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PubMed2003

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth.

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.

Adaptation, Physiological↗
PubMed2004

Partial purification, kinetic analysis, and amino acid sequence information of a flavonol 3-O-methyltransferase from Serratula tinctoria.

Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that accumulates in response to stress conditions, prompted us to purify and characterize the enzyme involved in its methylation. The flavonol 3-O-methyltransferase (3-OMT) was partially purified by ammonium sulfate precipitation and successive chromatography on Superose-12, Mono-Q, and adenosine-agarose affinity columns, resulting in a 194-fold increase of its specific activity. The enzyme protein exhibited an expressed specificity for the methylation of position 3 of the flavonol, quercetin, although it also utilized kaempferol, myricetin, and some monomethyl flavonols as substrates. It exhibited a pH optimum of 7.6, a pI of 6.0, and an apparent molecular mass of 31 kD. Its K(m) values for quercetin as the substrate and S-adenosyl-l-Met (AdoMet) as the cosubstrate were 12 and 45 microm, respectively. The 3-OMT had no requirement for Mg(2+), but was severely inhibited by p-chloromercuribenzoate, suggesting the requirement for SH groups for catalytic activity. Quercetin methylation was competitively inhibited by S-adenosyl-l-homo-Cys with respect to the cosubstrate AdoMet, and followed a sequential bi-bi reaction mechanism, where AdoMet was the first to bind and S-adenosyl-l-homo-Cys was released last. In-gel trypsin digestion of the purified protein yielded several peptides, two of which exhibited strong amino acid sequence homology, upon protein identification, to a number of previously identified Group II plant OMTs. The availability of peptide sequences will allow the design of specific nucleotide probes for future cloning of the gene encoding this novel enzyme for its use in metabolic engineering.

Amino Acid Sequence↗
PubMed1983

New applications of microbial products.

Microbial secondary metabolites are now being used for applications other than as antibacterial, antifungal, and antitumor agents. These applications include use against parasites (coccidia, helminths) and insects as well as for animal and plant growth stimulation, immunosuppression, uterocontraction, and other pharmacological activities. Further applications are possible in various areas of pharmacology and agriculture, a development catalyzed by the use of simple enzyme assays for screening prior to testing in intact animals or in the field.

Cloning, Molecular↗
PubMed2000

Activation of lymphocyte signaling by the R1 protein of rhesus monkey rhadinovirus.

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that exhibits a considerable degree of similarity to the human Kaposi's sarcoma-associated herpesvirus (KSHV). The R1 protein of RRV is distantly related to the K1 protein of KSHV, and R1, like K1, can contribute to cell growth transformation. In this study we analyzed the ability of the cytoplasmic tail of R1 to function as a signal transducer. The cytoplasmic domain of the R1 protein contains several tyrosine residues whose phosphorylation is induced in cells expressing Syk kinase. Expression of a CD8 chimera protein containing the extracellular and transmembrane domains of CD8 fused to the cytoplasmic domain of R1 mobilized intracellular calcium and induced cellular tyrosine phosphorylation in B cells upon stimulation with anti-CD8 antibody. None of the CD8-R1 cytoplasmic deletion mutants tested were able to mobilize intracellular calcium or to induce tyrosine phosphorylation to a significant extent upon addition of anti-CD8 antibody. Expression of wild-type R1 protein activated nuclear factor of activated T lymphocytes (NFAT) eightfold in B cells in the absence of antibody stimulation; expression of the CD8-R1C chimera strongly induced NFAT activity (60-fold) but only upon the addition of anti-CD8 antibody. We conclude that the cytoplasmic domain of R1 is capable of transducing signals that elicit B-lymphocyte activation events. The signal-inducing properties of R1 appear to be similar to those of K1 but differ in that the required sequences are distributed over a much longer stretch of the cytoplasmic domain (>150 amino acids). In addition, the induction of calcium mobilization was considerably longer in duration and stronger with R1 than with K1.

Amino Acid Sequence↗
PubMed2006

Human neural stem cells target experimental intracranial medulloblastoma and deliver a therapeutic gene leading to tumor regression.

