Production of protoplasts from the fungi Curvularia inaequalis and Trichoderma reesei

Summary Protoplast formation in Curvularia inaequalis was achieved using non-commercial and commercial snail gut enzymes or Trichoderma harzianum enzymes. The cells were grown for enzyme treatment on cellophane sheets or in liquid cultures for varying periods of time. The production of T. harzianum enzymes is discussed. The highest protoplast yields were 2.6x10⁷ protoplasts/ml enzyme solution. Protoplasts were shown to have zero to four nuclei. Protoplast regeneration was succesfully carried out in semisolid agar.     

Synthesis of methyl α-d-glucopyranosyl-(1–2)-α-d-galactopyranosyl-(1-3)-α-d-glucopyranoside and an acyclic analogue thereof for probing the carbohydrate-binding specificity of bacteriophage ϕX 174

The title trisaccharide was synthesized using methyl 1-thioglycoside building blocks. An acyclic analogue, methyl 3-O-(α-D-glycopyranosyl-oxyethyl)-α-D-glucopyranoside, which has an ethylene bridge in place of the galactosyl residue, was also synthesized.     

Accumulation of pyrophosphate in Escherichia coli. Relationship to growth and nucleotide synthesis

Pyrophosphate (PPi) content of Escherichia coli is increased manyfold when the growth of the cells is limited by inhibition of the synthesis of nucleotides. The accumulated PPi is immediately degraded when inhibition is released and growth allowed to resume. Other tested nutritional deficiencies (starvation of carbon source, sulfate or an amino acid) fail to induce PPi accumulation.     

Aspartate carbamoyltransferase from rat liver

1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, beta-benzyl-l-aspartate and beta-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The K(m) for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [(14)C]aspartate and an assay with [(14)C]carbamoyl phosphate. 2. The K(m) for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pK(e), was at pH9.0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag(+), Hg(2+), or Zn(2+). The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, beta-erythro-hydroxy-dl-aspartate and beta-benzyl-l-aspartate. The K(i) for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed.     

Some factors stimulating and inhibiting pancreatic deoxyribonuclease

Для выяснения роли ДН-азы в реакции трансформации исследовали действие различных сред, применяемых при трансформации пневмококков, на активность панкреатической ДН-азы. Эти виды среды резко повышали активность фермента. Далее, было установлено, что это повышение вызывается yeast-экстрактом, входящим в состав этих сред. С помщяю спектрального анализа и пламенного спектрофотометра в yeast-экстракте были обнаружены Mg²⁺, Ca²⁺, K⁺ и Na⁺. Изучалось действие этих ионов на активность ДН-азы и было установлено, что в присутствии Mg²⁺ (5×10^?3 m)иCa²⁺(вконцентрации 2×10^?4 m) резко повышается активность фермента, тогда как прибавление к этой системе KCl в концентрации 2×10^?1, 2×10^?2, 2×10^?3 и 2×10^?4 m оказывает угнетающее действие.—Обсуждается возможное влияние этих ионов на peaкцию трансформации.     

Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.     

Polymorphisms of the apolipoprotein and angiotensin converting enzyme genes in young North Karelian patients with coronary heart disease

The genes encoding apolipoproteins (apos) A-I, B, C-III and E as well as that encoding the angiotensin converting enyzme (ACE) have been proposed as candidate genes for coronary heart disease (CHD). We determined the common polymorphisms of the apo genes, previously found to influence serum lipid levels at the population level, and the insertion/deletion polymorphism of the ACE gene, recently reported to reflect the risk of myocardial infarction, in 82 very young (mean, 41 years) North Karelian Finns with symptomatic CHD and 50 controls of similar age. Patients with familial hypercholesterolemia had been excluded from this material. None of the polymorphisms examined, including the apo A-I promoter MspI, apo C-III SstI and apo B XbaI restriction fragment polymorphisms, a common variation of apo E (2, 3 and 4 alleles) and an ACE insertion/deletion (I/D) polymorphism, was significantly associated with the risk of premature CHD. Patients with CHD had a higher mean serum LDL cholesterol/HDL cholesterol ratio than controls (3.15±1.30 vs 2.72±0.98, P < 0.05), but no significant associations between the common apo gene polymorphisms and serum lipid levels were disclosed in either group. It is possible that other genetic loci than those proposed to be associated with accelerated atherosclerosis may be more important as risk factors of symptomatic CHD at the age of 40 years.