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At least 415 records · Page 23Linked to original sources
PubMed1985

Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

Acetyltransferases↗
PubMed2000

Search and discovery strategies for biotechnology: the paradigm shift.

Profound changes are occurring in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. The advances that have been made in the past decade in areas such as combinatorial chemistry, combinatorial biosynthesis, metabolic pathway engineering, gene shuffling, and directed evolution of proteins have caused some companies to consider withdrawing from natural product screening. In this review we examine the paradigm shift from traditional biology to bioinformatics that is revolutionizing exploitable biology. We conclude that the reinvigorated means of detecting novel organisms, novel chemical structures, and novel biocatalytic activities will ensure that natural products will continue to be a primary resource for biotechnology. The paradigm shift has been driven by a convergence of complementary technologies, exemplified by DNA sequencing and amplification, genome sequencing and annotation, proteome analysis, and phenotypic inventorying, resulting in the establishment of huge databases that can be mined in order to generate useful knowledge such as the identity and characterization of organisms and the identity of biotechnology targets. Concurrently there have been major advances in understanding the extent of microbial diversity, how uncultured organisms might be grown, and how expression of the metabolic potential of microorganisms can be maximized. The integration of information from complementary databases presents a significant challenge. Such integration should facilitate answers to complex questions involving sequence, biochemical, physiological, taxonomic, and ecological information of the sort posed in exploitable biology. The paradigm shift which we discuss is not absolute in the sense that it will replace established microbiology; rather, it reinforces our view that innovative microbiology is essential for releasing the potential of microbial diversity for biotechnology penetration throughout industry. Various of these issues are considered with reference to deep-sea microbiology and biotechnology.

Biotechnology↗
PubMed2004

UT-B1 urea transporter is expressed along the urinary and gastrointestinal tracts of the mouse.

Selective transporters account for rapid urea transport across plasma membranes of several cell types. UT-B1 urea transporter is widely distributed in rat and human tissues. Because mice exhibit high urea turnover and are the preferred species for gene engineering, we have delineated UT-B1 tissue expression in murine tissues. A cDNA was cloned from BALB/c mouse kidney, encoding a polypeptide that differed from C57BL/6 mouse UT-B1 by one residue (Val-8-Ala). UT-B1 mRNA was detected by RT-PCR in brain, kidney, bladder, testis, lung, spleen, and digestive tract (liver, stomach, jejunum, colon). Northern blotting revealed seven UT-B1 transcripts in mouse tissues. Immunoblots identified a nonglycosylated UT-B1 protein of 29 kDa in most tissues and of 36 and 32 kDa in testis and liver, respectively. UT-B1 protein of gastrointestinal tract did not undergo N-glycosylation. Immunohistochemistry and in situ hybridization localized UT-B1 in urinary tract urothelium (papillary surface, ureter, bladder, and urethra), prominently on plasma membranes and restricted to the basolateral area in umbrella cells. UT-B1 was found in endothelial cells of descending vasa recta in kidney medulla and in astrocyte processes in brain. Dehydration induced by water deprivation for 2 days caused a tissue-specific decrease in UT-B1 abundance in the urinary bladder and the ureter.

Animals↗
PubMed2003

[Enjoying research on lysozymes for 42 years].

I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.

Animals↗
PubMed2001

Chloride channels and their functional roles in smooth muscle tone in the vasculature.

Although evidence of important contributions by Cl- channels to agonist-induced currents have been reported in vascular smooth muscle cells, the functional roles played by Cl- channels in the smooth muscle contraction and in setting the membrane potential remain essentially obscure. All of the admittedly few papers published have focused on the physiological roles of Cl- channels in the contraction and membrane depolarization elicited by agonists. At present, it seems likely that in vascular cells: a) Cl- conductance contributes to membrane depolarization, with the subsequent contraction being due to Ca2+ release from the intracellular store sites, and b) Cl- movements through the membrane of the Ca2+ store sites also regulate Ca2+ release and Ca2+ uptake from/into the store sites. As a Ca2+-dependent Cl- current is most easily demonstrated under quasi-physiological conditions (by the perforated patch-clamp method), the contribution made by Cl- channels to smooth muscle function may be more important than previously thought. The development of the new, selective Cl--channel blockers as well as the identification and gene engineering of the channel molecules are essential if we are to advance our knowledge of the physiology and pharmacology of the Cl- channels residing in vascular smooth muscle cells.