PURPOSE: Medulloblastoma, a malignant pediatric brain tumor, is incurable in about one third of patients despite multimodal treatments. In addition, current therapies can lead to long-term disabilities. Based on studies of the extensive tropism of neural stem cells (NSC) toward malignant gliomas and the secretion of growth factors common to glioma and medulloblastoma, we hypothesized that NSCs could target medulloblastoma and be used as a cellular therapeutic delivery system. EXPERIMENTAL DESIGN: The migratory ability of HB1.F3 cells (an immortalized, clonal human NSC line) to medulloblastoma was studied both in vitro and in vivo. As proof-of-concept, we used HB1.F3 cells engineered to secrete the prodrug activating enzyme cytosine deaminase. We investigated the potential of human NSCs to deliver a therapeutic gene and reduce tumor growth. RESULTS: The migratory capacity of HB1.F3 cells was confirmed by an in vitro migration assay, and corroborated in vivo by injecting chloromethylbenzamido-Dil-labeled HB1.F3 cells into the hemisphere contralateral to established medulloblastoma in nude mice. In vitro studies showed the therapeutic efficacy of HB1.F3-CD on Daoy cells in coculture experiments. In vitro therapeutic studies were conducted in which animals bearing intracranial medulloblastoma were injected ipsilaterally with HB1.F3-CD cells followed by systemic 5-flourocytosine treatment. Histologic analyses showed that human NSCs migrate to the tumor bed and its boundary, resulting in a 76% reduction of tumor volume in the treatment group (P<0.01). CONCLUSION: These studies show for the first time the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to medulloblastoma.

Animals↗
PubMed1999

Turning over a new leaf. Tobacco.

Anticipating a diminishing market for cigarettes and other tobacco products in the future, researchers around the country are studying alternative uses for tobacco plants. The most promising field of research for tobacco involves the genetic engineering of tobacco plants to produce various substances such as industrial chemicals, pharmaceuticals, and consumer product ingredients. Tobacco has been called the "fruit fly of the plant kingdom" because of the ease with which it can be genetically engineered. There are countless possibilities for the use of tobacco, but current efforts are concentrating on engineering tobacco to produce vaccines, human enzymes, and plastics. Tobacco researchers have been successful in expressing bovine lysozyme, an enzyme with antibacterial properties, and insulin.

Biotechnology↗
PubMed2004

Genomic approaches to antibacterial discovery.

This chapter describes two key strategies for the discovery of new antibacterial agents and illustrates the critical role played by genomics in each. The first approach is genomic target-based screening. Comparative genomics and bioinformatics are used to identify novel, selective antibacterial targets of the appropriate antibacterial spectrum. Genetic technologies integral for the success of this approach, such as essentiality testing, are also described. An unprecedented number of novel targets have been discovered via this approach, and a plethora of examples are discussed. This section concludes with the case history of a target successfully progressed from identification by genomics, to high-throughput screening, and onto proof of concept in curing experimental infections. The second approach is based on screening for compounds with antibacterial activity and then employing a broad variety of newer technologies to identify the molecular target of the antibacterial agent. The advantage of this approach is that compounds already possess antibacterial activity, which is a property often challenging to engineer into molecules obtained from enzyme-based screening approaches. The recent development of novel biochemical and genomic technologies that facilitate identification and characterization of the mode of action of these agents has made this approach as attractive as the genomic target-based screening strategy.

Anti-Bacterial Agents↗
PubMed2004

Prohormone convertase 1 (PC1) processing and sorting: effect of PC1 propeptide and proSAAS.

Prohormone convertase 1 (PC1) is a serine proteinase responsible for the proteolytic processing of many precursor proteins within the regulated secretory pathway. The activity of PC1 is potentially regulated by two endogenous inhibitors, the PC1 propeptide and proSAAS. Here we have investigated the effect of proSAAS and propeptide-containing constructs on PC1 carboxy-terminal processing and activity. In AtT-20 cells, proSAAS expression inhibited both C-terminal PC1 processing and proopiomelanocortin (POMC) processing under pulse/chase conditions. SAAS CT peptide-propeptide chimeric constructs had no effect on the cleavage of PC1 and POMC under pulse/chase conditions. However, a construct containing the propeptide alone reduced C-terminal PC1 processing under pulse/chase conditions and also inhibited POMC processing. In contrast, experiments using HEK293 cells transiently expressing PC1 plus the respective constructs demonstrated significant inhibition of zymogen processing and decreased C-terminal processing of PC1 by the SAAS CT peptide portion of the chimera. Our results suggest that the PC1 propeptide expressed in trans is able to act as an endogenous inhibitor of PC1, but that SAAS CT peptide-containing/propeptide constructs cannot function as effective inhibitors of precursor maturation in the regulated pathway.

Animals↗
PubMed1981

Semisynthetic peptides and proteins.

Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.

Amino Acid Sequence↗
PubMed1998

[Preparation of monoclonal antibodies against hepatitis E and its application].

