Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Plasmid cloning vectors replicating in Escherichia coli,amino acid-producing coryneform bacteria and Methylobacillus sp.

Summary Four hybrid plasmids were constructed from the cryptic plasmid pAM330 (from Brevibacterium lactofermentum; 4.5 kb) and the broadhost-range plasmid pGV1106 (9.0 kb; Km^r Sm^r) isolated from Escherichia coli. All of them were mobilized from E. coli into the Gram-negative methylotrophic bacterium Methylobacillus sp. and two of these constructs (pCEM300 and pCEM400) were transferred by transformation into B. flavum and Corynebacterium glutamicum. Their kanamycin-resistance determinant coming from Gram-negative hosts was expressed in these Gram-positive bacteria. Both pCEM300 and pCEM400 are very stably maintained in B. flavum and represent suitable vectors for gene cloning in coryneform producers of amino acids.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

Production of milk-clotting enzymes by basidiomycetes

BasidiomycetesPhellinus chrysoloma, Kuehneromyces mutabilis andGanoderma applanatum produce extracellular milk-clotting enzymes. The enzymes are acid proteinases stable at 40°C and within pH 3–5.5. Only the enzyme preparation fromP. chrysoloma exhibits properties comparable with animal chymosin.     

Identification of thetrans-stilbene oxide-active glutathione transferase in human mononuclear leukocytes and in liver as GST1

A glutathione transferase from human mononuclear leukocytes with a high activity towardtrans-stilbene oxide (GT-tSBO) has been studied in liver and blood from fetus and adults and in blood from neonates. Using starch gel electrophoresis, different phenotypes of GST1 have been determined, GST1 0, GST1 1, and GST1 2. As judged from activity measurements and the fact that only those individuals who express the null allele of GST1, the GST1 0, which has a low activity towardtrans-stilbene oxide, it is concluded that the hepatic transferase GST1 is identical to GT-tSBO, as well as to hepatic transferase μ. In addition, it has been shown that the different genotypes of GST1 1 (GST1 1-1, GST1 1-0) and GST1 2 (GST1 2-2, GST1 2-0) can be separated by measuring the GT-tSBO activity in whole blood from the same individual. It is also demonstrated that GT-tSBO activity is much lower in fetal liver, approximately 10 times, compared with adult liver, while this activity seems to be unchanged in the blood from fetus and adults, as well as in neonates.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

ΔF508 frequency and associated haplotypes near the cystic fibrosis locus in the Yugoslav population

Summary Chromosomes from 19 unrelated Southern Yugoslav families in which cystic fibrosis (CF) occurs were analysed for the presence of the ΔF508 mutation, using polymerase chain reaction amplification followed by dot blot and polyacrylamide gel analysis. Of the 38 CF chromosomes, 15 (39.5%) carry the ΔF508 deletion. Restriction fragment length polymorphism haplotypes for KM19/PstI, XV2c/TaqI and J3.11/PstI marker loci were determined and are compared for a total of 34 N and 37 CF chromosomes.