Molecular breeding of a biotin-hyperproducing Serratia marcescens strain.

We previously reported that an acidomycin-resistant mutant of Serratia marcescens Sr41, SB304, and a mutant that was derived from SB304 and was resistant to a higher concentration of acidomycin, SB412, produced 5 and 20 mg of D-biotin, respectively, per liter of a medium containing sucrose and urea (N. Sakurai, Y. Imai, M. Masuda, S. Komatsubara, and T. Tosa, Appl. Environ. Microbiol. 59:2857-2863, 1993). In order to increase the productivity of D-biotin, the biotin (bio) operons were cloned from strains SB412, SB304, and 8000 (wild-type strain), and pLGM412, pLGM304, and pLGW101, respectively, were obtained through subcloning. These plasmids harbored 7.2-kb DNA fragments coding for the bioABFCD genes on a low-copy-number vector and were introduced into SB304, SB412, and 8000. Among the resulting recombinant strains, SB412(pLGM304) exhibited the highest D-biotin production (200 mg/liter) in the production medium. The plasmid was stably maintained in cells. Unexpectedly, SB412(pLGM412) grew very slowly, and the D-biotin productivity of this recombinant strain was not evaluated because pLGM412 was unstable.     

Amplifiedbcl-2/JH rearrangements in reactive lymphadenopathy

Using the polymerase chain reaction (PCR) to examine the occurrence ofbcl-2/JH joining produced by t(14;18) chromosomal translocation, amplified DNA was detected in 2 of 18 lymph nodes showing reactive lymphadenopathy. The PCR was repeated in these two lymphs nodes using the same DNA samples, but no amplification was detected at the second attempt. Thus the amplified DNA was considered to be derived from one copy of joinedbcl-2/JH in one cell, or from a few copies in a few clonal cells with the same joinedbcl-2/JH. These results suggest that false joining ofbcl-2/JH at the t(14;l8) junction may occur in reactive lymph nodes.     

Cytotoxicity Induced by Monoclonal Antibody to α-Fetoprotein Coadministered with Spleen Cells of Nude Mice

This paper describes an attempt to effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) in nude mice. A monoclonal antibody against α-fetoprotein, 80G, coadministered with spleen cells from other nude mice bearing HuH-7N (xenograft of human hepatoma cell line, HuH-7) significantly suppressed the growth of HuH-7N as compared to treatment with 80G alone. 80G with spleen cells from normal nude mice also had some suppressive effect. In contrast, no effect was observed with each spleen cells alone as well as 80G alone. These results suggest that further supply of effector cells could enhance ADCC activity in nude mice.     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.     

A Close Relationship between Increases in Galactosyltransferase Activity and the Accumulation of Galactolipids during Plastid Development in Cucumber Seedlings

Changes in the activity of UDP-galactose:diacylglycerol galactosyltransferase(UDGT), a key enzyme in galactolipid biosynthesis, during germinationwere investigated in cucumber (Cucumis sativus L. cv. Aonagajibai)seedlings. After germination, UDGT activity increased duringgrowth in darkness for 4 days, reaching 10 times the activityin ungerminated seeds. Illumination of 4-day-old dark-grownseedlings strongly stimulated the activity. By contrast, inseedlings grown continuously in darkness, the increase in UDGTactivity ceased after 4 days and the activity remained constantthereafter. A similar increase in the specific activity of UDGTwas observed i n the envelope fraction from seedlings, indicatingthat the increase in the enzymatic activity preceded synthesisof other proteins in the envelope membrane. Coincident withthe change in the enzymatic activity, here was an increase inlevels of monogalactosyl diacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), two major constituents of chloroplastmembrane lipids, in the germinated seedlings. Cycloheximideinhibited the light-mediated increase in the enzymatic activityby illumination of 4-day-old dark-grown seedlings, and, as aconsequence, it inhibited the accumulation of MGDG and DGDG.It was clear, therefore, that protein synthesis was necessaryduring this activation. Addition of a cytokinin, benzyladenine(BA), stimulated the increase in the UDGT activity. The increasein the UDGT activity caused by BA was accompanied by the accumulationof galactolipids, as in the case of the activation by light.These results suggest that activation of the final reactionin the synthesis of MGDG, which is catalyzed by the galactosyl-transferase,contributes to the accumulation of galactolipids during thedevelopment of the chloroplast membrane. (Received December 3, 1994; Accepted July 3, 1995)