OBJECTIVE: To study the particle of hepatitis E virus (HEV) and its significance in specific diagnosis for hepatitis E (HE). METHODS: Four strains of monoclonal antibodies (McAb) against HEV, 5B12, 5F1, 5B9 and 4G10, were prepared with HEV antigen (used as immunogen) expressed by genetic engineering and detected by indirect enzyme-linked immunosorbent assay (ELISA) with purified synthetic polypeptide antigen. RESULTS: The monoclonal anti-HEV prefared could produce inhibitory reaction with anti-HEV positive serum and combined specifically with HEV particle forming virus-antibody complex seen clearly under electron microscope. CONCLUSION: McAb against HEV so prepared with high specificity can be used to detect and identify HEV.

Antibodies, Monoclonal↗
PubMed2000

Analysis of tissue inhibitor of metalloproteinases-2 effect on pro-matrix metalloproteinase-2 activation by membrane-type 1 matrix metalloproteinase using baculovirus/insect-cell expression system.

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14) is known to activate pro-matrix metalloproteinase-2 (pro-MMP-2; progelatinase A) on the cell surface. To analyse the tissue inhibitor of metalloproteinases-2 (TIMP-2) effect on activation of pro-MMP-2 by MT1-MMP, we have expressed the full-size MT1-MMP (fMT1-MMP) and a transmembrane (TM)-domain-deleted soluble MT1-MMP (sMT1-MMP) in the baculovirus/Sf9 (Spodoptera frugiperda 9) insect-cell system, where neither endogenous gelatinolytic MMPs nor TIMP-2 are expressed. Both fMT1-MMP and sMT1-MMP expressed in the expression system were found not to contain the pro-domain and were able to activate the TIMP-2-free pro-MMP-2. Both in the insect cells and in vitro, activation of pro-MMP-2 by fMT1-MMP was enhanced at low concentrations of TIMP-2 and inhibited by its higher concentrations. The maximal enhancing effect was detected at 0.05 molar fraction of TIMP-2/fMT1-MMP. In contrast, activation of pro-MMP-2 by sMT1-MMP was dose-dependently inhibited by TIMP-2. These results demonstrate that the TM domain of MT1-MMP is not required for the ability to activate pro-MMP-2, but is required for the enhancing effect of TIMP-2 on pro-MMP-2 activation by recruiting pro-MMP-2 to the MT1-MMP-TIMP-2 complex as a cell-surface pro-MMP-2 receptor. Moreover, our data strongly suggest that the pro-domain of MT1-MMP is not required for the TIMP-2-mediated enhancing effect on pro-MMP-2 activation. In addition, the pro-MMP-2 in the MT1-MMP-TIMP-2-pro-MMP-2 ternary complex was not activated without external activator, but readily by addition of sMT1-MMP. This result demonstrates that MT1-MMP free of TIMP-2 would be the enzyme responsible for activation of the pro-MMP-2 in the ternary complex under physiological conditions.

Animals↗
PubMedDate not supplied

Surface fixation of hydrogels. Heparin and glucose oxidase hydrogelated surfaces.

The authors report a new photochemical technique for the fixation of hydrogels onto the surface of fabricated devices and sensors. The preparative method is based on two different types of photochemistry: photocross-linking due to intermolecular photodimerization of cinnamoyl groups, and photoinduced covalent bonding due to the photoreactivity of azido groups. The immobilization of heparin, useful as a thromboresistant coating, onto poly(ethylene terephthalate), and the immobilization of glucose oxidase onto the surface of a carbon fiber electrode, were demonstrated. In the former, immobilized heparin was found to be continuously released from the hydrogel, and the release rate was effectively controlled by the thickness of the hydrogel. In the latter, upon the addition of glucose, a spontaneous electrical response was observed to function as a glucose sensor. No delamination was found for either system after vigorous washing with water. Thus, durable surface hydrogelation, simultaneous immobilization of bioactive substances, and functioning of the systems were demonstrated.

Biocompatible Materials↗
PubMedDate not supplied

[Various aspects of structural studies of aspartate proteinases].

The paper is a brief account of aspartic proteinases' structural studies developed in V.A. Engelhardt Institute of Molecular Biology during the last 3 years. The work on porcine pepsin has been finalized after the refinement of the monoclinic crystal form at 1.8 A resolution performed in collaboration with the group of protein structure and function studies of the University of Alberta in Canada. An important structural property of chymosin which explains the enzyme specificity has been found. Protein engineering work on chymosin is being developed. The structural template for aspartic proteinases has been elucidated and on the basis of this template the model of HIV-1 protease molecule has been built. Some approaches to the design of HIV-1 protease inhibitors were elucidated.

Amino Acid Sequence↗