Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.     

Homing of intravenously and intralymphatically injected human dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells

Dendritic cells (DC) are professional antigen-presenting cells that can be generated in vitro from CD34⁺ peripheral blood progenitor cells by recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. Physiologically, immature DC in the periphery capture and process antigens, then mature to interdigitating DC and migrate to lymphoid organs, where they activate lymphocytes. However, it is not known if DC generated in vitro have the capacity to traffic in vivo to the lymphoid tissues, such as spleen and lymph nodes. We have investigated whether human radiolabeled DC differentiated in vitro migrate and localize to lymphoid tissues after intravenous and intralymphatic injection. The distribution and localization of the DC were evaluated in five patients with malignant melanoma using serial whole-body gamma camera imaging. Intravenously infused DC demonstrated transient lung uptake followed by localization in the spleen and liver for at least 7 days. DC injected into a lymphatic vessel at the dorsal foot were rapidly detected in the draining lymph nodes where they remained for more than 24 h. These data suggest that DC differentiated in vitro localize preferentially to lymphoid tissue, where they could induce specific immune responses. Received: 28 January 1999 / Accepted: 4 March 1999     

Diversity of Superoxide-Dismutases Among Clinical and Soil Isolates of Streptomyces Species

Comparison of the nature, activity, and cellular localization of superoxide-dismutases (SOD) from soil and clinical isolates of Streptomyces species was investigated to identify possible factors that could account for the pathological role of the strains isolated from human lesions. Results showed that all of the studied strains possessed a cytoplasmic Ni-SOD. This particular SOD, found in isolates from patients, could be a new taxonomic criterion to identify Streptomyces species with greater precision. A second minor SOD, assimilated to an Fe/Zn-SOD, was detected in some strains, but no relationship was established between the presence of this enzyme and the clinical origin of the strains. Received: 9 April 1999 / Accepted: 9 June 1999     

膜去极化和cAMP在胰岛细胞株HIT中活化CBPC端片段

在胰岛细胞株 Ｈ Ｉ Ｔ 细胞中,用瞬时转染法观察高 Ｋ＋ 导致的膜去极化与ｃ Ａ Ｍ Ｐ对 Ｃ Ｂ Ｐ Ｃ端片段转录活性的影响．发现二者均可诱导 Ｃ Ｂ Ｐ Ｃ端片段的转录活性增强,并有协同效应; Ｃ Ｂ Ｐ Ｃ端片段的突变体（ Ｓｅｒ １ ７７２ 突变为 Ａｌａ）表现相同的诱导特性,但其基本转录活性降低．说明膜去极化和 ｃ Ａ Ｍ Ｐ对 Ｃ Ｂ Ｐ Ｃ 端片段转录活性的诱导作用与 Ｐ Ｋ Ａ 磷酸化位点 Ｓｅｒ １ ７７２ 无关,而该位点的磷酸化对调节 Ｃ Ｂ Ｐ Ｃ 端片段的基本转录活性起重要作用．蛋白激酶 Ｃ 通路对 Ｃ Ｂ Ｐ Ｔｉ的转录活性无影响．     

In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

Summary Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.     

Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Delta) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Delta is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Delta helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Delta helicase activity and found that it appears to move in a 3' to 5' direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Delta or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Delta endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.     

Association of Influenza Virus Matrix Protein with Ribonucleoproteins

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.     

Intracellular signaling molecules involved in an inhibitory factor–induced decrease in fetal‐type AChR expression

The innervation‐induced down‐regulation of fetal‐type acetylcholine receptor (AChR) expression in developing muscle fibers has largely been attributed to nerve‐evoked muscle activity; however, there is increasing evidence that a neural trophic factor also contributes to this receptor down‐regulation. Previous studies from this laboratory have shown that neural extracts contain a factor which decreases fetal‐type AChR expression in skeletal muscle cell lines and therefore may account for the proposed inhibitory neurotrophic influence. The current study investigated possible intracellular signaling molecules involved in this receptor down‐regulation and demonstrated that activation of protein kinase C and p70^S6k appeared to be important in receptor down‐regulation. Decreases in AChR density were independent of myogenin. In addition, the receptor down‐regulation was independent of neuregulin, which also induces p70^S6k activity. These studies demonstrate that neural extracts contain an inhibitory factor which can down‐regulate fetal‐type AChR expression independently of nerve‐evoked muscle activity through intracellular signaling molecules which are known to regulate AChR expression. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 190–201, 2000