IAA, and nine analogues more easily oxidized by HRP, caused lipid peroxidation in liposomes, but not detectably in membranes of hamster fibroblasts, and were cytotoxic after HRP activation to varying degrees. Cytotoxicity was not correlated with activation rate. The hydrophilic vitamin E analogue, Trolox, inhibited cytotoxicity, whereas loading fibroblasts with vitamin E was ineffective, consistent with an oxidative mechanism in which radical precursors to damage are intercepted by Trolox in the aqueous phase. However, two known oxidation products were nontoxic (the 3-carbinol and 3-aldehyde, both probably produced from 3-CH2OO* peroxyl radicals via the 3-CH*2 [skatolyl] radical following decarboxylation of the radical cation). The skatolyl radical from IAA was shown by EPR with spin trapping to react with DNA; electrophoresis showed binding to occur. Treatment of hamster fibroblasts with 5-3H-IAA/HRP resulted in intracellular bound 3H. Together with earlier results, the new data point to unknown electrophilic oxidation products, reactive towards intracellular targets, being involved in cytotoxicity of the IAA/HRP combination, rather than direct attack of free radicals, excited states, or membrane lipid peroxidation.