Formation of O6-methyldeoxyguanosine at specific sites in a synthetic oligonucleotide designed to resemble a known mutagenic hotspot

Four synthetic oligodeoxyribonucleotides of the sequence 5'-CCG1TG2G3G4ATATGGGCTG-3' were constructed with a 1',2'-[3H]deoxyguanosine located at one of the four sites indicated (1, 2, 3, or 4). This sequence was derived from a region of the Escherichia coli xanthine-guanine phosphoribosyltransferase gene where position 4 is a site frequently mutated by N-methyl-N'-nitrosourea as compared to sites 1-3. These four oligomers were alkylated in both single- and double-stranded form with N-methyl-N'-nitrosourea, and the relative amount of O6-methyldeoxyguanosine (O6-MedGuo) formed at each position was quantitated. Up to a 5-6-fold greater formation of O6-MedGuo was observed at positions 3 and 4 as compared to positions 1 and 2. This uneven distribution was only observed in oligomers in the double-stranded form, suggesting that secondary structure was an important determinant in generating the uneven distribution of O6-MedGuo. Comparisons between the extent of O6-MedGuo formation and mutation frequency at the four positions suggest that a difference in the formation of promutagenic adducts at specific sites is just one of the factors involved in the generation of mutagenic "hotspots." The novel method developed was applied to the study of formation of O6-MedGuo at specific sites; however, it should be suitable for studying the formation and repair of DNA adducts generated by a variety of chemicals in a wide variety of DNA sequences.     

Iron-supplemented bovine serum as an alternative to fetal bovine serum in the CHO/HGPRT mutation assay

Iron-supplemented bovine calf serum (ICS) was found to be a viable alternative to fetal bovine serum (FBS) in the growth promotion and cloning efficiency of Chinese hamster ovary (CHO) cells that are used in the HGPRT mutation assay. Suspension cultures of CHO cells had an average generation time of 11.5 h in ICS and 13.6 h for cells maintained in FBS. This slight difference was due to lot variability on the part of FBS and could be eliminated by routine quality control measures. The average cloning efficiencies for CHO cells cloned in either ICS or FBS were 107% and 88%, respectively, and these values were not statistically different. No appreciable difference was noted in the spontaneous mutation rates of cells cloned in either ICS or FBS. Furthermore, the use of ICS in mutagenicity studies with genotoxic agents shows the serum to be at least equal or superior to FBS in the detection of both direct-acting mutagens and promutagens. These data suggest that ICS is an appropriate serum to be used in the CHO/HGPRT test system. Since ICS is more readily available and considerably less costly than FBS, a substantial reduction in the cost of the assay can be realized.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.     

Changing responsiveness of luteal cells of the marmoset monkey (Callithrix jacchus) to luteotrophic and luteolytic agents during normal and conception cycles

Dispersed marmoset luteal cells were incubated for 2 h and progesterone production measured after exposure to hCG, cloprostenol, dibutyryl cAMP, PGF-2 alpha, PGF-2, adrenaline or melatonin. The cells were studied on Days 6, 14 and 20 after ovulation in conception and non-conception cycles. Luteal cells from Day 14 non-pregnant marmosets were compared with human luteal cells taken in the mid-luteal phase. All the treatments stimulated progesterone production including cloprostenol, which is luteolytic when administered to the marmoset in vivo, but the degree of response varied with the stage of the cycle or pregnancy and between marmoset and human luteal cells. In the marmoset, overall analysis of the effect of the treatments showed that, on Day 6 after ovulation, there was no significant effect of any of the treatments in cells from pregnant or non-pregnant animals. In contrast, luteal cells from non-pregnant animals on Day 14 showed a significant response to the treatments (F (8,41) = 2.79, P less than 0.0145) whereas cells from pregnant Day-14 animals were responsive; in cells from pregnant animals, the control production of progesterone was high and already equivalent to the levels stimulated by the treatments. By Day 20, cells from pregnant animals produced lower control concentrations of progesterone than did those on Day 14 and there was a significant overall effect of the treatments (F (8,33) = 3.78, P less than 0.003). These results show that the marmoset CL gains responsiveness to treatment between Days 6 and 14 after ovulation in the non-pregnant cycle. In pregnancy, on Day 14, 2 days after attachment of the embryo, the high control concentrations of progesterone and absence of response to treatment suggest that an embryo message may have affected the CL, providing an endogenous stimulus.     

Effect of transforming growth factor-beta on insulin-like growth factor 1- and dexamethasone-induced proliferation and differentiation in primary cultures of pig preadipocytes.

The purpose of this study was to examine the effects of a known inhibitor, transforming growth factor-beta1 (TGF-beta1) versus the known stimulators insulin-like growth factor-1 (IGF-1) and dexamethasone (DEX) on pig preadipocyte differentiation in serum and serum-free primary cultures. In cultures with serum, preadipocyte and nonpreadipocyte replication was increased (p < 0.02) by IGF-1 and by TGF-beta1 (p < 0.05; p < 0.001). IGF-1 (10 nM) enhanced preadipocyte differentiation (p < 0.05) in serum-supplemented (1% pig serum) cultures, whereas TGF-beta1 (15 pM) reduced preadipocyte differentiation (p < 0.01) in the presence and absence of IGF-1. Furthermore, GPDH (SN-glycerol-3-phosphate dehydrogenase) specific activity (marker that indicates differentiation) was decreased (p < 0.05) by adding TGF-beta1 to serum-free cultures, but TGF-beta1 had little effect in serum-supplemented cultures. DEX significantly enhanced GPDH activity and fat cell cluster number, whereas pretreatment with TGF-beta1 eliminated the DEX enhancement. We have shown for the first time that TGF-beta can decrease (p < 0.01) the cellular secretion of IGF-1 by pig adipose tissue cells and counter the effects of exogenous IGF-1. These studies indicate that TGF-beta1 may not inhibit adipocyte development in the initial growth phase, but may inhibit differentiation and/or hypertrophy (lipid filling) at a later stage of development.     

