Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers

 The cyanobacterium, Aphanocapsa halo-phytia MN-11, was immobilized in calcium alginate gel and coated on light-diffusing optical fibers (LDOF) for sulfated extracellular polysaccharide production. Results indicated that sulfated extracellular polysaccharide production depends on the number of immobilized cells and the light intensity. In addition, the production rate reached 116.0 mg (mg dry cells)^-1 day^-1 when the cells that were immobilized on LDOF were incubated under a light intensity of 1380 cd sr m^-2 at a cell concentration of 1.0×10⁸ cells/cm³ gel. Cells immobilized on LDOF produced about ten times more sulfated extracellular polysaccharide than those immobilized in calcium alginate beads only (11.7 mg(mg dry cells)^-1day^-1). Received: 31 March 1995/Revised last revision 12 June 1995/Accepted 26 July 1995     

Enzyme complex structure and location of enzymes in the phthalate degradative pathway in Rhodococcus erythropolis

A. SUEMORI, K. NAKAJIMA, R. KURANE AND Y. NAKAMURA. 1996. Rhodococcus erythropolis strain S1 formed enzymes essential to the degradation of phthalate when grown in phthalate-minimal medium. The reaction responsible for the dihydroxylation of the phthalate-benzene ring was concluded to be catalysed by membrane-associated phthalate 3,4-dioxygenase (PO). Of the other enzymes involved, 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH) and 3,4-dihydroxyphthalate 2-decarboxylase (PC) appeared likely to be membrane-bound, while protocatechuate 3,4-dioxygenase appeared to be present in the cytoplasm. Based on the data, the membrane-bound PO and PH apparently form an enzyme complex, which is associated with the NADH-regenerating system.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Structure and Chromosomal Assignment of the Human S1-5 Gene (FBNL) That Is Highly Homologous to Fibrillin

The human S1-5 gene (fibrillin-like; FBNL) was originally isolated from a subtractively enriched cDNA library established from a subject with Werner syndrome (WS). We isolated genomic clones containing the entire S1-5 gene and determined its genomic structure including the exon–intron organization. The gene spanned approximately 18 kb of genomic DNA and consisted of 12 exons. Its expression was abundant in all tissues examined except brain and peripheral leukocytes, where it was undetectable. In addition, we have mapped S1-5 by fluorescencein situhybridization to chromosome 2p16, a position that excludes it as a candidate for WS. Our data should facilitate an understanding of the function and regulation of S1-5 in human tissues.     

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability

Tetrahymena thermophila could still swim after incubation of the cell body at 40°C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to ajust the pH level when using medium-circulating-fermenters.