珍贵橙色束丝放线菌中新颖香草酰氨鼠李糖糖苷化合物

从珍贵橙色束丝放线菌(Actinosynnema pretiosum)的琼脂平板发酵产物中分离得到一个新的糖苷化合物,通过理化性质和谱学分析(MS、1D-NMR、2D-NMR)确定为香草酰氨4-O-鼠李糖苷(3-甲氧基4-O-鼠李糖苷-苯甲酰胺).     

Two piperazic acid-containing cyclic hexapeptides from Streptomyces alboflavus 313

Two novel cyclic hexapeptides, designated NW-G10 (1) and NW-G11 (2), were isolated from the fermentation broth of Streptomyces alboflavus 313. Their relative structures were elucidated on the basis of extensive spectroscopic analysis, and the absolute configurations of several constituent amino acids were determined by Marfey’s method. NW-G10 (1) and NW-G11 (2) exhibited significant activity against Gram-positive bacteria, such as Bacillus cereus, Bacillus subtilis and Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA), but they are not active against gram negatives.     

Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides.

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.     

Risk from genetically engineered and modified marine fish

In support of the emerging industries of warmwater marine fish mariculture, genetic engineering and classical genetic improvement programmes have been initiated for a variety of exclusively marine fish. These programmes have the potential to perturb allele and genotype frequencies, or introduce novel alleles and genes into conspecific wild populations. Despite concerns to the contrary, the following hypothesis remains to be falsified: laboratory induced allele frequency/genotype changes and novel alleles or genes have a negligible probability of being selectively favoured in wild populations under natural selection, and accordingly, without sustained large scale releases, have little potential for ecological impact     

Structure and conformation of a novel genetically engineered polysaccharide P2

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.     

Glucose-regulated insulin production from genetically engineered human non-beta cells

In this report we describe development and characterization of four human cell lines that are able to secrete insulin and C-peptide in response to higher concentrations of glucose. These cell lines have been developed by stably and constitutively expressing human proinsulin with a furin-cleavable site, whereas expression of furin is regulated by glucose concentration. These cell lines have been cloned and, therefore, the transgene in each cell is located in an identical location of the genome leading to a uniform expression. Cloning has also allowed us to identify cell lines with more desirable properties such as higher basal insulin secretion and/or better glucose responsiveness. We have further shown that the insulin produced by these cells is biologically active and induces normoglycemia when injected in diabetic animals. Our objective in initiating these studies was to identify a cell line that could serve as a surrogate beta cell line for therapeutic intervention in type I diabetic patients.     

Reversible hydrogels from self-assembling genetically engineered protein block copolymers

A series of triblock protein copolymers composed of a central water-soluble polyelectrolyte segment flanked by two coiled-coil domains was synthesized by genetic engineering methods. The copolymers self-assembled into reversible hydrogels in response to changes in temperature, pH, and the presence or absence of denaturating agent (guanidine hydrochloride, GdnHCl). Hydrogel formation was concentration-dependent, and the concentration needed for hydrogel formation correlated with the oligomerization state of the coiled-coil domains in the protein copolymers. The morphology of the hydrogels, as determined by scanning electron microscopy (SEM), indicated the presence of porous interconnected networks. The thermal stabilities and self-assembling properties of the protein copolymers were successfully controlled by manipulating the amino acid sequences of the coiled-coil domains. The stimuli responsiveness and reversibility of the hydrogel self-assembly suggest that these protein copolymers may have potential in biomedical applications.     

Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli

Genetically engineered E. coli, which express both a Hg2+ transport system and metallothionein, were tested for their ability to remove mercury from wastewater. The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6. Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation. EDTA also stimulated mercury uptake rather than inhibiting it. A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake. The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water. These results indicated that the induced cells had a high affinity and specificity for mercury.     

Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas

Pseudomonas putida and P oleovorans have been extensively studied for their production of medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). These bacteria are incapable of metabolizing triacylglycerols (TAGs). We have constructed recombinant P. putida and P. oleovorans that can utilize TAGs as substrates for growth and mcl-PHA synthesis. A recombinant plasmid, pCN51lip-1, carrying Pseudomonas lipase genes was used to electrotransform these organisms. The transformants expressed TAG-hydrolyzing activity as shown by a rhodamine B fluorescence plate assay. The genetically modified organisms grew in TAG-containing medium to a cell dry weight of 2-4 g/l. The recombinant P. putida produced mcl-PHA at a crude yield of 0.9-1.6 g/l with lard or coconut oil (Co) as substrate. While P. oleovorans transformant did not produce mcl-PHA, a mixed-culture fermentation approach with the wild-type and recombinant strains afforded polymer production from Co at a crude yield of 0.5 g/l. Compositional analysis by gas chromatography/mass spectrometry showed that beta-hydroxyoctanoate (31-45 mol %) and beta-hydroxydecanoate (28-35 mol %) were the dominant repeat units of the TAG-based PHA. The number-average and weight-average molecular masses of the PHAs as determined by gel permeation chromatography were 82-170 x 10(3) g/mol and 464-693 x 10(3) g/mol, respectively. The recombinant approach can greatly increase the number of organisms that can be used to produce PHA from fat and oil substrates.     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).     

Evaluating the risk of releasing genetically engineered organisms

This article considers the question of a priori assessment of the safety of releasing recombinant DNA engineered organisms. Now and for the foreseeable future, decisions to release such an organism must be based on the results of limited, case-by-case risk assessment studies. The criteria calling for the termination of release programs must be agreed upon in advance of these studies. There is no justification for excluding classes of release organisms from risk assessment. Theory is useful in suggesting a hierarchy of risks, raising the questions that have to be addressed in case-by-case risk assessment and providing protocols for the standardization and execution of these studies. We do not believe that theory can be used to argue categorically for or against the safety of specific releases of recombinant DNA engineered organisms.     

Release of genetically engineered micro-organisms into the environment

Summary Genetically engineered micro-organisms (GEM) at present are the subject of much public attention. They are being considered for biological control, frost protection of plants, and other applications. There is a need to test such organisms before release to the environment. Examples of GEM includePseudomonas fluorescens into which have been cloned -toxin genes ofBacillus thuringiensis, ice-minus bacteria, and other organisms modified by addition, rearrangement and/or deletion of genetic material. Prior to release, the survival, fate, and effects of GEM in the environment must be established. Because organisms, once released, cannot be recalled or always controlled, it is imperative that a full understanding of the risks be known. Predictive ecology must include the new sub-discipline of molecular microbial ecology, if the need for information prior to release of GEM is to be met. One of the most important aspects of deliberate release which must be considered is the ability to detect and monitor GEM in the environment. It has been discovered that micro-organisms can undergo dormancy, i.e. enter a viable but non-recoverable stage in the natural environment. New techniques have been developed, employing immunofluorescent/epifluorescent microscopy, coupled with 5S rRNA sequencing, which allow accurate non-genetic detection of GEM. These techniques have been employed in aquatic systems. Charateristics of GEM important in release to the environment include ability to colonize surfaces, transfer genetic material and persist in specific environments. Clearly, the effects of GEM on the environment cannot be precisely predicted, unless the organisms have been so debilitated that they cannot persist in any natural habitat and cannot exchange genetic material with any other organism. It must be recognized that micro-organisms are extremely diverse and versatile. Uniformly applied, standard regulations governing deliberate release of GEM to the environment cannot be applied in the same way as for regulation of chemicals or medical devices. Case-by-case regulation appears to be the best approach for the immediate future. The implications of each organism, in terms of its own biology, will have to be considered.

