Chromosome aberrations (CA), sister-chromatid exchanges (SCE) and mitogen-induced blastogenesis in cultured peripheral lymphocytes from 48 control individuals sampled 8 times over 2 years

Confidence in the measurement of positive effects determined by monitoring of environmentally or occupationally exposed individuals can be enhanced by a knowledge of the normal variability in these endpoints in the general population. Confounding effects can be determined and study interpretation improved by correlation of this variability with various lifestyle factors such as sex and age of donor, smoking and drinking habits, viral infections, exposure to diagnostic X-rays, etc.

8 blood samples were taken from each of 24 male and 24 female volunteers over a period of 2 years. Questionnaires pertaining to lifestyle were completed at the time of each sampling. Whole blood was cultured and slides prepared for CA or SCE analysis. Separated mononuclear cells were cultured with a range of phytohaemagglutinin concentrations and the maximum level of mitogen-induced blastogenesis was determined by measurement of [³H]thymidine uptake.

There was a significant effect of both year and season of sampling for all 3 endpoints. No significant effects in any of the 3 endpoints were found with respect to sex or age of donor nor any of the other lifestyle factors, although SCE frequency and mitogen-induced blastogenesis were nearly always higher in females than males. These results point to the need for concurrent sampling of controls with exposed populations.     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low-oxygen conditions

Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

A YAC contig across the fragile X site defines the region of fragility.

The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.     

Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France     

Effects of extremely low frequency (ELF) electric fields on cell growth and DNA repair in human skin fibroblasts

A number of physical and chemical agents in the environment have been studied for their ability to induce or alter DNA repair mechanisms in human cells. We have investigated the effects of 60 Hz, 1000 V/cm electric fields on DNA repair in normal human fibroblasts in vitro. An examination was done on the ability of electric fields suspected to cause damage which could be repaired by thymine dimer excision and measurable by the bromodeoxyuridine photolysis assay. The thymine dimer assay with enzyme-sensitive site analysis was used to measure the cells' capacity for removing ultraviolet light (u.v.)-induced pyrimidine dimers; during exposure to electric field 24 hr before u.v. irradiation; 24 hr after u.v. irradiation; and up to 48 hr continuously after u.v. irradiation. Cell growth and cell survival following electric field exposure were also studied. Within the limits of these experiments, it was found that exposure to such electric fields did not alter cell growth or survival, and no DNA repair or alteration in DNA excision repair capacity was observed as compared with unexposed control cultures.     

Amino Acid Analysis Demonstrates that Increased Plasma Free Tryptophan Causes the Increase of Brain Tryptophan During Exercise in the Rat

Rats were trained to run on a horizontal treadmill for 2 h at 20 m/min. This activity considerably increased plasma free tryptophan (TRP) (+70%) but did not alter plasma total TRP levels and had little or no effect on plasma concentrations of the other large neutral amino acids (LNAAs) that compete with TRP for entry into the brain. Brain TRP levels increased by 80%. The only other brain LNAA to be affected by exercise was threonine, which rose moderately. The results indicate that increased plasma free TRP was specifically responsible for the increase of brain TRP after 2 h of exercise. Brain lysine was also increased whereas glycine, alanine, and gamma-aminobutyric acid were decreased. The differences between the present findings and those previously obtained following 2 h immobilization stress are discussed.     

The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.