Animals↗
PubMed2000

Telomerase and the cellular lifespan: implications of the aging process.

The aging process has multiple causes. However, there is now substantial evidence consistent with the hypothesis that (i) all normal mammalian somatic cells have a finite capacity to replicate and (ii) that gradual cell turnover throughout the lifespan of a mammal eventually exhausts this finite capacity. This results in a gradual accumulation of senescent (irreversibly post-mitotic) cells with increasing age. These cells display a radically different phenotype to their growing counterparts, which has the potential to compromise tissue function. Perhaps the best evidence for this is seen in Werner's syndrome, a rare genetic disease, in which patients display most of the features of accelerated aging, together with a profoundly compromised replicative lifespan in certain tissue lineages. Several classes of human cells are now known to count divisions by monitoring the progressive attrition of chromosomal ends (telomeres), leading to the activation of a p53-p21waf-dependent G1 checkpoint. Ectopic expression of telomerase has been shown to prevent senescence in several cell types and offers the potential for interventions in the aging process based on tissue engineering, gene therapy or homeografts. However, this telomere-driven senescence mechanism seems to be absent from rodents, which use telomere-independent means (perhaps based upon p14arf) to count divisions. Similar senescence pathways are now being reported in humans, and this, coupled with the demonstration of tissue-specific telomeric loss rates, has the potential to render strategies based on the use of telomerase dependent on the characteristics of the target tissue. Werner's syndrome may provide strong clues regarding the potential limitations and prospects of such future treatments.

Aging↗
PubMed1995

Pharmacological approaches to reduce perioperative transfusion requirements in the aged.

Although iron deficiency is undoubtedly the commonest cause of anaemia even in elderly people, the aetiology is not always clear owing to various underlying diseases. Correction of anaemia is sometimes needed before surgery. The use of drugs that may influence blood coagulation, such as aspirin (acetylsalicylic acid), should be checked. Perioperative allogenic blood transfusion can often be avoided by the use of autologous blood and improved surgical techniques. Autologous blood donations are preferable in cases of planned surgery. Epoetin (recombinant human erythropoietin) in combination with iron supplementation facilitates the donation of autologous blood, even in elderly patients. Another method of avoiding allogenic blood transfusion is the collection and reuse of the blood a patient sheds in operations. During and/or after surgery, many haemostatic agents are available. Moreover, recent developments in gene engineering have enabled the utilisation of recombinant cytokines and coagulation factors. Further work remains to be done to define the proper use of these agents.

Aged↗
PubMedDate not supplied

[The efficacy and outlook for the study of live vaccines for the prevention of melioidosis].

The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B. pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis. B. pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis. The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled. The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine.

Animals↗
PubMed1999

[The safety problems of new vaccines].

The paper characterizes the etiological factors that produce side effects and postvaccinal complications. It presents a classification of the types of sides effects. The study of the immunological safety of vaccines is a new trend in assessing the quality of vaccines and their standardization. Due to the design of new-generation vaccines (gene-engineering, synthetic, DNA vaccines, etc.), their safety gain in their particular significance. It is necessary to make a strict government surveillance over the design, testing of these agents. Possible ways of reducing the adverse effects of vaccines.

Humans↗
PubMed1999

[B-cell, T-cell surface phenotypes and epitopes in autoimmune thyroid diseases].

