alpha-Amylase inhibitor from fungus Cladosporium herbarum F-828

A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.     

Nδ-benzoyl-l-ornithine and Nδ-benzoyl-l-γ-hydroxyornithine from Vicia pseudo-orobus

Two new non-protein amino acids, N^δ-benzoyl-l-ornithine and N^δ-benzoyl-l-γ-hydroxyornithine have been characterized from the seeds of Vicia pseudo-orobus.     

Genetic studies on site-specific endodeoxyribonucleases in Bacillus subtilis: multiple modification and restriction systems in transformants of Bacillus subtilis 168

Summary We transformed B. subtilis 168 with DNA from B. subtilis IAM1231, IAM1192 and ATCC6633. When we examined the restriction activities of the transformants in vivo and in vitro using phage 105C we found the following: (1) Cells of either IAM1231 or IAM1192 have two modification and restriction systems (Bsu1231(1)-system and Bsu1231(II)-system in IAM1231, and Bsu1192(I)-system and Bsu1192(II)-systems in IAM1192), and cells of ATCC6633 have only one system (Bsu6633-system). (2) The restriction enzymes of all of these five systems are site-specific endonucleases. (3) The nucleotide sequence specificities of the enzymes involved in Bsu1231(I)-system, Bsu1192(I)-system and Bsu6633-system are the same; and those of Bsu1231(II)-system and Bsu1192(II)-system are the same. The sequence specificities of these two groups are different from each other and also different from those of the Bsu168-system of B. subtilis 168, the BsuR-system of B. subtilis R and the Bsu1247(I)-and Bsu1247(II)-systems which are systems of B. subtilis IAM1247. (4) Transformants possessing four different modification and restriction systems (Bsu1231(I)-, Bsu1247(I)-, BsuR- and Bsu168-systems) were constructed. (5) Transformation of two derivatives of 168 that were m _R ⁺ r _R ⁺ by DNA from IAM1231 produced 16 transformants that had the Bsu1231(II) restriction system, but had lost the BsuR system. Transformation of a derivative of 168 that was m _1247(II) ⁺ r _1247(II) ⁺ by DNA from m _1231(II) ⁺ r _1231(II) ⁺ -or m _R ⁺ r _R ⁺ -derivative of 168 produced about 100 each of transformants that had the Bsu1231(II)-restriction system or the BsuR-restriction system. But all these transformants lost the Bsu1247(II)-system.     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

A new site-specific endonuclease StuI from Streptomyces tubercidicus

A new sequence-specific endonuclease, StuI, produced by Streptomyces tubercidicus KCC S-0054, was identified and partially purified. StuI recognizes the hexanucleotide "palindromic" sequence (Formula: see text), and cleaves it at the middle, producing blunt ends.     

Relationship of ploidy and chromatin condensation in the rat liver,moreover a comparison of the nuclear texture in sections and touch preparations

Summary In touch preparations and tissue sections from normal rat hepatocytes and -amanitine- and actinomycind-poisoned liver cells the extent of chromatin condensation relatively to the degree of ploidy and the chromatin distribution were studied by means of computer aided cytophotometry. It could be found, that the relationship of the condensed and decondensed chromatin is independent of the degree of ploidy and first of all dependent upon the intoxicant. Therefore, the extent of chromatin condensation can be utilized also in sections as additional parameter for automated cell screening.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 105     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Fibrin membrane endowed with biological function VI. Immobilization of multienzymes of urea cycle

Four enzymes in urea cycle and inorganic pyrophosphatase were immobilized simultaneously into a matrix of fibrin fiber formed from fibrinogen by the concerted action of thrombin and blood coagulation Factor XIII. The immobilized multienzyme system not only had an ability to carry out urea cycle continuously at least over several hours, but also had a greatly improved efficiency over the corresponding soluble system.     

A quantitative analysis of orbital soft tissue in Graves' disease based on B-mode ultrasonography

Using B-mode ultrasonography, an attempt was made to measure the volume of extraocular muscles and retrobulbar fat in 31 patients (62 orbits) with Graves' disease. None of the patients had exophthalmometric measurements greater than 21 mm or had eye symptoms. The mean value of muscle volume of Graves' patients was significantly larger than that of normal controls (6.48 +/- 2.70 cm3 and 3.25 +/- 1.30 cm3, respectively, p less than 0.001). All of the patients had extraocular muscle swelling, although 2 of them had no extraocular muscle change for their unilateral eye. The extraocular muscle volume increased as the degree of the proptosis increased. The fat volume tended to increase in parallel with the degree of the proptosis. In the Graves' group with obvious proptosis (Hertel reading: 19--21 mm), the fat volume increased more significantly than in any other group. The ratio of extraocular muscle volume to retrobulbar fat volume was significantly higher in Graves' disease, but it did not increase as the degree of the proptosis increased. A significant correlation between proptosis and muscle volume plus fat volume was observed. No significant difference of the extraocular muscle volume was observed between the patients untreated and treated with antithyroid drugs. The data show a uniform enlargement of the extraocular muscles in Graves' disease and also suggest an involvement of increased retrobulbar fat volume in a group of obvious exophthalmos. The degree of the proptosis is in aclose proportion ot the quantitative change of the orbital soft tissue.