Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by ¹H and ¹³C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.     

The posibility of soil micromycetes produced the abscisic acid

The typical soil micromycetes Aspergillus niger and Cladosporium cladosporioides from the family moniliaceae were investigated with emphasis on production of ABA into the culture medium. The both fungi were cultivated in a static liquid Czapek — Dox medium and agar Czapek — Dox medium. Aspergillus niger and Cladosporium cladosporioides showed ability to produce ABA. Analytical detection of ABA from the culture medium was performed by TLC combinated with biotest and HPLC with spectroscopy.     

Evergreen leaf respiration acclimates to long-term nocturnal warming under field conditions

Acclimation of plant respiration rates (R) to climate warming is highly variable and many results appear contradictory. We tested the recently suggested hypotheses that pre‐existing, long‐lived leaves should exhibit a relatively limited ability for R to acclimate to climate warming, and that acclimation would occur via changes in the short‐term temperature sensitivity of respiration. Seedlings of a subalpine, evergreen tree species (Eucalyptus pauciflora) were grown under naturally fluctuating conditions within its natural distribution. We used a free air temperature increase (FATI) system of infra‐red ceramic lamps to raise night‐time leaf temperatures by 0.3±0.1, 1.3±0.1, and 2.2±0.1 °C above ambient for 1 year. Light‐saturated assimilation rates and plant growth did not change with nocturnal FATI treatments. Leaf R measured at prevailing temperatures did not differ between FATI treatments. Within each FATI treatment, nocturnal leaf R was highly sensitive to artificial temperature changes within minutes, and also correlated strongly with natural nocturnal and seasonal temperature variation. The corresponding values of Q₁₀ of R varied according to time scale of measurements, but did not vary between FATI treatments. Instead, acclimation of R to nocturnal FATI occurred through changes in the base rate of respiration.     

Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Characterization of sialic acids containing lipopolysaccharide from Rhizobium meliloti M 11 S

Abstract The lipopolysaccharide (LPS) of Rhizobium meliloti strain M 11 S was isolated and analyzed. It contained fatty acids (3-hydroxymyristic, palmitic, stearic, arachidic acids) and sugars: glucose, galactose, glucosamine, 3-deoxy- d -mannooctulosonic acid and sialic acids (NeuAc, 9- O -acetyl-NeuAc) identified by combined gas-liquid-chromatography/ mass spectrometry (GLC-MS).