Autoimmune thyroid diseases (AITD) are categorized as organ specific autoimmune diseases, including Graves' disease (GD), Hashimoto's thyroiditis and idiopathic myxoedema. In this article, B-, T-cell surface phenotypes and epitopes in AITD were reviewed. It has been reported that the proportion and absolute number of B and T lymphocyte subpopulation or CD4/8 ratio in peripheral blood from AITD was not different from normal subjects. Those of CD5+ B cells and gamma delta T cells known as autoreactive clones were, however, increased in peripheral blood from GD. In the thyroid tissue of AITD, B-cells with activated markers were predominantly infiltrated rather than T-cells. The CD4/8 ratio and the proportion of CD8+CD11b+ T (suppressor T) cells was decreased in contrast to the increase of CD8+CD11b- T (cytotoxic T) cells. The CD4+4B4+ T (helper/inducer) cells were more predominant than CD4+2H4+ T (suppressor/inducer) cells in thyroid tissue from GD. These abnormality might cause the cell destruction and the enhanced autoantibody production in the tissue of AITD. Thyroglobulin, thyroid peroxidase and thyroid stimulating hormone receptor are known as major autoantigens in AITD. Recently, B-cell and T-cell epitopes on these autoantigen molecules have been defined, using monoclonal antibodies, proteolytic or synthetic peptides with molecular gene engineering. These epitope analysis would be contributed to the autoimmune tolerance in AITD.

Autoimmune Diseases↗
PubMedDate not supplied

[The DNA of recombinant plasmids acquires mutagenic activity in a Bacillus subtilis culture after the insertion of the human genome Alu repeat].

In our previous work the mutagenic activity of recombinant plasmids (pBR322 carrying the human Alu repeat alone or in combination with preproinsulin or apo AI gene) in competent Bacillus subtilis culture was demonstrated. In present work it was shown that among seven tested plasmids only three revealed mutagenic activity (pBR322 and two Alu repeat-containing constructions). It seems that mutagenic activity is not inherent to any recombinant molecule in applied test system but depends on its structure. For example, Alu repeat of human genome may attach mutagenic properties to plasmids which either had no such properties, or lost them after gene engineering manipulations.

Alu Elements↗
PubMed2000

[Cloning and expression of the lux-operon of Photorhabdus luminescens, strain Zm1: nucleotide sequence of luxAB genes and basic properties of luciferase].

A chromosomal fragment of bacteria Photorhabdus luminescence Zm1, which contains the lux operon, was cloned into the vector pUC18. The hybrid clone containing plasmid pXen7 with the EcoRI fragment approximately 7-kb was shown to manifest a high level of bioluminescence. By subcloning and restriction analysis of the EcoRI fragment, the location of luxCDABE genes relative to restriction sites was determined. The nucleotide sequence of the DNA fragment containing the luxA and luxB genes encoding alpha- and beta-subunits of luciferase was determined. A comparison with the nucleotide sequences of luxAB genes in Hm and Hw strains of Ph. luminescence revealed 94.5 and 89.7% homology, respectively. The enterobacterial repetitive intergenic sequence (ERIC) of 126 bp typical for Hw strains was identified in the spacer between the luxD and luxA genes. The lux operon of Zm1 is assumed to emerge through recombination between Hm and Hw strains. Luciferase of Ph. luminescence was shown to possess a high thermal stability: its activity decreased by a factor of 10 at 44 degrees C for 30 min, whereas luciferases of marine bacteria Vibrio fischeri and Vibrio harveyi were inactivated by one order of magnitude at 44 degrees C for 1 and 6 min, respectively. The lux genes of Ph. luminescence are suggested for use in gene engineering and biotechnology.

Amino Acid Sequence↗
PubMedDate not supplied

[The outlook for the development of live vaccines for the prevention of melioidosis].

The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B. pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis. The most immunogenic B. pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions. In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level. The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.

Animals↗
PubMed1998

[Antifertile effect of immunization with anti-idiotypic antibodies to porcine zona pellucida].

