Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp. KOD1.

The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcussp. KOD1, was expressed in Escherichia coli. The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form. A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity. The reaction product of this DNase activity was mononucleotides. The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively. In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity. The protein exhibited no DNase activity in the presence of Zn2+ion, which was one of the most preferable divalent cations for ATPase activity. Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP.Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity.     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

Pcal_0768, a hyperactive 4-α-glucanotransferase from <Emphasis Type="Italic">Pyrobacculum calidifontis</Emphasis>

Genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame, Pcal_0768, corresponding to a putative 4-α-glucanotranferase belonging to glycoside hydrolases (GH) family 77. We have produced, in Escherichia coli, and purified recombinant Pcal_0768 which exhibited high disproportionation (690 U mg^?1) activity. To the best of our knowledge, this is the highest ever reported activity for any member of family GH77. Maltooligosaccharides, when used as sole substrates, were disproportionated into linear maltooligohomologues. The analysis of the reaction end products revealed no evidence for the production of cycloamyloses. Catalytic activity of the enzyme remained unchanged in the presence or the absence of ionic and nonionic detergents. γ-cyclodextrin, an inhibitor of 4-α-glucanotransferases, did not show any inhibitory effect on Pcal_0768 activity. These properties make Pcal_0768 a potential candidate for starch processing industry.     

The use of a proteinaceous “cushion” with a polystyrene‐binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a “cushion” between the PS‐affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS‐affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527–534, 2016     

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.     

Synchronous Growth and Sporulation of Bacillus megaterium

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.     

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Esterastin: a potent inhibitor of lysosomal acid lipase

The effect of a fungal metabolite, esterastin, on lysosomal acid lipase purified from rabbit liver was studied. Esterastin inhibited the enzyme activity very strongly (IC50, about 80 nM). The inhibition of acid lipase by esterastin was competitive with respect to the substrate and the inhibition constant for esterastin was 90 nM. Esterastin was less inhibitory to other lipolytic enzymes, such as pancreatic lipase and carboxylesterase. Thus esterastin is a potent new inhibitor of lysosomal acid lipase.