Assessing the risk of invasive success in Pinus and Banksia in South African mountain fynbos

Abstract. South African mountain fynbos has been severely invaded by trees and shrubs introduced from other mediterranean-climate regions. Management of these invasions should involve controlling current invaders and screening future introductions. Invasion windows are described and functional groups are defined for pines based on life history attributes important for invasion in the fire-prone mountain fynbos. The most successful invasive pines here (Pinus halepensis, P. pinaster and P. radiata) are fire-resilient and have small seeds, low seed-wing loadings, short juvenile periods, moderate to high degrees of serotiny and relatively poor fire-tolerance as adults. Other species with these attributes, especially from mediterranean-climate regions, wouldbe high-risk introductions. Taxa in other functional groups have not become major weeds even with widespread man-aided dissemination. Experience with pine invaders was used to define functional groups in western Australian Banksia species (Proteaceae), shrubs and trees which include taxa with similar attributes to fynbos invaders (e.g. Hakea and Pinus spp.). Banksias have only recently been introduced to the Cape, and are likely to be increasingly cultivated for the cut flower market. Tall serotinous shrubs with many small seeds per plant, short juvenile periods and low fire tolerance were identified as high risk introductions. This group includes thicket-forming species which maintain very large viable seed banks, e.g. Banksia burdettii, B. hookeriana and B. leptophylla. Low sprouting shrubs with few large seeds per plant and long juvenile periods are unlikely to become invasive in mountain fynbos. The approach of defining functional groups based on life history attributes and invasion windows is valuable for predicting the probability of invasive success. Chance interactions suchas an opportunistic dispersal mutualism between Pinus pinea and an introduced squirrel sometimes confound these predictions and underscore the idiosyncracies inherent in biological invasions.     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

Time-dependent changes in cardiovascular regulation caused by chronic tobacco smoke exposure

The purpose of this study was to determine whether chronic tobacco smoke exposure for less than 2 mo alters cardiovascular regulation. One group of male Sprague-Dawley rats was administered tobacco smoke from low-nicotine cigarettes (group A, 1 mg/cigarette) for 4-6 wk, while a second group (B) served as a sham control by receiving only puffs of room air. Reflex adjustments in mean arterial blood pressure (MAP) after bilateral common carotid occlusion (BCCO) were compared between the two groups. In the anesthetized control state, no significant difference existed for the cardiovascular parameters measured in the two groups. However, MAP increases after BCCO were significantly greater in the smoke-treated animals (P less than 0.05) compared with the sham-treated group. At 10, 20, 30, and 40 s after BCCO, MAP increases above preocclusion values were 66, 45, 42, and 38% for group A and 35, 26, 24, and 22% for group B, respectively. Additionally, the time required to reach maximum MAP after BCCO was significantly less (P less than 0.05) for the smoke-treated vs. sham-treated animals (8.5 +/- 0.2s for group A, 11.2 +/- 0.3s for group B). MAP changes during BCCO were significantly different (P less than 0.05) between the treatment groups after cervical vagotomy. It is concluded that chronic tobacco smoke exposure in experimental animals for periods as short as 4-6 wk alters the reflex regulation of the cardiovascular system.     

PHYSIOLOGICAL AND OPTICAL PROPERTIES OF RHIZOSOLENIA FORMOSA (BACILLARIOPHYCEAE) IN THE CONTEXT OF OPEN-OCEAN VERTICAL MIGRATION1

Cultures of Rhizosolenia formosa H. Peragallo were studied to assess whether or not physiological and optical characteristics of this large diatom were consistent with the ability to migrate vertically in the open ocean. Time-course experiments examined changes in chemical composition and buoyancy of R. formosa during nitrate (N)–replete growth, N starvation, and recovery. Cells could maintain unbalanced growth for at least 53 h after depletion of ambient nitrate. Increases in C:N and carbohydrate: protein ratios observed during N starvation reversed within 24 h of reintroduction of nitrate to culture medium. Buoyancy was related to nutrition: Upon N depletion, the percentage of positively buoyant cells decreased to 4% from 11% but reverted to 9% within 12 h of nitrate readdition. Cells took up nitrate in the dark. Nitrogen-specific uptake rates averaged 0.48 d^?1; these rates were higher than N-specific growth rates (0. 15 d^?1), indicating the potential for luxury consumption of nitrate, which can be stored for later use. Measurements of photosynthesis vs. irradiance, chlorophyll-specific absorption (a_ph*(λ)), and pigment composition showed that cells may be adapted for growth under a wide range of irradiances. Values of a_ph*(λ) were lower for N-depleted cells than for N-replete cells, and N-depleted cells had higher ratios of total carotenoids to chlorophyll a. Aggregation of chloroplasts was more pronounced in N-depleted cells. These are possibly photoprotective mechanisms that would be an advantage to N-depleted cells in surface waters. Compounds that absorb in the ultraviolet region were detected in N-replete cells but were absent in N-depleted cultures. Overall, these results have important implications for migrations of Rhizosolenia in nature. Cells may survive fairly long periods in N-depleted surface waters and will continue to take up carbon; then they can resume nitrate uptake and revert to positive buoyancy upon returning to deep, N-rich water. Uncoupled uptake of carbon and nitrogen during migrations of Rhizosolenia is a form of new production that may result in the net removal of carbon from oceanic surface waters.