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Enantioselective reduction of acetyldimethylphenylsilane by Trigonopsis variabilis (DSM 70714)
(1987)
Growing and resting cells of the yeast Trigonapsis variabilis (DSM 70714) can be used for the enantioselective reduction of the organosilicon compound acetyldimethylphenylsilane (J) to give optically active (R)-(1-hydroxyethyl)dimethylphenylsilane [(R)-2] in good yields. The enantiomeric purity of the isolated product was determined tobe 62-86% ee depending on the substrate concentration used. Both substrate and product caused an inhibition of the reaction at concentrations higher than 0.35 and 0.5 g/1, respectively. Besides, higher substrate and product concentrations led to increased formation of the by-product 1,1,3,3-tetramethyl-1,3-diphenyldisiloxane. Considering the limiting substrate and product concentrations, it was possible to use the same biomass at least 5 times without significant loss of enzyme activity. 3-Methyl-3-phenyl-2-butanone (5) and acetyldimethylphenylgermane (7), which represent carbon and germanium analogues of 1, were also found to be accepted as substrates by Trigonapsis variabilis (DSM 70714). The reduction rates of the silicon {1) and germanium compound {7) were much higher than the transformation rate of the corresponding carbon analogue 5.
Mouse L-cells were transfected by electropenneabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). 1be DNA concentration used was 1 l'gfml, the field strength was 10 kV fcm, the duration of the pulse was S ~s. Transfeetion yield was optimal at a temperature of 4°C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seoonds) or Ionger (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 o C. The yield of transfectants in response to 10 pulses was up to 1000 per 106 cells (using 3.3 pg DNA per cell). The inßuence of the growth phase of the cells on the transfection yield and I or the subpopulation of the mouse L--ceU line used was shown. Furthennore the clone yield depended on the DNA per ceU ratio within a very small range.
Thyrotropin releasing hormone (TRH, I-pyroglutamyl-l-histidyl-l-prolinamide) was the fIrst hypothalamic releasing SUbstance to be isolated, chemically characterized and synthetized /1/. The studies to date have revealed that the thyrotropin release from the pituitary gland is only one of the numerous actions of TRH. In addition to its endocrine actions (TSH and prolactin release) this tripeptide has central nervous system actions totally unrelated to its effects on the hypothalamo-pituitary axis. This review aims to summarize the studies on the central nervous system' actions of TRH with special emphasis on the autonomic pharmacology of this peptide.
A literature review has shown that the daily intakes of various N -nitroso-precursor classes in a typical European diet span five orders of magnitude. Amides in the form of protein, and guanidines in the form of creatine and creatinine, are the nitrosatable groups found most abundantly in the diet, approaching Ievels of 100 g/day and 1 gjday, respectively. Approximately 100 mg of primary amines and amino acids are consumed daily, whereas aryl amines, secondary amines and ureas appear to lie in the 1-10 mg range. The ease of nitrosation of each precursor was estimated, the reactivities being found to span seven orders of magnitude, with ureas at the top and amines at the bottom of the scale. From this infonnation and an assessment of the carcinogenicity of the resulting N-nitroso derivatives, the potential health risk due to gastric in vivo nitrosation was calculated. The combined effects of these risk variables were analysed using a simple mathematical model: Risk = [daily intake of precursor] x [gastric concentration of nitrite]\(^n\) x [nitrosatability rate constant} x [carcinogenicity of derivative]. The risk estimates for the various dietary components spanned nine orders of magnitude. Dietary ureas and aromatic amines combined with a high nitrite burden could pose as great a risk as the intake of preformed dimethylnitrosamine in the diet. In contrast, the risk posed by the in vivo nitrosation of primary and secondary amines is probably negligib1y small. The risk contribution by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. Thus three priorities for future work are a comprehensive study of the sources and Ievels of arylamines and ureas in the diet, determination of the carcinogenic potencies of key nitrosated products to replace the necessarily vague categories used so far, and the development of short-term in situ tests for studying the alkylating power or genotoxicity of N-nitroso compounds too unstable for inclusion in long-term studies.
The potential health risk posed by the endogenous formation of N-nitroso compounds (NOC) from nitrosation of dietary ureas, guanidines, amides, amino acids and amanes (primary, secondary and aromatic) was estimated according to the model:
Risk = ( daily intake of precursor] X (gastric concentration of nitrite ]n X [nitrosatability rate constant] X [cilrcinogenicity of derivative].
The daily intakes ofthese compound classes span five orders ofmagnitude (100 g/day amides, top; 1-10 mg/day secondary amines, ureas, bottom); the nitrosation rate constants span seven orders of magnitude (aryl amines, ureas, top; amides, secondary amines, bottom); and the carcinogenicity estimates span a 10 000-fold range from 'very strong' to 'virtually noncarcinogenic'. The resulting risk estimates likewise span an enormous range (nine orders of magnitude ): dietary ureas and aromatic amines combined with high nitrite concentration could pose as great a risk as the intake of preformed N-nitrosodimethylamine in the diet. In contrast, the risk posed by the in-vivo nitrosation of primary and secondary amines is probably negligible. The risk contributed by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes.
