HIV viral sequences in seronegative people at risk detected by in situ hybridisation and polymerase chain reaction.

A study was conducted to assess the occurrence of latent infection with the human immunodeficiency virus (HIV) among seronegative people at high risk of infection. The presence of HIV genomes was analysed by molecular techniques in two seronegative children born to mothers infected with HIV and in three regular sexual partners of seropositive drug addicts. The adults were selected from a seronegative cohort at high risk of infection because of their sexual contacts and the children selected because of impaired growth. HIV retroviral sequences were detected in four of the five subjects directly at the cellular level by in situ hybridisation in peripheral blood mononuclear cells. HIV genomic sequences were confirmed by in vitro amplification of viral DNA with the polymerase chain reaction technique. The existence of a latent viral infection state in these seronegative subjects indicates the unreliability of standard serological analysis in people who have been in regular contact with infected patients.     

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.     

Curing of the killer character of Saccharomyces cerevisiae with acridine orange

Acridine orange, an intercalating dye usually employed in the curing of bacterial plasmids, was tested for its ability to cure K1 and K2 killer strains (laboratory and wine strains). The results showed a high curing percentage of the killer character. This was demonstrated by the loss of M1 or M2 dsRNAs (responsible for toxin production and resistance to it) and because the meiotic products exhibited non-Mendelian segregation. The curing percentages varied, depending on the strain but not on the killer type, and showed similar efficiency as compared with other known curing agents.     

Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

There is evidence that three inhibitors of Na,K-ATPase activity--ouabain, K-free extracellular fluid, and vanadate--inhibit renin secretion by increasing Ca2+ concentration in juxtaglomerular cells, but in the case of vanadate, it is uncertain whether the increase in Ca2+ is due to a decrease in Ca2+ efflux (inhibition of Ca-ATPase activity, or inhibition of Na,K-ATPase activity, followed by an increase in intracellular Na+ and a decrease in Na-Ca exchange) or to an increase in Ca2+ influx through potential operated Ca channels (inhibition of electrogenic Na,K transport, followed by membrane depolarization and activation of Ca channels). In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free fluid, and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, a Ca channel blocker. Basal renin secretory rate averaged 7.7 +/- 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by K-free fluid, by 0.5 mM vanadate, and by K-depolarization (increasing extracellular K+ to 60 mM). Although 0.5 microM methoxyverapamil completely blocked the inhibitory effect of K-depolarization, it failed to antagonize the inhibitory effects of ouabain, of K-free fluid, and of vanadate. A concentration of methoxyverapamil two hundred times higher (100 microM) completely blocked the inhibitory effects of vanadate, but still failed to antagonize the effects of ouabain and of K-free fluid. Collectively, these observations demonstrate that vanadate-induced inhibition of renin secretion cannot be attributed entirely to Na,K-ATPase inhibition, since in the presence of methoxyverapamil, the effect of vanadate differed from the effects of either ouabain (a specific Na,K-ATPase inhibitor) or K-free fluid. Moreover, it cannot be attributed entirely to a depolarization-induced influx of Ca2+ through potential-operated Ca channels, since methoxyverapamil antagonized K-depolarization-induced inhibition of renin secretion much more effectively than it antagonized vanadate-induced inhibition.     

Persistence of drug-induced chromosome aberrations in peripheral blood lymphocytes of the rat

We have studied the persistence of pre-clastogenic lesions, detected as induced chromosomal aberrations, in rat peripheral lymphocytes at various time intervals after acute treatment with 3 different antineoplastic drugs: cyclophosphamide (CPA), 5-fluorouracil (5-FU) and adriamycin (AM). Single i.p. doses were administered to groups of rats and heart blood samples from each group were taken after 3, 12, 24 or 48 h or weekly up to 20 weeks later. The cytogenetic analysis was performed on lymphocytes cultured for 33 h after sampling. The results for CPA exposure (10 mg/kg) show that the yield of chromosome aberrations is maximal 3 h after the treatment (20 times the control level). For up to 8 weeks the values remain about 6 times the baseline; afterwards a decrease is observed and the control level is reached after 20 weeks. For 5-FU (50 mg/kg) a remarkable increase (13-fold) in chromosomal damage is observed at the first sampling time. Within 48 h the effect is drastically reduced but persistent (3 times the control level), and the level returns to spontaneous values 1 week later. AM treatment (2 mg/kg) induced an increase of about 8 times the control level at 3 h post exposure. The clastogenic effects remained at a detectable level for 1 week (about 6 times the control level at all sampling times); 2 weeks after the treatment the control level was found. A parallel analysis was performed on bone marrow cells. In this tissue the clastogenic effects of the treatments were maximal, as in lymphocytes, at the first sampling time (20-25 times the control level) and were no longer detectable within 72 h after exposure, irrespective of the administered drug.     

Possible role of 06-methylguanine-DNA-transferase in the response of patients with primary tumor of the brain to chemotherapy using chloro-ethyl-nitroso-urea: results of a current study

We have started a study to measure the MT activity in surgical specimens from high grade human malignant gliomas, with the dual aim to (i), know whether lack of activity can be demonstrated in these tumors, and (ii), relate the measured levels of MT to the histology of the tumors and to the response of patients to chemotherapy with 1-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosourea (CCNU). To date, 12 Gliomas have been assayed. In 11 tumors, MT activities ranging from 30 to 150 fmoles/mg protein have been measured. The only negative specimen derived from a patient who had received radiotherapy before surgery. At the present stage of the study, therefore, we have no unequivocal evidence for the existence of MT-deficient Gliomas.     

A nonlinear three-compartiment model for the administration of 2′,3′-dideoxycytidine by using red blood cells as bioreactors

A non-linear three-compartment model is proposed to describe a new strategy for the administration of 2′,3′-dideoxycytidine (ddCyd) in the treatment of HIV infections. The drug is injected after having been encapsulated in a non-diffusible form (ddCMP) into erythrocytes. Nummerical solutions show that by this treatment the highest ddCyd blood concentration is strongly reduced and in turn its toxicity, while long-lasting therapeutic effect is assured. The model is compared with experimental data in vitro.     

Neurotrophic factor-like activity in Drosophila.

The NGF-family of neurotrophic factors are structurally similar peptides with related functional properties. So far, this family of neurotrophic factors has only been identified in the vertebrate nervous system. We have determined that cultured Drosophila embryonic cells produce and secrete into medium, an activity which stimulates neurite outgrowth of embryonic chick sensory ganglia. This Drosophila activity can be blocked by antibodies to mouse NGF, indicating an immunological relationship between the Drosophila factor, mouse NGF and possibly other vertebrate neurotrophic factors. Addition of mouse NGF to Drosophila embryonic cells in culture results in increased cell number and enrichment of the neuronal phenotype, indicating that Drosophila cells have the ability to respond to the vertebrate factor. In addition, poly(A)+RNA extracted from Drosophila contains a single 1.4 Kb band which cross-hybridizes with a mouse NGF cRNA probe. These results indicate that vertebrate neurotrophic factor-like functions may operate in a genetically defined invertebrate species.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.