OBJECTIVE: To evaluate the antifertile effect of anti-idiotypic antibodies to PZP. METHODS: The New Zealand rabbits were immunized by 17D3 monoclonal antibody against PZP to produce anti-idiotypic antibodies (Ab2) which were then purified by immuno-affinity chromatography. RESULTS: The mice immunized with Ab2 showed decrease of pregnancy rate and a statistically significant reduction in the number of embryos as compared to controls (P < 0.05). Histological examination of ovaries showed that Ab2 interferes only with late follicular development. CONCLUSIONS: Ab2 to 17D3mAb could alleviate ovarian damage and suppress partial fertility. Clearly, further research is required to screen epitope of inner image of Ab2 and to express it by gene engineering.

Animals↗
PubMed2001

[Phagotherapy in terms of bacteriophage genetics: hopes, perspectives, safety, limitations].

The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change some bacteriophage: properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.

Animals↗
PubMedDate not supplied

[Persistence of Escherichia coli recombinant strains in experimental animals].

Recombinant E. coli strains, obtained by gene engineering techniques and capable of producing human alpha-interferon and HIV proteins, were studied. The cultures under study were completely eliminated from the body of experimental animals (mice) in 48 hours, and generalization of the infectious process took place. The study revealed that these recombinant strains had low virulence and were weakly adhesive, nontoxigenic and weakly toxic. Thus, the recombinant strains under study could be classified with class 3-4 of danger according to the "Classification of Strains of Industrial Microorganisms by the Degree of Their Danger".

Agglutination Tests↗
PubMed2002

Effect of semi-synthesized quercetin water-soluble derivatives on recombinant human phosphatidylinositol 3-kinase p110beta catalytic subunit.

AIM: To study the effect of semi-synthesized quercetin water-soluble derivatives sodium quercetin-7-sulfate (SQMS) and disodium quercetin-7,4 -disulfate (SQDS) on recombinant human phosphatidylinositol 3-kinase (PI3-K) p110 beta catalytic subunit. METHODS: Recombinant human PI3-K p110 beta catalytic subunit was expressed by gene engineering. PI3 -K was assayed by incubating recombinant PI3-K p110beta with phosphatidylinositol-4,5-bisphosphate and [gamma-32P]ATP; the [32 P]-radiolabeled lipids were extracted with chloroform and methanol, assessed by thin layer chromatography and visualized by autoradiography. RESULTS: Wortmannin, a specific inhibitor o f PI3-K, showed inhibition on recombinant PI3-K p110beta catalytic subunit in a concentration-dependent manner (2.5 - 20 nmol/L); SQMS and SQD S showed inhibition on recombinant PI3-K p110beta catalytic subunit in a concentration-dependent manner (2.5 - 20 micromol/L). CONCLUSION: Semi-synthesized quercetin water-soluble derivatives were a type of inhibitors of PI3-K. The recombinant PI3-K p110beta catalytic subunit might be used as a molecular target for simpler filtrating and development of more effective inhibitors of PI3-K.

Androstadienes↗
PubMed2002

[PCR cloning of 3'-terminal repeat fragments of Clostridium difficile toxin A and toxin B gene].

OBJECTIVE: To explore a method to rapidly detect the presence of Clostridium difficile. METHODS: PCR was used to amplify DNA fragments from the highly conserved and repeated domains of the 3'-terminal of Clostridium difficile toxin A gene and toxin B genes. RESULTS: The fragments 960 bp and 1,851 bp in length were respectively amplified from toxin A and toxin B, while the control bacteria presented no distinct results. CONCLUSION: The 960 bp and 1,851 bp fragments are specific for Clostridium difficile and PCR can be used to detect Clostridium difficile from the stool. As the peptides encoded by these conserved domains have high hydrophobicity and strong antigenicity, manufacture of the protein vaccine by gene engineering is possible on the basis of these conserved domains.

Bacterial Proteins↗