Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

Engineering of closed ecological system in space and inter-organismal interactions.

Space agriculture is a concept of synthesis and operation of closed ecological system for controlling living environment and supplying materials in order to sustain life and to meet metabolic needs of space crew. It revitalizes metabolites and other excretion of crew for their recycled usage. It is an advanced concept for life support for long and large scaled manned space missions, where open loop system for materials cycle is not feasible to apply. Several issues, such as cost-benefit analysis with considering benefits of amenity and psychological factors of crew, are discussed in this essay, together with reliability and productivity of biological systems. Studies on plant physiology for inter-organismal interaction contribute to design work for space agriculture, and associate with our engagements to our future, sustainable development of our civilization both on the earth and extending to outer space.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Initiation of Cyclin B Degradation by the 26S Proteasome upon Egg Activation

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH₂ terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.     

Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.     

Morphogenesis and cell wall changes in maize shoots under simulated microgravity conditions.

Various plant organs show a spontaneous curvature on a three-dimensional clinostat. Changes in the cell wall metabolism underlying the curvature were examined in maize shoots. In coleoptile nodes, no differences were detected in either the level or the composition of cell wall polysaccharides between the convex and the concave halves. However, the convex side showed a higher activity of (1 --> 3),(l --> 4)-beta-glucan breakdown, which appears to be associated with the curvature. In the elongating region of coleoptiles, the accumulation of wall polysaccharides occurred in the convex side. There was no significant difference in the glucanase activity between both sides. Thus, the spontaneous curvature in different regions of maize shoots may be brought about through different mechanisms under simulated microgravity conditions.     

Studies on subfractions of hemoglobin A1b and hemoglobin A1c in diabetic subjects

Hemoglobin (Hb) obtained from the hemolysate of normal subjects and diabetic patients was separated into HbA1a1, HbA1a2, HbA1b, HbA1c and HbA0 (major Hb) by Bio-Rex 70 cation exchange column chromatography. The glycosylated Hbs were further separated reproductively by cation exchange high performance liquid chromatography (HPLC), using 50 mM sodium phosphate buffer pH 5.80 with 0-0.2 M NaCl linear gradient system. HbA1b and HbA1c were separated into two subfractions (HbA1b1 and HbA1b2) and three subfractions (HbA1c1, HbA1c2, HbA1c3), respectively. The percentages of each subfraction except HbA1c1 in diabetic patients were significantly higher than those in normal subjects. Furthermore, HbA1c1, HbA1c2 and HbA1c3 correlated well with fasting blood glucose levels in the prior 5 month period, while subfractions in HbA1b revealed no significant correlation with blood glucose levels. The percentages of each subfraction of HbA1c in patients either with diabetic cataracts or with diabetic neuropathy were almost the same as those in the patients without complications. However, the percentages of each of the three groups were markedly higher than those of the normal subjects. These results suggest that glycosylation of hemoglobin in diabetic patients may be increased in various sites of the molecule in parallel with the blood glucose levels during the preceding 4-5 months.     

Inhibitory effects of active vitamin D preparations on PTH secretion in rats

To study the effects of various vitamin D preparations on PTH secretion, serum calcium and urinary excretion of cAMP were monitored in conscious perfused rats, and the influences of a bolus iv injection of the preparations on these parameters were examined. Three hours after the administration of 0.25 microgram/kg (0.6 nmol/kg) of 1 alpha, 24(R)-dihydroxycholecalciferol [1 alpha, 24(OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized (PTX) rats (50% of initial value; p less than 0.05) with no change in the concentration of serum calcium (total and ionized). In PTX rats supplemented with bovine PTH (1 U/h), the vitamin D preparation showed no significant effects either on the urinary excretion of cAMP or on serum calcium. These effects were rather specific for active vitamin D preparations, i.e. 1 alpha, 25(OH)2D3 (0.25 micrograms/kg) and 1 alpha OHD3 (1.25-6.25 micrograms/kg). However, 24,25(OH)2D3 (up to 25 micrograms/kg) had no significant effect on these parameters. These results suggest that, in rats, active vitamin D preparations specifically inhibit PTH secretion without causing a significant increase in the serum calcium concentration, reflecting a direct feedback mechanism between active vitamin D metabolite and the parathyroid glands.