Differentiated rat glial cell strain in tissue culture.

Rat glial tumors, induced by injections of N-nitrosomethylurea, were plated and propagated in culture. Among a few cell strains obtained, one clone contains S-100 protein, which is unique to brain in vertebrates. Stationary-phase cultures contain approximately ten times more S-100 protein per cell than exponentially growing cells. When injected into newborn rats, cells producing S-100 grew as a glial tumor, which contained S-100 protein.

CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.

Decreased lymphocyte adenosine deaminase activity in acute lymphocytic leukemia children and their parents.

Adenosine deaminase has been measured in the lymphocytes of children with acute lymphocytic leukemia and in those of their parents. The activity was found to be significantly lower in both acute lymphocytic leukemia children in remission and relapse and in the parents of acute lymphocytic leukemia children when compared with normal adults and children of similar ages.

Decreased lymphocyte adenosine deaminase activity in tumor patients.

Adenosine deaminase has been measured in the lymphocytes of individuals with various types of solid tumors. The mean activity was found to be significantly lower when compared with control individuals of a similar age.

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris.

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.

Preparation and characterization of monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris.

Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.

The effects of desferrioxamine and ascorbate on oxidative stress in the streptozotocin diabetic rat.

Oxidative stress and protein glycation are closely related processes that may contribute to the development of complications in diabetes mellitus. Treatment with antioxidants could protect against these processes at a biochemical level, and we have therefore investigated the effects of ascorbate and desferrioxamine treatment in the streptozotocin diabetic rat. Diabetic animals were given ascorbate 1 g/l in drinking water or desferrioxamine 6 mg/kg/day by subcutaneous injection and were killed after 6 weeks. In diabetic animals, oxidative stress was increased as shown by increased levels of conjugated dienes (CD) in plasma and malondialdehyde (MDA) in plasma, erythrocyte membranes, and urine. In addition, there was depletion of the nutritional antioxidants ascorbate, alpha-tocopherol, and retinol. Insulin treatment returned all of these parameters to normal. Ascorbate supplementation or desferrioxamine treatment alone failed to reduce oxidative stress, but a combination of both interventions restored MDA, CD, and antioxidant vitamins to control values. Both ascorbate and desferrioxamine also reduced HbA1c and glycated albumin levels. Treatment with antioxidants can reduce both oxidative stress and protein glycation and may help to reduce the risk of developing diabetic complications. However, ascorbate can have both prooxidant and antioxidant effects in vivo, and its use in pharmacological doses should be approached with caution.

Oxidative stress in haemodialysis.

Patients with chronic renal failure, including those receiving regular long-term haemodialysis, have a high incidence of premature cardiovascular disease. Oxidative stress, which occurs when there is excessive free-radical production or low antioxidant levels, has recently been implicated as a causative factor in atherogenesis. The aim of this study was to determine if chronic renal failure and haemodialysis were associated with increased oxidative stress. Serum malondialdehyde was measured as a marker of lipid peroxidation in 15 patients with conservatively managed chronic renal failure (CRF), 15 patients with CRF undergoing regular haemodialysis and 15 healthy controls. Selenium, glutathione peroxidase and antioxidant vitamins were also measured. Malondialdehyde was elevated in dialysis patients in comparison to CRF and control groups (dialysis 1.16 +/- 0.08 mumol/l, CRF 0.94 +/- 0.07, controls 0.66 +/- 0.10). Antioxidants, including vitamin C, selenium and glutathione peroxidase, were decreased in dialysis patients and to a lesser extent in the CRF group (vitamin C-dialysis 16.43 +/- 3.76 mumol/l, CRF 34.5 +/- 8.6, controls 56.11 +/- 7.41; selenium-dialysis 0.77 +/- 0.07 mumol/l, CRF 0.69 +/- 0.06, controls 1.09 +/- 0.06: glutathione peroxidase-dialysis 101 +/- 5 U/l, CRF 160 +/- 11, controls 290 +/- 10). These findings indicate oxidative stress in patients with CRF which is further exacerbated by haemodialysis, as evidenced by increased lipid peroxidation and low antioxidant levels. This stress may play a role in the development of atherosclerosis in these groups.

Homocysteine and B-group vitamins in renal transplant patients.

Increased plasma homocysteine is an independent risk factor for cardiovascular disease. We have investigated homocysteine and B-group vitamin levels in renal transplant patients. Fasting blood was collected from 55 renal transplant recipients with good renal function and 32 age/sex matched control subjects. Total homocysteine was increased in transplant recipients in comparison to controls (10.9+/-1.5 vs. 6.7+/-1.3 micromol/l, P < 0.001). There was no difference in homocysteine between patients receiving cyclosporin (n = 39, homocysteine 11.0+/-1.5 micromol/l) and patients receiving prednisolone + azathioprine (n = 16, 10.8+/-1.6 micromol/l, mean+/-S.D.), although there was a significant correlation between homocysteine and serum cyclosporin concentration in the sub-group of patients receiving that immunosuppressive regimen (r = 0.42, P < 0.05). Levels of B-group vitamins were similar in patients and controls. Plasma homocysteine is increased in renal transplant recipients even in the presence of minor degrees of renal impairment and normal levels of B-group vitamins.

Comparative aspects of plasma antioxidant status in sheep and goats, and the influence of experimental abomasal nematode infection.

This paper provides, for the first time, comparative data on the plasma antioxidant status of two ruminant species, namely sheep and goats. In addition, the influence of experimental infection with Teladorsagia circumcincta on antioxidant status in the same two species is compared and contrasted. In general terms, antioxidant status was significantly higher in uninfected kids than in lambs. Differences in protein sulphydryl groups and vitamin E concentrations were particularly noteworthy; trends were similar, however, for albumin, vitamin A and total antioxidant capacity (TAC). Parasitological results, based on worm burden, faecal egg counts and peripheral blood eosinophil numbers, confirmed that goat kids were more susceptible than lambs to experimental T. circumcincta infection. "Trickle infection" had a variable impact on both total and individual antioxidant status; particularly during the early weeks, the trend was for reduced values in lambs and increased values in kids, as compared with uninfected controls. Subsequent challenge infection was associated with a transient decrease in TAC and albumin in trickle-infected animals of both species, and in appropriate control animals. The observed differences in plasma antioxidant capacity between sheep and goats may have important implications in terms of the comparative resilience of sheep and goats to parasite infection.