Immune response of Japanese flounder Paralichthys olivaceus to outer membrane protein of Edwardsiella tarda

The humoral and cellular immune responses of Japanese flounder, Paralichthys olivaceus, were investigated following intraperitioneal injection with outer membrane protein (OMP) of Edwardsiella tarda in Freund's incomplete adjuvant (FIA). The specific serum antibody titre against OMP of E. tarda were measured using ELISA for 14 weeks, and the total serum antibody concentrations were also determined according to the sandwich ELISA standard model constructed using purified IgM. Both of the specific and total antibodies had an increase and reached their peaks 4 weeks after immunization. Simultaneously, the percentages of sIg + lymphocytes in blood, spleen, pronephros and mesonephros were detected by flow cytometry. It was shown that the percentages of sIg + lymphocytes in all lymphoid organs reached their peak levels 4 weeks after immunization, and then decreased gradually. To investigate the protection against infection, three challenges were performed in the same way at day 14, 30 and 100 after immunization, fish challenged at day 30 showed a higher relative percentage survival (RPS) of 71 compared to the 14-day group (30) and 100-day group (53), which indicated a positive correlation between the survival and the levels of the antibody.     

藻－菌生态系统代谢功能的生态学研究

在室内模拟条件下,研究了一些生态因子对藻－菌（Ａ＋Ｂ）生态系统代谢有机碳（Ｃ６Ｈ１２Ｏ６）、ＮＨ３－Ｎ和无机磷（ＩＰ）的影响．研究结果表明,当藻－菌生态系统中藻（Ａ）或菌（Ｂ）的起始数量一定时,其代谢Ｃ６Ｈ１２Ｏ６的速率,随与之组合的Ｂ或Ａ的起始数量增加（数量比则相应降低）而增加．在光照和黑暗条件下,Ａ＋Ｂ系统代谢上述３种营养物质的速率均有一定的差异．黑暗下Ｃ６Ｈ１２Ｏ６的平均代谢速率较光照下高１２．３％（Ｐ＜０．０５）,ＩＰ和ＮＨ３－Ｎ的平均代谢速率则分别较光照下低１４．４％（Ｐ＜０．０５）和１６．２％（Ｐ＜０．００１）．在Ａ＋Ｂ系统和Ａ、Ｂ单培养物中,３种营养物质的代谢速率均随有机负荷量增加而增加,而且Ａ＋Ｂ系统的代谢速率分别高于单培养的Ａ和Ｂ,其中ＮＨ３－Ｎ代谢尤为显著．文章还就生态系统结构与功能的关系问题进行了讨论．     

Transforming growth factor-beta1 enhanced vascular endothelial growth factor synthesis in mesenchymal stem cells

Angiogenesis is essential for transplantation of mesenchymal stem cells (MSCs). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors identified to date. Elevated VEGF levels in MSCs correlate with the potential of MSCs transplantation. As an indirect angiogenic agent, transforming growth factor-β1 (TGF-β1) plays a pivotal role in the regulation of vasculogenesis and angiogenesis. However, the effect of TGF-β1 on VEGF synthesis in MSCs is still unknown. Besides, the intracellular signaling mechanism by which TGF-β1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of MSCs to TGF-β1 stimulated the synthesis of VEGF. Meanwhile, TGF-β1 stimulated the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, Ly 294002, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt significantly attenuated the VEGF synthesis stimulated by TGF-β1. Additionally, U0126, a specific inhibitor of ERK1/2, also significantly attenuated the TGF-β1-stimulated VEGF synthesis. These results indicated that TGF-β1 enhanced VEGF synthesis in MSCs, and the Akt and ERK1/2 activation were involved in this process.     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

An indigoidine biosynthetic gene cluster from Streptomyces chromofuscus ATCC 49982 contains an unusual IndB homologue

A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.     

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.     

Modification of ricin A chain, by addition of endoplasmic reticulum (KDEL) or Golgi (YQRL) retention sequences, enhances its cytotoxicity and translocation

 A pKK expression system in Escherichia coli was used to produce recombinant ricin A chain (rRTA) and rRTA modified by addition of organelle-specific amino acid retention sequences, including KDEL (an endoplasmic reticulum, ER, lumen retention signal), KKMP (an ER membrane retention signal), YQRL (a trans-Golgi network retention signal) and KFERQ (a lysosome-targeting signal) to the C terminus of rRTA. The toxicities of these RTA mutants were assessed in Jurkat cells following fluid-phase endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytotoxic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This difference did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles have been employed to assess translocation of rRTA and mutant rRTA. RTA-KDEL and RTA-YQRL respectively exhibited 6.7-fold and 6.1-fold more protection against papain digestion in reconstituted ER vesicles and 2.2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, than unmodified rRTA. These mutants were reassociated with ricin B chain to form holotoxins. The mutant RTA-KDEL and RTA-YQRL holotoxins were 3.8-fold and 1.5-fold more cytotoxic for target cells, respectively, than ricin produced using unmodified rRTA. Our results suggest that both ER and the trans-Golgi network may play important roles in the intracellular trafficking and translocation of ricin A chain. Received: 14 August 1997 / Accepted: 14 October 1997     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.