Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.     

Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus.

A complex "replication competent" holoenzyme form of DNA polymerase alpha (RC-alpha) was purified 10,000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-alpha form could partially replicate a double-stranded oligo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-alpha was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-alpha required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-alpha with the neutralizing anti-DNA polymerase alpha monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386-8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-alpha preparation following removal of specific polypeptides strongly suggested that the capacity of RC-alpha to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.     

Effects of 2.8-GHz microwaves on restrained and ketamine-anesthetized rats

Summary To compare the effects of ketamine anesthesia and mild restraint on microwave-induced thermal and cardiovascular changes, sixteen Fischer 344 rats were irradiated in two states:1) unanesthetized, restrained, and2) ketamine-anesthetized (150 mg/kg, I.M.). Individual animals were exposed in H orientation to far-field continuous-wave 2.8-GHz microwaves. Irradiation was conducted at a power density of 60 mW/cm² (whole-body average specific absorption rate of 14.4 W/kg) to cyclicly increase colonic temperature from 38.5 to 39.5° C. Colonic and subcutaneous temperatures, aortic blood pressure, and heart rate were continuously monitored. The time required for colonic temperature to increase 1° C was significantly longer in the anesthetized state; however, the time to return to baseline was similar under both conditions. Heart rate and blood pressure significantly increased during irradiation in the unanesthetized state, but remained virtually unchanged in the anesthetized state. The subcutaneous temperature increase during exposure was significantly greater in the anesthetized state. The differences in responses of anesthetized and mildly restrained animals should be considered when conducting experiments on thermoregulatory responses to microwave irradiation.     

Thermal responses to 5.6-GHz radiofrequency radiation in anesthetized rats: effect of chlorpromazine

Anesthetized rats were exposed to 5.6-GHz continuous wave radiofrequency radiation at an average power density of 60 mW/cm2 (average specific absorption rate 12 W/kg). Exposure was performed to raise colonic temperature from 38.5 to 39.5 degrees C. Following acute administration of chlorpromazine, body temperature exhibited a faster return to baseline temperature when exposure was discontinued. When exposure was initiated at 38.5 degrees C and continued until lethal temperatures resulted, chlorpromazine-treated animals exhibited significantly shorter survival times than saline-treated animals. Thus, although chlorpromazine enhanced thermo-regulatory efficiency at colonic temperatures below 39.5 degrees C, the drug caused increased susceptibility to terminal radiofrequency radiation exposure. The present results, when compared to previous studies of irradiation at 2.8 GHz, indicate that the effects of chlorpromazine on thermal responses to RFR during intermittent and terminal exposure are similar at both 2.8 and 5.6 GHz.     

Ca2+ release from mitochondria induced by prooxidants

A variety of chemically different prooxidants causes Ca2+ release from mitochondria. The prooxidant-induced Ca2+ release occurs from intact mitochondria via a route which is physiologically relevant and may be regulated by protein ADP-ribosylation. When the released Ca2+ is excessively cycled by mitochondria they are damaged. This leads to uncoupling, a decreased ATP supply, and a decreased ability of mitochondria to retain Ca2+. Excessive Ca2+ cycling by mitochondria will deprive cells of ATP. As a result, Ca2+ ATPases of the endoplasmic (sarcoplasmic) reticulum and the plasma membrane are stopped. The rising cytosolic Ca2+ level cannot be counterbalanced due to damage of mitochondria which, under normoxic conditions, act as safety device against increased cytosolic Ca2+. It is proposed that prooxidants are toxic because they impair the ability of mitochondria to retain Ca2+.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.