Reactivity of an HIV gag gene polypeptide expressed in E. coli with sera from AIDS patients and monoclonal antibodies to gag

A segment of the gag gene of the human immunodeficiency virus (HIV) (HTLV-IIIB strain), the virus which causes acquired immunodeficiency syndrome (AIDS), has been cloned into the bacterial expression vector, pCQV2, and mapped to the right-hand portion of the gag gene containing the carboxyl-terminal portion of p24 and the amino-terminal portion of p15. Nucleic-acid sequencing of the insert-vector junctions further defined the 5'-terminal nucleotide of HIV sequence as nucleotide 997 and the 3'-terminal nucleotide as 1696. When used in an enzyme-linked immunosorbent assay (ELISA) with sera from HIV-infected patients, the cloned antigen reacted with a subset of sera which were positive on a standard ELISA using whole virus as antigen. Western-blot screening of these sera with whole virus indicated that all p24-positive sera were positive with the clone, suggesting that the carboxyl-terminal portion of p24 contains a highly antigenic epitope(s). A serum which was p24-negative p15-positive by Western blot analysis was also highly reactive, indicating that a p15 epitope is present in the cloned antigen. Epitope mapping with a series of monoclonal antibodies to gag resulted in positive ELISA with 2 of 3 anti-p24, 0 of 1 anti-p15, and 0 of 1 anti-p17 Western-blot-positive monoclonal antibodies, suggesting that one of the anti-p24 monoclonal antibodies reacts with epitopes amino-terminal to those coded from nucleotide 997, two anti-p24 monoclonals react with epitopes carboxyl-terminal to those coded from nucleotide 997, and the anti-p15 monoclonal reacts with epitopes carboxyl-terminal to those coded from nucleotide 1696.     

Predictors of success in hypertensives treated with biofeedback-assisted relaxation

This paper describes differences in response in seventeen patients with essential hypertension who participated in a treatment program consisting of electromyograph biofeedback assisted relaxation training. Responders were found to have higher treatment values of urinary and plasma cortisol, Trait Anxiety and forehead muscle tension compared to treatment failures. Responders also sustained greater decreases in plasma, and urinary cortisol after treatment. These data are discussed in light of the ability to predict which hypertensive patients may be most benefitted by a relaxation based treatment.We would like to thank Dr. Charles Spielberger for his permission to use the State-Trait Anxiety Inventory. We thank Michael Robinson for assistance with statistical analysis.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

The effect of biofeedback-assisted relaxation training on blood pressure and selected biochemical parameters in patients with essential hypertension

The effect of EMG biofeedback-assisted relaxation on blood pressure and selected biochemical parameters was evaluated in 38 patients with essential hypertension. Training consisted of 8 weeks of biofeedback and home practice of relaxation exercises. Mean blood pressure decreased in the experimental group from 144/90 to 133/84 mm Hg while the control group remained unchanged. Statistically significant decreases in the experimental group also occurred in muscle tension levels, in plasma aldosterone, and in urinary cortisol. Both aldosterone and cortisol are secreted by the adrenal cortex. It was concluded that the technique taught to the experimental group produced a reduction in skeletal muscle tension and a decrease in stress responding mediated by the adrenal cortex.This work was supported by the Northwestern Ohio Heart Association Grant No. 93298. It was presented as a citation paper at the meetings of the Biofeedback Society in 1979 and 1980.     

CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental autoimmune encephalomyelitis

The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.