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[Inhibitory effect of AG1109 on recombinant human protein kinase CK2 holoenzyme].

BACKGROUND & OBJECTIVE: Protein kinase CK2 is an evolutionarily conserved serine/threonine kinase that is upregulated and can serve as an oncogene in many cancer cells. Targeted inhibition of CK2 could be useful in the treatment of many cancers. To search specific CK2 inhibitors we investigated the direct effect of tyrphostin AG1109 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS: Recombinant human protein kinase CK2 alpha and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. The two subunits were mixed at the same molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of [gamma-32P]ATP or [gamma-32P]GTP into the substrate in various conditions. RESULTS: The recombinant human CK2 was the second messengers (Ca2+, cAMP, and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG1109 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 9.7 mumol/L, which was slightly more effective than 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide (A3), which were both known CK2 special inhibitors. Kinetic studies of AG1109 on recombinant human CK2 showed that the inhibition was mixed (competitive is dominant) with GTP and noncompetitive with casein. CONCLUSIONS: AG1109 is an effective inhibitor of recombinant human protein kinase CK2 holoenzyme. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.

Casein Kinase II↗

Basic principles on toe-to-hand transplantation.

Within the last three decades, toe-to-hand transplantation has become a well-established method for function and appearance reconstruction after trauma and in congenital hand anomalies. An otherwise healthy and cooperative patient is the ideal candidate for toe transplantation after trauma. In such patient, even primary toe transplantation is possible, if the stump is clean and viable. If secondary reconstruction after completed wound healing is considered, emphasis should be laid on tissue sacrifice during the acute management of non-replantable amputations at the hand. Specific considerations regarding selection of toe(s) to be transplanted, technique of toe harvest and inset, sequence of transplantations if more than one digit is to be reconstructed such as in the metacarpal hand, and postoperative regimen are important to achieve satisfying functional and aesthetic results on both recipient and donor sites. A trimmed great toe is ideal for thumb reconstruction if the amputaiton is located at or distal to the middle metacarpal shaft. However, in more proximal amputations a second toe may be more suitable as it allows transmetatarsal harvest without increasing donor site morbidity. Distal finger reconstruction with partial toe or second toe warp around flap gives most gratifing result to those patients who are critically concerned about their body images and also those who need distal fingers for jobs or recreation activities. Combined second and third toe or third and fourth toe transplantations are particular useful in metacarpal hand reconstruction to provide tripod pinch. The role of toe-to-hand transplantation in the new millenium assuming progress in tissue engineering, gene transfer, and the development of new immunosuppressive drugs is discussed.

Amputation, Traumatic↗

[Mesenchymal stem cells and related factors].

Mesenchymal stem cells possess the ability to differentiate into osteoblasts, chondroblasts, lipoblasts, myoblasts and so on, which can be used in the formation of hematopoietic microenvironment, tissue repairing and gene therapy. Growth factors such as TGF-beta, IGF-I, BMP and FGF can influence on the differentiation of MSC and they cooperate with each other. MSCs support hematopoiesis by secreting cytokines including G-CSF, SCF, LIF, M-CSF, IL-6, IL-11 and are related to some diseases. MSC would demonstrate important effect on gene engineering.

Animals↗

Immunological Problems of Diagnostics and Therapy in Hematology and Oncology.

Immunology takes reliable place in the system of diagnostics and therapy of oncological and haematological diseases. It should be noted that serodiagnostics of tumors achieved its methodological limit, since all tumor-associated antigens are already known, and the search of new serum immunological markers seems to fail. New markers have attracted attention of investigators: cytoplasmic and surface proteins and glycoproteins, being products of different genes, which control cell viability, such as Pgp170, p53, Bcl-2, CD95 (Fas/APO-1), Her-2/neu and others. All these proteins may be identified by monoclonal antibodies. At present, the identification of these biomarkers by immunohistochemical methods is beginning to use for individualization of therapy. New direction in oncohematology is biotherapy of tumors. Tumor biotherapy means the treatment of oncological patients with vaccines, immunomodulators, cytokines, monoclonal antibodies and so on. Cancer vaccinotherapy took new design due to the achievements in molecular biology and gene engineering. The great success was achieved in the therapy of tumors and leukemias by interferon preparations. Again, the great expectations seem to be in the field of cancer therapy with immunomodulators. Thus, the achievements in the treatment of oncohaematological diseases are as usual related to the achievements in immunology.

