Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids

The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively. C. paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R. glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced. The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments. A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

Hydrazonopropionic acids, a new class of hypoglycemic substances. 4. Hypoglycemic effect of 2-(3-methyl-cinnamylhydrazono)-propionate in the rat and guinea pig

The hydrazone-compound 2-(3-methyl-cinnamylhydrazono)-propionate (MCHP) significantly lowered the blood glucose concentration in fasted guinea pigs and rats. A significant decrease of blood glucose levels was observed in fasted guinea pigs already after an intraperitoneal injection of 20.5 mumol/kg MCHP, while much higher doses (about 1000 mumol/kg) were necessary to produce a hypoglycemic effect in the fasted rat. After oral administration MCHP (82.0 mumol/kg) significantly decreased the blood glucose concentration in guinea pigs. Furthermore MCHP caused a dose-dependent increase of plasma free fatty acid concentrations in guinea pigs and rats. In addition, MCHP decreased the concentrations of blood ketone bodies, plasma cholesterol and intrahepatic acetyl-coenzyme A in the guinea pig. All of these findings appear to be due to a reduced fatty acid utilization in the presence of MCHP resulting presumably in an intramitochondrial deficiency of acetyl-CoA. At hypoglycemic effective doses the intramitochondrial and cytoplasmatic redox ratios as well as the hepatic ATP/ADP ratio were not influenced by MCHP in fasted guinea pigs. Even at large doses (123 mumol/kg) MCHP decreased the activity of monoamino oxidase in guinea pigs only by less than 15%. Furthermore MCHP showed under our experimental conditions no relevant influence on the activity of various liver enzymes in plasma, the plasma concentration of creatinine, the plasma triglyceride-glycerol level and on the intrahepatic triglyceride-glycerol concentration of fasted guinea pigs. It is concluded that MCHP meets basic requirements for a potential oral antidiabetic agent.     

Influence of a 9-double bond on stereospecific microbial 4,5-reductions

By stereospecific microbial reduction with Rhodosporidium rubrum or Rhodotorula glutinis, 17 alpha-cyano-methyl-4-estren-17 beta-ol-3-one was metabolized to 17 alpha-cyanomethyl-5 alpha-estrane-3 beta,17 beta-diol (50%) and 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha,17 beta-diol (30%). By Clostridium paraputrificum the same substrate was reduced stereospecifically to 17 alpha-cyanomethyl-5 beta-estrane-3 alpha, 17 beta-diol (70%). When the corresponding 9-dehydrogenated compound 17 alpha-cyanomethyl-4,9-estradien-17 beta-ol-3-one (STS 557, a new progestagen) was fermented, yeasts failed in 5 alpha-reducing the 4-double bond. Still Clostridium paraputrificum formed the expected 5 beta-reduced metabolite 17 alpha-cyanomethyl-5 beta-estr-9-ene-3 alpha,17 beta-diol (60%). Structures were elucidated by n.m.r. and mass spectra and partly by circular dichroism. By oxidation of the metabolites, the corresponding 3-oxo compounds 17 alpha-cyanomethyl-5 alpha-estran-17 beta-ol-3-one, 17 alpha-cyanomethyl-5 beta-estran-17 beta-ol-3-one and 17 alpha-cyanomethyl-5 beta-estr-9-en-17 beta-ol-3-one were prepared. The evident influence of the 9-double bond on reduction of the 4-en-3-oxo compound STS 557 preventing 5 alpha-reduction but permitting 5 beta-reduction is discussed in view of the distinctly diminished metabolism of this progestagen in mammals.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

Farbstoffgehalt und Grösse der Augen bei verschiedenen Mutanten von Drosophila melanogaster

Zusammenfassung Quantitative Untersuchungen über den Farbstoffgehalt der Drosophilaaugen haben schon wiederholt gezeigt, daß die Werte bei bestimmten Mutanten von der Erwartung abweichen. So fand man regelmäßig bei den rotäugigen Mutanten v bzw. cn weniger Pterin und bei der braunäugigen Mutante bw weniger Ommochrom als bei Wildfliegen.Wir haben diese Befunde zunächst mit Hilfe einer vereinfachten Extraktions- und Meßtechnik nachgeprüft und bestätigt. Die genauere Analyse ergab dann aber, daß das Farbstoffdefizit der Mutanten v, cn und bw lediglich darauf beruht, daß diese Tiere kleinere Augen haben als die Wildfliegen. Die Augenverkleinerung ist jedoch nicht, wie gelegentlich vermutet wurde, die Folge einer polyphänen Wirkung der Gene v, cn und bw, sondern nur eine besondere Eigenschaft bestimmter Fliegenstämme, die heute in fast allen Laboratorien gehalten werden.Die Erscheinung selbst beruht auf der Wirkung augenverkleinernder Modifikationsgene, die bei diesen Stämmen zufällig mit den Farbgenen gekoppelt sind, durch geeignete Kreuzungen aber eliminiert werden können. Unsere so erhaltenen neuen v-, cn- und bw-Stämme besitzen nicht nur ebenso große Augen wie die Wildfliegen, sondern enthalten auch die theoretisch erwarteten Mengen an Augenfarbstoffen. Der Zusammenhang zwischen der Größe der Augen und ihrem Farbstoffgehalt hat u. a. zur Folge, daß die Männchen, die ja stets kleinere Augen haben als die Weibchen, bei allen Mutanten weniger Augenpigment besitzen als jene.Der Farbstoffgehalt der Augen hängt außerdem von der Zucht-temperatur ab. Fliegen, die sich bei 18° C entwickeln, besitzen weniger Pterin aber mehr Ommochrom als solche, die bei 26° C aufgezogen werden. Auch die Melaninsynthese im Integument der Tiere wird durch Temperaturerniedrigung begünstigt; aus 18°-Zuchten stammende Fliegen sind deutlich dunkler als die entsprechenden 26°-Tiere.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.