Nitrosation of dietary components has been combined with the 4-(para-nitrobenzyl)pyridine (NBP) colorimetric test for screening alkylating agents and with the Ames test for the detection of mutagenic activity. This allowed the investigation of short-hved nitrosation products of dietary components which generate electrophilic degradation products requiring no metabolic activation (natural amino acids and some derivatives, ureas, guanidines, primary alkyl and aryl amines). In a first system, precursor, nitrous acid and NBP were present simultaneously. All amino acids tested, except glutamic acid and glutamine, gave positive results. The reactivities spanned more than three orders of magnitude, with the aromatic amino acids and methionine the most active; two primary amines, tryptamine and histamine, were also strongly reactive. All guanidines tested, except the amino acid arginine, gave negative results. A second system consisted of two phases: NBP was added only after destruction of residual nitrite and adjustment of the pH to neutrality. This system was useful for the study of ureas, which are stable in acid but not in neutral media. The range of responses covered more than two orders of magnitude. Most amino acids and primary amines also gave positive results, but could be assessed only after analysing the kinetics of the competing reactions and choosing appropriate reaction times. In a third system, Salmonella typhimurium strain TA1OO replaced NBP. Representatives of the class of amino acids, ureas, the primary amine tryptamine, and aniline became higbly mutagenic upon nitrosation. Methylguanidine was only weakly mutagenic under the present assay conditions. The results indicate that further studies with unstable nitrosation products of dietary components are required to understand more thoroughly the role of endogenous nitrosation in gastric cancer.
Newly isolated lymphocytes from mousespleensexpress the c-fos oncogene even in the absence of mitogen with maximal mRNA levels 60 min post preparation of single cell suspension, whereas c-myc mRNA Ievels increase only after mitogenic Stimulation with maximal mRNA Ievels 6 h post Stimulation. The half-lives of c-fos mRNA are generally very short; they increase from 14 min (after 30 min of culture) to 70 min (after 2 h of culture). The half-lives of c-myc mRNA decrease from 50 min (at 2 and 6 h post stimulation with concanavalin A) to 12 min (at 48 h post stimulation). The c-fos gene transcription is already tumed on in time-0 lymphocytes 10 min after disruption of the organ structure of the spleens and is down-regulated after 2 h and later. In nuclear run-on experiments with nonstimulated lymphocytes there is already significant transcription of the first exon of c-myc, but almost no elongation of the transcript to exon 2 and 3. In concanavalin A-treated lymphocytes elongation is stimulated about 5-fold within 6 h and returns to background levels at 48 h post Stimulation. · The nuclear run-on analyses of nonactivated lymphocytes showed a signal for RNA complementary to c-myc mRNA detected with a probe specific for the exon 1/intron 1 boundary of c-myc, which disappeared with increasing time of concanavalin A Stimulation. This anti-sense transcription may play a role in regulating the elongation of cmyc transcripts.
According to more recent studies on memory development in young children, preschoolers and kindergarteners are able to demonstrate surprisingly good memory skills in natural as weH as in laboratory-type settings. This finding is not consistent with the results of a study by Istomina (1975), conducted in 1948, leading to the concJusion that (a) preschoolers do not use voluntary remembering, and (b) children generally recall better in play situations than in typical experimental settings. In this study, two experiments were conducted to replicate Istomina's research. In the first, it was shown that Istomina's findings were replicable when methodological problems in the procedure were ignored. Experiment 2 improved methodologically upon Istomina's experimental methods and did not produce results to support her concJusions. Four- and 6-year-olds showed voluntary memory in play activities as weH as in laboratory-type settings, and remembered equally weB in both contexts. The results did not support the assumption that memory performance in young children can be substantially facilitated by motivating contexts.
Nucleotide sequence of the sfaA gene coding for the S fimbrial protein subunit of Escherichia coli
(1987)
The sfaA gene of the uropathogenic Escherichia coli 06 strain 536, which is responsible for the determination of the S fimbrial protein subunit, was sequenced. The structural gene codes for a polypeptide of 180 amino acids including a 24-residue N-terminal signal sequence. A size of 15.95 kDa was calculated for the processed SfaA protein. The nucleotide and deduced amino acid sequences show significant homology to those of the F1C fimbria and, to a lesser extent, of the mannose- sensitive hemagglutinating fimbria (FimA, PilA). Only week homology toP fimbriae subunits (F72 , Pap) was found.
High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody
(1987)
A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.