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PubMed2000

In Vitro Amidating Processing of Products Expressed by Gene Engineering.

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.

Journal Article↗
PubMed1998

[Gene engineering studies on sleep].

Recent advancements in molecular engineering techniques have enabled us to study sleep of animals lacking or overproducing any protein molecule we are interested in. Abnormalities in sleep have already been observe in knockout mice lacking the gene for prion protein, 55 kD-receptor of tumor necrosis factor, etc. We also started a project to examine the effect of the loss or the overproduction of prostaglandin D synthase in sleep. The enzyme is responsible for biosynthesis of prostaglandin D2, the most potent endogenous substance to promote sleep reported so far. We generated both knockout and transgenic mice of prostaglandin D synthase. We are currently analysing the sleep of these mutant mice.

Animals↗
PubMedDate not supplied

[Genomics and gene engineering: rationale to the development of new means of tuberculosis control].

The review contains information on modern approaches to the development of antituberculosis vaccines and remedies. Data on the comparative effectiveness of different subunit and DNA vaccines against tuberculosis are presented. The use of comparative and structural genomics for the search and characterization of new Mycobacterium tuberculosis genes, whose products may prove to be important antigens for the development of vaccines or target proteins for remedies against tuberculosis, is considered.

Adjuvants, Immunologic↗
PubMed2000

[Molecular diagnosis and new gene engineering in hematological malignancies].

Advancement molecular diagnostics in hematological malignancy has provided us with a broad menu of new assays and techniques. By integrating the data gleaned from these techniques we can formulate a more rational broad-based diagnosis. Hematological malignancies have traditionally been classified by morphological criteria. However, molecular advances have provided new insights in the genetic backbone of chimeric genes. These malignancies have shown chromosome abnormalities of translocation with chimeric genes, and revealed the rearrangement of chimeric genes by PCR analysis and quantitative PCR system. This review summarized the recent technology for detecting chimeric genes along with concepts of laboratory performance.

Adult↗
PubMed1993

Retroviral-mediated transfer of the murine interleukin-3 gene engineered for intracellular retention results in a myeloproliferative syndrome but is associated with circulating interleukin-3 levels.

Myeloid leukemias have been shown to secrete as well as respond to cytokines such as interleukin-3 (IL-3) with an increased growth rate and may therefore become self-stimulatory through an external autocrine mechanism. In vitro evidence that IL-3 is functional within the intracellular compartment has been obtained through modification of the murine IL-3 gene to encode for the amino acids SEKDEL on the carboxyl terminus of the protein, resulting in preferential intracellular retention. The ability of bone marrow-derived hematopoietic progenitor cells to increase their proliferative capacity through intracellular mechanisms was investigated in vivo using retroviruses containing the wild-type or SEKDEL-modified IL-3 gene, transcriptionally regulated by the retroviral long terminal repeat (LTR) or by the SV40 early promoter, in lethally irradiated, bone marrow-reconstituted mice. Bone marrow cells exposed to the N2KDEL virus containing the SEKDEL-modified IL-3 gene were shown by bioassay to retain large amounts of IL-3 intracellularly, and the presence of an integrated provirus containing the SEKDEL sequences was demonstrated by polymerase chain reaction (PCR) in the spleen and bone marrow of these animals. Transduction with all four types of IL-3 viruses resulted in dramatic increases in the circulating white blood cell (WBC) count; this myeloproliferative state occurred within several weeks following bone marrow transplantation (BMT), when viruses expressing the IL-3 or modified gene were under transcriptional regulation of the viral LTR, and approximately 2 months post-BMT, when they were under control of the SV40 internal promoter. Serum levels of IL-3 were measured in transplanted animals and found to be markedly increased in each case in which WBC elevation was observed, including mice receiving marrow transduced with constructs containing the IL-3 gene modified for intracellular retention. No animals were observed in which myeloproliferation occurred without secretion. From these experiments, it seems unlikely that exclusively intracellular mechanisms are a major contributor to the development of the myeloproliferative syndrome observed in these animals.

Amino Acid Sequence↗
PubMed2000

Design and gene engineering synthesis of an extremely thermostable protein with biological activity.

A novel strategy for designing and synthesizing extremely thermostable biologically active proteins is proposed. The design concept is based on combining a rigid and extremely hydrophobic peptide unit with a biologically active peptide unit. The cell adhesive peptide sequence, Arg-Gly-Asp (RGD), as a functional peptide unit was incorporated into the elastin-based rigid polyhexapeptide, whose repeating unit is Ala-Pro-Gly-Val-Gly-Val (APGVGV). The designed fusion gene was expressed in E. coli, and the resulting protein, designated ER4, was purified with affinity chromatography. The ER4-coated cell culture plate showed sufficient cell adhesive activity through the RGD sequence on the surface of ER4. The thermostability of ER4 was demonstrated by estimating the remaining cell adhesive activity after autoclaving at 120 degrees C for 20 min, and it retained over 90% of cell adhesive activity compared with native ER4.

