Comparison of dose-response relationships for ethyl methanesulfonate and 1-ethyl-1-nitrosourea in Drosophila melanogaster spermatozoa

The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents. 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine. Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site. To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa. For both mutagens the dose response curve was linear and extrapolated to the origin. The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3). Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS. In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G. If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS. In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS. It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis.     

Calcium-loaded 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid blocks cell-to-cell diffusion of carboxyfluorescein in staminal hairs of Setcreasea purpurea

The effect of microinjected calcium-loaded 1,2-bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (CaBAPTA) on cell-to-cell diffusion of carboxyfluorescein (CF) was examined in staminal hairs of S. purpurea Boom. The CaBAPTA was microinjected into the cytoplasm of the staminal hairs either with CF or prior to a subsequent microinjection of CF. The cell-to-cell diffusion of CF along the hair was monitored using enhanced-fluorescence video microscopy. Cytoplasmic streaming stopped in cells treated with CaBAPTA, indicating that intracellular Ca²⁺ had increased. Cell-to-cell diffusion of CF was blocked in cells treated with Ca-BAPTA. An inhibition of cytoplasmic streaming and cell-to-cell diffusion was observed in the cells adjoining the CaBAPTA-microinjected cell, indicating that the Ca-BAPTA appeared to pass through plasmodesmata. While cytoplasmic streaming resumed 5–10 min after CaBAPTA treatment, cell-to-cell diffusion did not resume until 30–120 min later. These data support an involvement of calcium in the regulation of cell-to-cell communication in plants.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid - CF carboxyfluorescein This work was supported by Professional Staff Congress-City University (PSC-CUNY) of New York grant No. 667180 and U.S. Department of Agriculture grant No. 87-CRCR-1-244.     

Mechanism of cadmium uptake by activated sludge

Summary The significance of metabolic activity in cadmium uptake by unacclimated activated sludge was studied. Below 30 mg/l cadmium in solution, biosorption was found to follow the Freundlich isotherm, which is the most common pattern for physico-chemical adsorption. More than 95% of total cadmium uptake was achieved within 5 min metal-sludge contact time. Biosorption increased strongly when the initial cadmium concentration in solution was raised from 10 to 100 mg/l, whereas in the same concentration range the metabolic activity of the sludge, as measured by respiratory activity and extracellular protein production, was very significantly inhibited. The addition of nutrients at low but significant levels failed to increase cadmium uptake in 2 h contact time, while in 24 h the addition of nutrients caused the biosorption to increase by only 5–10% without any significant growth of the biomass. Biosorption was found to increase with temperature between 5° C and 40° C, in correlation with an increase in the metabolic activity of the sludge. Pretreatment of the sludge with metabolic inhibitors (NaN₃ and UV rays) appeared to cause only a very slight decrease (5–10%) of biosorption. These results suggest that metabolic uptake of cadmium was low and that adsorption to the surface of the cells was the major mechanism of uptake.Offprint requests to: S. S. Sofer     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Preservative-mediated changes of the surface properties ofEscherichia coli

The effectiveness of two commonly used preservatives, sodium benzoate and potassium disulfite, was evaluated in terms of their bactericidal activity and capacity to induce changes in the surface properties ofEscherichia coli isolated from commercial food preserves. Preservative treatment over a five-week test period resulted in controlling the multiplication of these organisms and causing a decline in cell-surface hydrophobicity, hemagglutinating ability and adherence capacity to rat intestinal cells ofE. coli isolates. A loss in motility was also exhibited.     

Linkage of cutaneous malignant melanoma/dysplastic nevi to chromosome 9p, and evidence for genetic heterogeneity.

We examined the relationship between cutaneous malignant melanoma/dysplastic nevi (CMM/DN) and chromosome 9p in 13 pedigrees with two or more living cases of invasive melanoma. We used two highly informative (CA)n repeats, D9S126 and IFNA, previously implicated in familial malignant melanoma (MLM), to conduct linkage analysis. Three analyses were performed: (1) CMM alone--all individuals without either confirmed melanoma or borderline lesions were considered unaffected (model A); (2) CMM/DN with both variable age at onset and sporadics (model B); and (3) CMM affecteds only--all individuals either without confirmed melanoma or with borderline lesions were designated "unknown" (model C). There was significant evidence for linkage to IFNA in all three models. For CMM alone, the maximum lod score (Zmax) was 4.36 at theta = .10 for model A and 3.39 at theta = .10 for model C. For CMM/DN (model B), Zmax = 3.05 at theta = .20. There was no significant evidence for linkage between CMM alone or CMM/DN and chromosome 9p marker D9S126. In addition, there was significant evidence for heterogeneity when a homogeneity test allowing for linkage to chromosome 9p or chromosome 1p or neither region was used. These results suggest that there is an MLM susceptibility locus on chromosome 9p but that familial melanoma is heterogeneous and not all families with CMM/DN are linked to a locus in this region.     

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.     

Spicebush [Lindera benzoin (L.) Blume var.benzoin,Lauraceae]: A tea,spice, and medicine

The essential oils of fresh leaves, twigs, and/or fruits of spicebush cultivated in Oregon (field distilled) and Delaware (laboratory distilled) are examined by GC/MS. The oil of leaves of spicebush is notably high in 6-methyl-5-hepten-2-one (1.94% in Oregon to 34.83 ± 9.69% in Delaware). β-caryophyllene (15.26% in Oregon to 48.44 ± 1.35% in Delaware), and/or (E)-nerolidol (10.20% in Oregon to 12.05 ± 2.02% in Delaware). The oil of the twigs of spicebush is notably high in 1,8-cineole (45.41 ± 0.35% in Delaware), while the oil of the fruits is notably high in a-phellandrene (64.62 ± 0.66% in Delaware) and β-phellandrene (11.23+0.17% in Delaware).     

Sex chromosomes,micronuclei and aging in women

Several studies on aneuploidy and aging have shown a significant increase in the loss of chromosomes in both males and females with age. Others have observed a significant increase in micronucleus formation in lymphocytes with age. The objectives of this investigation were to determine the relationship between sex chromosome loss and increased micronucleus frequencies with age, to establish sex chromosome loss frequencies unbiased by cellular survival factors or slide preparation, and to determine the effect of smoking on sex chromosome loss. Blood samples were obtained from 8 newborn females and 38 adult females ranging in age from 19 to 77. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Two thousand binucleated cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. Slides were then hybridized with a 2.0 kb centromeric X chromosome-specific probe labeled with biotinylated dUTP, and detected with fluorescein-conjugated avidin. All micronucleated cells were relocated and their X chromosome status was determined. We found the X chromosome to be present in 72.2% of the micronuclei scored; additionally our results show a significant increase with age in the number of micronuclei containing an X chromosome.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.