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Resumen En el campo de la biotecnología, en rápida expansión, causa preocupación la liberación deliberada al medio ambiente de microorganismos manipulados genéticamente. Se estan llevando a cabo estudios para determinar el destino y el efecto de tales microorganismos en el medio ambiente y especialmente su impacto ambiental. Estos estudios indican que la detección directa de dichos microorganismos, no cultivables pero que permanecen viables en un entorno natural, se puede realizar empleando microscopía de inmunofluorescencia/epifluorescencia junto con secuencias 5S rARN utilizadas como etiquetas, lo que proporciona un sistema eficaz para rastrear estos organismos en muestras del medio ambiente. La utilización de microcosmos y los estudios realizados hasta el momento indican que, efectivamente, los microorganismos sufren una transformación que los mantiene viables aunque no recuperables, en un estado similar a la dormancia, por lo que se requieren métodos de detección directa para controlar la liberación al medio ambiente de microorganismos producidos mediante ingeniería genética. Este fenómeno ocurre también frecuentemente en otros microorganismos liberados al ambiente, entre ellos posibles patógenos. Los estudios realizados sobreVibrio cholerae proporcionan un ejemplo interesante tanto de los métodos empleados como de los resultados obtenidos. Las conclusiones obtenidas indican que el organismo esta ampliamente distribuido en el entorno natural, pero que solo puede ser detectado mediante microscopía directa debido al estado viable, aunque no vultivable, en que se mantiene en el ambiente. Ensayos en animales muentran que se pueden recolectar células deV. cholerae a partir de muestras del medio ambiente y recuperarse como células viables despues de haber sido inyectadas en el íleo de conejos, habiendose demostrado una respuesta ileal positiva para aquellas cepas que son enterotóxicas. Obviamente las ideas actuales acerca de la persistencia y función de los microorganismos en el ambiente necesitaran ser modificadas a medida que se vayan desarrollando nuevos métodos y nuevos conceptos en ecología microbiana.

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Résumé Le développement rapide de la biotechnologie a fait naître des préoccupations concernant le relâchement délibéré dans l'environment de micro-organismes génétiquement manipulés. Des études sont en cours pour déterminer le sort et les effects de ces micro-organismes dans la nature. Les travaux entrepris montrent que la microscopie d'immuno-fluorescence et d'épifluorescence, associée à la détérmination de séquences-marqueurs de r-ARN 5S, permet de détecter des micro-organismes non-cultivables, mais demeurés viables dans les milieux naturels. Les études déjà réalisées sur divers microcosmes indiquent que des micro-organismes peuvent effectivement se trouver dans un état viable, mais non-cultivable, qui ressemble à une dormance, ce qui exige, pour contrôler le relâchement dans la nature d'organismes génétiquement manipulés, l'emploi de méthodes de détection directe. Ce phénomène se produit fréquemment aussi dans le cas de beaucoup d'autres organismes, y compris des pathogènes potentiels, qui sont relâchés dans l'environnement. Des études réalisées avecVibrio cholereae fournissent un exemple de méthodes utiles et de résultats intéressants. Il résulte de ces études queV. cholereae est largement distribué dans l'environnement naturel et ne peut souvent y être détecté que par microscopie directe, à cause de l'état viable, mais non-cultivable, sous lequel il se maintient dans la nature. Les études sur l'animal montrent qu'on peut collecter à partir d'échantillons de milieu naturel des cellules deV. cholereae viables mais noncultivables, après avoir donné, dans le cas des souches entéro-toxigènes, une réponse intestinale positive. Il est clair que les idées courantes sur la persistance et la fonction des micro-organismes dans l'environnement demandent à être modifiées en fonction de l'émergence de nouvelles méthodes et de nouveaux concepts en écologie microbienne.

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Invited paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki: 12–16.8.1985. Session 4     

Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.     

Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented—by either chemical functionalization or genetic engineering—with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.     

Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein

In the interphase nuclei of cultured cells, chromatin is compacted and organized in higher-order structures through the condensation and decondensation processes. Chromosomes in the interphase nucleus are known to occupy distinct territories. The chromosome territory-interchromatin compartment model premises that the interchromatin compartment is separated from compact higher-order chromatin domains and expands in between these chromatin-organized territories. Chromatin in cultured cells is compacted under some conditions, such as the stress of heat shock and high osmolarity, and Src-mediated nuclear tyrosine phosphorylation. We report here that a novel arginine-rich cationic protein is generated by frameshift mutation of enhanced green fluorescent protein (EGFP). The arginine-rich cationic protein is highly hydrophilic and contains potential arginine-based nuclear localization signals. Expression of the arginine-rich cationic protein shows its predominant localization to the nucleus and induces striking chromatin condensation in the interphase, which might be involved in interchromatin spacing or euchromatinization. Thus, the arginine-rich cationic protein as a new tool would be useful for dissecting chromatin architecture dynamics.