3T3 Cells↗
PubMed2006

Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Reverse engineering gene networks to identify key drivers of complex disease phenotypes.

Diseases such as obesity, diabetes, and atherosclerosis result from multiple genetic and environmental factors, and importantly, interactions between genetic and environmental factors. Identifying susceptibility genes for these diseases using genetic and genomic technologies is accelerating, and the expectation over the next several years is that a number of genes will be identified for common diseases. However, the identification of single genes for disease has limited utility, given that diseases do not originate in complex systems from single gene changes. Further, the identification of single genes for disease may not lead directly to genes that can be targeted for therapeutic intervention. Therefore, uncovering single genes for disease in isolation of the broader network of molecular interactions in which they operate will generally limit the overall utility of such discoveries. Several integrative approaches have been developed and applied to reconstructing networks. Here we review several of these approaches that involve integrating genetic, expression, and clinical data to elucidate networks underlying disease. Networks reconstructed from these data provide a richer context in which to interpret associations between genes and disease. Therefore, these networks can lead to defining pathways underlying disease more objectively and to identifying biomarkers and more-robust points for therapeutic intervention.

Animals↗
PubMed1996

Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity.

Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of the squash-type protease inhibitor family, is composed of 29 amino acids and shows strong inhibition of trypsin by its compact structure. To study the structure-function relationship of this inhibitor using protein engineering methods, we constructed an expression system for CMTI-I as a fused protein with porcine adenylate kinase (ADK). A Met residue was introduced into the junction of ADK and CMTI-I to cleave the fusion protein with CNBr, whereas a Met at position 8 of authentic CMTI-I was replaced by Leu. Escherichia coli JM109 transformed with the constructed plasmid expressed the fused protein as an inclusion body. After cleavage of the expressed protein with CNBr, fully reduced species of CMTI-I were purified by reversed-phase HPLC and then oxidized with air by shaking. For efficient refolding of CMTI-I, we used 50 mM NH4HCO3 (pH 7.8) containing 0.1% PEG 6000 at higher protein concentration. Strong inhibitory activity toward trypsin was detected only in the first of three HPLC peaks. The inhibitor constant of CMTI-I thus obtained, in which Met8 was replaced by Leu, was 1.4 x 10(-10) M. The effect of replacement of Met with Leu at position 8 was shown to be small by comparison of the inhibitor constant of authentic CMTI-III bearing Lys at position 9 (8.9 x 10(-11) M) with that of its mutant bearing Leu at position 8 and Lys at position 9 (1.8 x 10(-10) M). To investigate the role of the well conserved hydrophobic residues of CMTI-I in its interaction with trypsin, CMTI-I mutants in which one or all of the four hydrophobic residues were replaced by Ala were prepared. The inhibitor constants of these mutants indicated that those with single replacements were 5-40 times less effective as trypsin inhibitors and that the quadruple mutant was approximately 450 times less effective, suggesting that the hydrophobic residues in CMTI-I contribute to its tight binding with trypsin. However, each mutant was not converted to a temporary inhibitor.

Chromatography, High Pressure Liquid↗
PubMed1999

Stem cell technology and bioceramics: from cell to gene engineering.

Mesenchymal stem cells reside in bone marrow and, when these cells are incorporated into porous ceramics, the composites exhibit osteo-chondrogenic phenotypic expression in ectopic (subcutaneous and intramuscular) or orthotopic sites. The expressional cascade is dependent upon the material properties of the delivery vehicle. Bioactive ceramics provide a suitable substrate for the attachment of the cells. This is followed by osteogenic differentiation directly on the surface of the ceramic, which results in bone bonding. Nonbioactive materials show neither surface-dependent cell differentiation nor bone bonding. The number of mesenchymal stem cells in fresh adult bone marrow is small, about one per one-hundred-thousand nucleated cells, and decreases with donor age. In vitro cell culture technology can be used to mitotically expand these cells without the loss of their developmental potency regardless of donor age. The implanted composite of porous ceramic and culture-expanded mesenchymal stem cells exhibits in vivo osteo-chondrogenic differentiation. In certain culture conditions, these stem cells differentiate into osteoblasts, which make bone matrix on the ceramic surface. Such in vitro prefabricated bone within the ceramic provides immediate new bone-forming capability after in vivo implantation. Prior to loading of the cultured, marrow-derived mesenchymal stem cells into the porous ceramics, exogenous genes can be introduced into these cells in culture. Combining in vitro manipulated mesenchymal stem cells with porous ceramics can be expected to effect sufficient new bone-forming capability, which can thereby provide tissue engineering approaches to patients with skeletal defects in order to regenerate skeletal tissues.

