Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.     

Oxygen consumption by portal vein-drained organs and by whole animal in conscious growing swine

A method was developed to measure simultaneously the O2 consumption (VO) by the whole animal and by the hepatic portal vein-drained organs (PVDO), including the gastrointestinal tract, spleen, and pancreas in conscious 3.5- to 4-month-old swine. The method was used to determine (i) the effect of feeding on hepatic portal vein blood flow rate (Qpv) and VO by PVDO and by the whole animal, and (ii) the significance of PVDO on the oxidative demand in the pig. Chronic cannulas were placed in the hepatic portal vein, carotid artery, and ileal vein. The Qpv was determined by an indicator dilution technique employing continuous constant infusion of 1% p-aminohippuric acid into the ileal vein. The VO2 by PVDO was estimated by multiplying Qpv by arterial-portal vein O2 difference measured with an arterial-venous O2 difference analyzer connected to the carotid artery and portal vein cannulas. Whole animal VO2 was measured with an open circuit indirect calorimeter. In seven pigs (3.5- to 4-month-old, 37.4 +/- 0.8 kg) trained to be fed once daily, feeding (1.2 kg of feed mixed with 1.2 liter of H2O) caused postprandial (6 hr) Qpv to increase more than 34 +/- 15% above the preprandial value of 34.5 +/- 4.2 ml.min-1.kg-1 body wt. The postprandial VO2 by PVDO was elevated more than 46 +/- 12% above the value of 1.52 +/- 0.20 ml.min-1.kg-1 body wt observed during the preprandial period. Whole animal VO2 increased 45 +/- 9 and 33 +/- 7% above the preprandial value of 6.23 +/- 0.57 ml.min-1.kg-1 body wt for the first 6 hr and the 7 to 12 hr after feeding, respectively. Although PVDO represent only 5% of body weight, they used 25% of whole body VO2. The study clearly illustrates the significance of PVDO on the whole animal oxidative demand in conscious growing swine.     

The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification (R+/M+) and abortive infection (Hsp+)

pTR2030 is a conjugative plasmid which encodes resistance to bacteriophage in lactococci by a mechanism that aborts the phage infection (Hsp+). Subcloning and in vivo deletion events showed that two independent mechanisms of resistance are located on a 13.6-kilobase Bg/II fragment cloned in pSA3; one mechanism is responsible for the abortive infection, and the other incodes a restriction modification system. The introduction of pTR2030 or the recombinant plasmid pTK6 resulted in the loss of a resident restriction modification plasmid in Lactococcus lactis NCK202 which was not previously identified.     

Mouse DNA methylase. Intracellular location and degradation

DNA methylase extracted with low salt from mouse Krebs II ascites cell nuclei has been degraded stepwise by trypsin treatment. Degradation, accompanied by a limited reduction in size of the native enzyme, leads to the progressive introduction of several nicks so that, eventually, fragments of 14, 18, 24 and 28 kD are released on denaturation. This illustrates the domain structure of the enzyme. In contrast to ascites cell nuclear extracts, preparations from liver nuclei are already nicked and the major from of the enzyme contains a 100 kD fragment though the native molecular weight is unchanged. Newborn mouse liver contains more undegraded enzyme that is mostly firmly-bound within the nucleus. Trypsin treatment increases the de novo activity of the enzyme and prevents its aggregation in the absence of salt, even in the presence of high concentrations of native DNA.     

Soil N mineralization and nitrification in relation to nitrogen solution chemistry in a small forested watershed

Spatial variations in soil processes regulating mineral N losses to streams were studied in a small watershed near Toronto, Ontario. Annual net N mineralization in the 0–8 cm soil was measured in adjacent upland and riparian forest stands using in situ soil incubations from April 1985 to 1987. Mean annual rates of soil N mineralization and nitrification were higher in a maple soil (93.8 and 87.0 kg.ha^–1) than in a pine soil (23.3 and 8.2 kg.ha^–1 ). Very low mean rates of mineralization (3.3 kg.ha^–1) and nitrification (3.4 kg.ha^–1) were found in a riparian hemlock stand. Average NO₃-N concentrations in soil solutions were 0.3–1.0 mg.L^–1 in the maple stand and >0.06mg.L^–1 in the pine stand. Concentrations of NO₃–N in shallow ground water and stream water were 3–4× greater in a maple subwatershed than in a pine subwatershed. Rapid N uptake by vegetation was an important mechanism reducing solution losses of NO₃–N in the maple stand. Low rates of nitrification were mainly responsible for negligible NO₃–N solution losses in the pine stand.     

In-vivo effects of ivermection onRhipicephalus appendiculatus: the influence of tick feeding patterns and drug pharmacokinetics

Sublethal effects seen amongstRhipicephalus appendiculatus feeding on ivermectin-treated rabbits were diverse and dependent both on drug dose, pharmacokinetics and tick feeding patterns: changes in drug formulation, the time of infestation relative to treatment, and the tick instar used, profoundly influenced acaricidal activity. Death was a sequel to paralysis only if tick feeding was interrupted for sufficient time to produce irreversible dehydration. Concurrent pharmacokinetic investigations revealed that, for the larvae ofR. appendiculatus, the mean critical lethal dose of ivermectin imbibed over a 5-day engorgement period was 3500 g/kg. This quantity of ivermectin was achieved in the blood-meals of larvae feeding on rabbits treated subcutaneously with a single dose of Ivomec injection (MSD)^*800 g/kg, provided infestation took place within 24 h of treatment. At lower drug doses, or if larval infestations were delayed for>24 h post-treatment, the quantity of circulating ivermectin (and thus imbibed by the tick larvae) fell below 3500 g/kg and an increasing percentage of larvae successfully engorged and detached. More than 90% of such larvae moulted to the nymphal stage. Nymphae and larvae exhibited similar susceptibility to ivermectin on treated rabbits which could be explained by similar feeding patterns. However, adult female and male ticks were markedly less susceptible and interpretation of ivermectin-induced effects was more complex.     

A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.

The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Hydro-sedimentology of the Johnstone River estuary

This paper examines the physical consequences of increased catchment sediment yields on the sediment budget and the hydrodynamic setting of the South Johnstone River estuary in North Queensland. A combined study involving hydrological monitoring, assessment of sediment sources, estimation of riverine sediment budget and hydro-sedimentological numerical modelling for estuarine sediment transport is currently underway. Initial field and laboratory observations indicate that the sediment delivery from highly erosion-prone sugar cane cultivations in the tropical catchment has increased dramatically during the last 10 years. This has subsequently given rise to elevated flood levels in both the lower and upper catchment areas, as well as significant modifications to the river bed morphology.