Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.     

Dimorphism of both head and prothoracic horn morphologies in male Copris acutidens (Coleoptera: Scarabaeoidea)

Allometric scaling of male horn morphologies of Copris acutidens, including the head and prothoracic horns, was analyzed. The analyses of scaling relationships indicated a discontinuous increase in head horn length and prothoracic horn height but a linear increase in prothoracic horn length relative to body size. The different results for length and height of the prothoracic horn suggest that the height is functionally more important for ensuring mates in the nests. Furthermore, both the head and prothoracic horns were dimorphic, and this characteristic was not found in the scarabaeoid beetle Onthophagus. Similarity in the switch point values for the head and prothoracic horns suggests that the dimorphism of the two kinds of horns may result from the same developmental threshold mechanism.     

Microhabitat use by larvae of the endangered dragonfly <Emphasis Type="Italic">Sympetrum pedemontanum</Emphasis><Emphasis Type="Italic">elatum</Emphasis> (Selys) in Japan

Sympetrum pedemontanum (Müller in Allioni) (Odonata: Libellulidae), which is distributed widely in the Eurasian continent and its neighboring islands, is listed as a Least Concern species in the International Union for Conservation of Nature Red List (2015). In Japan, however, the population of its subspecies S. pedemontanum elatum (Selys) has been rapidly decreasing since the 1970s. In order to conserve this subspecies, it is important to understand the seasonal microhabitat use by its larvae. However, this has been a difficult task because larvae of S. pedemontanum elatum often coexist with those of a common congener, S. eroticum (Selys), and cannot be morphologically distinguished from the latter. Thus, in this study, we first established a molecular technique based on the polymerase chain reaction to accurately identify each species. In the subsequent field survey in 2015 with its application in the Sakasegawa River, Hyogo Prefecture, we found that S. pedemontanum elatum larvae hatch in stagnant water and subsequently advance into weakly flowing water. Our results indicated a change in the microhabitats during the larval developmental process, reflecting the need for a continuous spectrum of stagnant, transitional, and flowing water. Such aquatic environments with a spectrum of water conditions are disappearing in Satoyama, a rural farming area in Japan. This has endangered species such as S. pedemontanum elatum and Oryzias latipes (Beloniformes: Adrianichthyidae) by depriving them of their favorable habitats. For their conservation, it is necessary to develop methods to recover the traditional aquatic environments in Satoyama.     

Drug-selected complete restoration of superoxide generation in Epstein-Barr virus-transformed B cells from p47 phox -deficient chronic granulomatous disease patients by using a bicistronic retrovirus vector encoding a human multi-drug resistance gene (MDR1) and the p47 phox gene

Chronic granulomatous disease (CGD) is a group of disorders characterized by the failure of phagocytes to produce superoxide. One-third of the cases of CGD in the USA and Europe results from defects in the gene encoding p47 ^phox , a cytoplasmic component of NADPH oxidase for superoxide generation. In this study, we constructed the bicistronic retrovirus vector Ha-MDR-IRES-p47, which carries cDNAs for a human multi-drug-resistance gene (MDR1) and p47 ^phox . The amphotropic retroviral producer cells were generated, and the supernatant of the producer cells was used to transduce Epstein-Barr virus-transformed B (EBV-B) cells, established from B cells of p47 ^phox -deficient CGD patients, as an in vitro model of gene therapy for p47 ^phox -deficient CGD. The transduced cells expressed both P-glycoprotein and p47 ^phox protein, and the expression levels were increased after appropriate vincristine selection. The levels of superoxide production in the vincristine-selected cells were increased to a level similar to normal EBV-B cells. This result suggests that it is possible to achieve 100% correction of the CGD defect in p47 ^phox -deficient EBV-B cells by using the bicistronic vector. This strategy could be employed not only in vitro, but also in vivo, in the gene therapy of a number of inherited diseases. Received: 8 June 1998 / Accepted: 5 August 1998     

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

 Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene. Received: 24 February 1998 / Revised: 11 May 1998     

Expression of tissue-type plasminogen activator and its inhibitor couples with development of capillary network by human microvascular endothelial cells on matrigel

Human omental microvascular endothelial (HOME) cells seeded on Matrigel begin to migrate within 1 h, forming honeycomb-like structures and capillary-like networks within 18 h. Cross-sections of the capillary networks show them to be tube-like structures. Northern blot analysis showed that tissue-type plasminogen activator (t-PA) mRNA synthesis increased from the initial state at 0 h after seeding on Matrigel, reaching a steady state after 4 h. This elevated cellular t-PA mRNA level decreased markedly at 24 h. In contrast, the cellular plasminogen activator inhibitor-1 (PAI-1) mRNA level demonstrated biphasic curves during the 24 h after seeding on Matrigel: the PAI-1 mRNA level was increased eightfold initially at 4 h over that at O h, then declined, and again secondarily increased to greater than tenfold at 18 h. Cellular levels of both 72 kD type IV collagenase and tissue inhibitor of metalloproteinase (TIMP-2) mRNA were increased only a slightly within 2–4 h. These elevated mRNA levels were maintained for 18 h, while the TIMP-1 mRNA level increased up to 18 h, reaching around three times the level at O h. However, on collagen-coated dishes, cellular levels of t-PA, PAI-1, 72 kD type IV collagenase, TIMP-1, and TIMP-2 mRNA were not greatly changed during incubation for 24 h. On Matrigel, the cellular t-PA mRNA level at 18 h after seeding was greatly increased when treated with specific anti-transforming growth factor-β (TGF-β) antibody. In contrast, both PAI-1 and TIMP-1 mRNA levels at 18 h were reduced in the presence of anti-TGF-β antibody. Development of the capillary network on Matrigel was inhibited in the presence of anti-t-PA antibody. Epidermal growth factor (EGF) enhanced t-PA gene expression and TGF-β inhibited its expression in HOME cells cultured on collagen-coated dishes. On the other hand, TGF-β enhanced cellular expression of the PAI-1 gene. The formation of a capillary network by HOME cells on Matrigel appears to be balanced by angiogenic EGF and anti-angiogenic TGF-β through modulation of PA activity. © 1995 Wiley-Liss, Inc.     

Enzymatic synthesis of mannobioses and mannotrioses by reverse hydrolysis using α-mannosidase from Aspergillus niger

Various manno-oligosaccharides including and

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were formed when a highly concentrated mannose solution was incubated in the presence of α-mannosidase from Aspergillus niger. and

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were isolated by activated carbon chromatography followed by high performance liquid chromatography using an amino-silica column. In addition to the above oligosaccharides,

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, and

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were also isolated.     

Application of the paired label radioantibody technique to detection and subtyping of hepatitis B surface antigen.

A sensitive radioimmunoassay has been developed for the detection and subtyping, in regard to w and r specificities, of hepatitis B surface antigens (HBsAg). The binding of monospecific rabbit antibodies directed to either w or r determinant, labeled with 131I and 125I, respectively, by HBsAg was determined in the same experiment, taking advantage of the fact that these specificities are in general, expressed mutually exclusively. HBsAg in the test sample were bound to solid phase guinea pig anti-HBsAg antibody tube before the test. The ratio of percentage uptake of 125I anti-r to that of 131I anti-w for 28 normal sera without antigen was 0.69 0.18. The ratio for seven sera containing HBsAg with w determinant was less than 0.1, and that for 34 with r determinant was more than 10; 100-fold difference was noted between values of w and r antigens.     

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.