Adult↗
PubMed1996

Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Amino Acid Sequence↗
PubMedDate not supplied

[Investigation of the effect of various buffer solutions used for microinjection of gene-engineering constructs on preimplantation development of mouse embryos].

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl2 and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.

Animals↗
PubMedDate not supplied

[Experimental evaluation of the biological safety of gene-engineered bacteria using a model strain Bacillus subtilis interferon-producing strain].

The in vitro and in vivo evaluation of the biological and ecological safety of genetically modified bacteria (GMB) was carried out on B. subtilis recombinant strain 2335/105, capable of producing human interferon alpha-2, used as experimental model. As shown in this investigation made with the use of bacteriological analysis and polymerase chain reaction, the oral administration of GMB to calves, chickens and white mice produced no disturbances in the microbial ecology of the gastrointestinal tract of warm-blooded animals and did not lead to the appearance of spontaneous transformants. The present work is the first experimental evaluation of the biological safety of genetically modified microorganisms, used as the component of Subalin, a probiotic preparation intended for use in veterinary practice.

Animals↗
PubMed2001

Mapping critical sites in collagen II for rational design of gene-engineered proteins for cell-supporting materials.

Collagen II is the most abundant protein of cartilage and forms a network of fibrils extended by proteoglycans that enables cartilage to resist pressure. The surface of the collagen fibril serves as a platform for the attachment of collagen IX, growth factors, and cells. In this study we examined the mechanism of the interaction of chondrocytes with recombinant versions of procollagen II, in which one of the four blocks of 234 amino acids that define repeating D periods of the collagen triple helix has been deleted. Analysis of the attachment of chondrocytes to collagen II variants with deleted D periods indicated that the collagen II monomer contains randomly distributed sites critical for cell binding. However, as was shown by spreading and migration assays, the D4 period, which is between residues 703 to 936, contains amino acids critical for cell motility. We also showed that binding, spreading, and migration of chondrocytes through three-dimensional nanofibrillar collagenous matrices are controlled by an interaction of the collagen triple helix with beta1 integrins. The results of this study provide a basis for the rational design of a scaffold containing genetically engineered collagen with a high density of specific sites of interaction.

Biocompatible Materials↗
PubMed2004

Transcriptional networks: reverse-engineering gene regulation on a global scale.

A major objective in post-genome research is to fully understand the transcriptional control of each gene and the targets of each transcription factor. In yeast, large-scale experimental and computational approaches have been applied to identify co-regulated genes, cis regulatory elements, and transcription factor DNA binding sites in vivo. Methods for modeling and predicting system behavior, and for reconciling discrepancies among data types, are being explored. The results indicate that a complete and comprehensive yeast transcriptional network will ultimately be achieved.

Computational Biology↗
PubMed1990

Alteration of the specificity of the Streptomyces subtilisin inhibitor by gene engineering.

We have altered the amino acid at the center of the reactive site (methionine 73) of Streptomyces subtilisin inhibitor (SSI) by site-directed and cassette mutagenesis. Replacement by lysine or arginine resulted in trypsin inhibitory activity, replacement only by lysine gave inhibition of lysyl endopeptidase, and replacement by tyrosine or tryptophan resulted in inhibition of alpha-chymotrypsin. The four mutant SSIs retained their native activity against subtilisin BPN'. Thus by altering only one amino acid residue at the reactive site of SSI to the substrate specificity of the respective protease we could successfully change its inhibitory profile.

Amino Acid Sequence↗
PubMed2002

Engineering gene expression and protein synthesis by modulation of nuclear shape.

The current understanding of the relationships between cell shape, intracellular forces and signaling, nuclear shape and organization, and gene expression is in its infancy. Here we introduce a method for investigating gene-specific responses in individual cells with controlled nuclear shape and projected area. The shape of the nuclei of primary osteogenic cells were controlled on microfabricated substrata with regiospecific chemistry by confining attachment and spreading of isolated cells on adhesive islands. Gene expression and protein synthesis were altered by changing nuclear shape. Collagen I synthesis correlated directly with cell shape and nuclear shape index (NSI), where intermediate values of nuclear distension (6 < NSI < 8) promoted maximum synthesis. Osteocalcin mRNA, a bone-specific differentiation marker, was observed intracellularly by using reverse transcription in situ PCR at 4 days in cells constrained by the pattern and not detected in unconstrained cells of similar projected area, but different NSI. Our data supports the concept of gene expression and protein synthesis based on optimal distortion of the nucleus, possibly altering transcription factor affinity for DNA, transport to the nucleus, or nuclear matrix organization. The combination of microfabricated surfaces, reverse transcription in situ PCR, and NSI measurement is an excellent system to study how transcription factors, the nuclear matrix, and the cytoskeleton interact to control gene expression and may be useful for studying a wide variety of other cell shape/gene expression relationships.

Active Transport, Cell Nucleus↗