A lipase from
Serratia marcescens was selected as an asymmetric hydrolytic enzyme for
trans-3-(4-methoxyphenyl)glycidic acid methyl ester (±)-MPGM, a key intermediate in the synthesis of diltiazem ...hydrochloride that is useful as a coronary vasodilator. This lipase has high enantioselectivity (
E=135) and was applied to the industrial production of the optically active intermediate of diltiazem using two-phase reaction system of organic solvent–water. Introduction of enzymatic reaction into the chemical synthetic route of diltiazem reduces the number of processes from nine to five. Analyses of the secretion mechanism of the lipase from
S. marcescens cell membrane revealed that lipase (LipA), metalloprotease (PrtA), cell surface protein (SlaA) and flagellin are secreted via ABC-transporter, which is a common secreting mechanism in Gram-negative bacteria other than N-terminal signal peptide-dependent secreting mechanism. Molecular cloning of both the
lipA gene, which codes the lipase protein, and
lipBCD genes, which code the secretion device proteins, enable the production of the lipase by the self-cloning strain 140-fold as compared to the wild type strain. Immobilization of the lipase on a hollow fiber type membrane reactor contributes to the repeated use of enzyme and to efficient separation of the reaction product. Thus, enzymatic reaction and product separation are achieved simultaneously.
The present study examined initial changes in non-traumatic osteonecrosis of the femoral head (ONF) on T1- and T2-weighted MR images, and fat suppression images. The subjects were 57 renal transplant ...recipients (37 males and 20 females), whose median age at the time of transplantation was 31.5 years old (range, 10 to 58 years). Twelve patients developed band patterns (sign of established ONF) at an early postoperative period. Among them, 4 joints of 3 patients had a localized, faint signal abnormality in fat suppression images, where band pattern was confirmed later in T1- and T2-weighted images. In all the 57 patients, no bone marrow edema preceding to ONF was observed. Bone marrow edema would not be the cause of ONF in renal transplant patients. Early changes depicted in our fat suppression images would be useful information in the studies on pathogenesis of ONF.
Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical ...synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.
Background and Aims: Increased numbers and enhanced functions of peripheral neutrophils have been observed in obstructive jaundice. However, the effects of obstructive jaundice on the bone marrow, ...that is neutrophil production and acquisition of neutrophil chemotactic activity, have been poorly understood. In the present study, differentials of bone marrow cells and chemotactic activity of bone marrow neutrophils were evaluated in bile duct‐obstructed rats.
Methods: Male Wistar rats underwent either bile duct obstruction for 10 days or bile duct obstruction for 4 days followed by 6 days’ internal biliary drainage. Differentials of peripheral blood and bone marrow cells were sequentially determined. Chemotactic activity of peripheral and bone marrow neutrophils was evaluated with a modified Boyden method using interleukin‐8 (recombinant rat Gro‐β) as a chemoattractant.
Results: Numbers of peripheral neutrophils significantly increased after bile duct obstruction. Significant increases in the myeloid/erythroid (M/E) ratio of bone marrow cells were observed after bile duct obstruction. The neutrophil proliferative pool (promyelocytes and myelocytes) increased initially, followed by an increased neutrophil storage pool (metamyelocytes, bands, and segmented neutrophils). The M/E ratio as well as the neutrophil proliferative and storage pools normalized after internal biliary drainage. Chemotactic activity was enhanced in both peripheral and bone marrow neutrophils after bile duct obstruction, and enhanced chemotaxis was alleviated with internal biliary drainage.
Conclusion: The present results strongly suggest the principal role of the bone marrow in increasing the number of neutrophils and their chemotactic activity during obstructive jaundice.
The crystal structure of polyporusterone A, from Polyporus umbellatus F. was determined by X-ray diffraction analysis. The crystals are orthorhombic, space group P212121, Z=4, unit-cell dimension ...a=17.968(2), b=26.201(5), c=6.227(1) Å. The structure, (+)-2β, 3β, 14α, (20R, 22R)-pentahydroxy-(24S)-methyl-5β-cholest-7-en-6-one, was solved from diffractometric data by direct methods and refined by least-squares calculations to R=0.053 (2345 observed independent significant reflections (I>3σ(I)). All the hydroxyl groups are involved in a hydrogen-bonding network. The NMR data indicate that the side-chain has the same conformation in both the crystalline state and solution.
The effect of obstructive jaundice on neutrophil function has not been extensively studied. Therefore, the present study aimed at evaluating the effect of obstructive jaundice on human neutrophils.
...Twelve patients with obstructive jaundice due to common bile duct obstruction underwent endoscopic biliary drainage. Neutrophil functions (chemotaxis and superoxide anion generation) were evaluated before and 7 days after drainage.
Neutrophil chemotaxis in response to FMLP (formyl-methionyl-leucyl-phenylalanine) or interleukin-8 was abnormally increased before drainage, and was normalized after drainage. Similarly, enhanced superoxide anion generation in response to FMLP or phorbol myristate acetate before drainage was alleviated after drainage.
The results suggest neutrophil overactivity in patients with obstructive jaundice. The ameliorating effect of biliary drainage on neutrophil overactivity might play a role in the prevention of postoperative complications.
The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) ...exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.
We have isolated a lipase-overproducing mutant, GE14, from
Serratia marcescens 8000 after three rounds of
N-methyl-
N′-nitro-
N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of ...extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (
lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the
lipA gene without an amino acid substitution. Promoter analysis using
lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the
lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number
lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the
S. marcescens lipase.
In a first experiment, 28 specific pathogen-free chickens aged 3 weeks showed clinical signs 1 to 5 days after intramuscular inoculation with
Erysipelothrix rhusiopathiae. Twelve of 28 birds died 2 ...to 4 days after inoculation. Macroscopically, the liver, spleen and kidneys were seen to be enlarged and congested. Histologically, fibrinous thrombus formation, seen in the hepatic sinusoids, renal glomerular capillaries and small pulmonary blood vessels, was a characteristic feature. In addition, the liver showed marked congestion, increase of mononuclear cells and heterophils in the sinusoids, hyperplasia of sinusoidal lining cells, and vacuolar changes in hepatic cells. The spleen showed fibrinous exudation of the lymphoid follicles and ellipsoids with lymphocytic depletion, and hyperplasia of ellipsoidal reticular cells. There was oedema, congestion and cellular infiltration in the interstitium of the kidney. The bursa of Fabricius and thymus showed marked lymphocytic depletion. In a second experiment, the blood chemical values (uric acid, glutamic-oxalacetic transaminase, lactate dehydrogenase and γ-glutamyl transpeptidase) of birds inoculated intramuscularly with
E. rhusiopathiae were significantly higher than those of uninfected controls. The blood prothrombin times and activated partial thromboplastin times of the inoculated group were significantly greater than those of the control group. The pathological and haematological findings demonstrated that
E. rhusiopathiae induced disseminated intravascular coagulation in the chickens.
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