Journal Article↗

New Principles of Development of Preparations for Immunoprophylaxis.

In the development of new vaccine the fate of preparation depends on three main factors: potential to decrease morbidity and benefits from vaccine use; the risk of post vaccinal complications and possible damage from vaccination; the cost of vaccine and economic advantages. By basic parameters, dealing with safety and effectiveness, home immunological preparations, first of all vaccines used in framework of vaccination calendar, meet all the requirements of WHO. However, absolutely safe vaccines don't exist in the world. All the above may be an evidence of the need to find new approaches to the development of more improved vaccines. Gene-engineering technology is a revolutionary event in vaccinology, it made it possible to develop recombinant vaccines, possessing weak reactogenity and sufficient effectivity. Anti-idiotypic vaccines didn't go out the sphere of experimental approaches. Any protein vaccine, being introduced to the body, is undergone to digestion. Forming peptides interact with histocompatibility antigens and acquire immunogenic properties. The development of new class vaccines is based on this principle. Synthetic peptide vaccines are safe and low reactogenic. In spite of a large amount of experimental peptide vaccines, there is no still any vaccine of such a type in the practice of health services. Meanwhile, WHO gave recommendations dealing with development and control of peptide vaccines, suggesting that such vaccines will appear in the next future. The same recommendations were given for so called DNA vaccines.

Journal Article↗

[The use of interferon in the combined therapy of juvenile rheumatoid arthritis].

The efficacy of recombinant gene engineering alpha 2-interferon (reaferon) was studied and compared in 60 patients suffering from verified juvenile rheumatoid arthritis (JRA). Reaferon was shown to possess good tolerance and to produce an adequate therapeutic effect. The combined use of reaferon and methotrexate permits potentiating the therapeutic effect of interferon and avoiding side effects seen with methotrexate used alone. Besides, it makes it possible to reduce the incidence of respiratory infections which are often associated with exacerbation of the underlying disease when treated by conventional methods.

Administration, Rectal↗

[The basic and applied study on the epidermal growth factor].

This article reviews the results of the basic research about epidermal growth factor and its receptor, and the development of the novel drug, EGF eyedrop, that containing chemically synthesized EGF gene, the construction of EGF expression vector, the transformation of the host cells, the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the alpha factor leading sequence in the yeast cells. The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the concentration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (> 10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4.6 and it has correct N-terminal amino acids sequences, immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety, ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.

Animals↗

[Cloning and sequence analysis of active fragments of human thrombospondin-1].

OBJECTIVE: To lay a foundation for researches on the expression of gene engineering anti-tumor drugs. METHODS: On the basis of mRNA sequence of human thrombospondin-1(TSP-1), two pairs of primers were designed and synthesized. Two active fragments TSP-1, called TSP-1-I and TSP-1-III, were obtained by using reverse transcription-polymerase chain reaction(RT-PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purified and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs. RESULTS: Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP-1-I is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6 x 10(3) and the TSP-I III is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5 x 10(3). CONCLUSION: The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP-1 of Genbank.

Adenocarcinoma↗

[Production of recombinant fragments of the Clostridium tetani neurotoxin for the development of new immune-prophylaxis preparations against tetanus].

Tetanus belongs to dangerous infection diseases, whose effective prevention can be ensured by vaccines. The acting substance of tetanus vaccines, presently in use, is a partially purified and deprived-of-lethal-action Clostridium tetani neurotoxin. The construction of a subunit preparation on the basis of toxin fragments obtained through gene engineering could be a method aimed at promoting the quality of the used tetanus vaccines. With this goal in mind, we built, within the present case study, the expressing genetic constructions and obtained, in the pure form, an extensive tetanus-vaccine chain with its C-terminal (Hc) fragment, hydride peptides, containing the Hc-fragment and C-terminal fragment of toxin B C. difficile, as well as Hc-fragment and S3 collagen-binding domain of collagenase C. histolyticum. The thus obtained proteins can be used in testing their immunogenic and protective properties, while the conducted study could be a basis for further research of a new-generation vaccine against tetanus and other human infection diseases.

Binding Sites↗

[Immuno-therapeutic study of anti-idiotype minibody (single chain Fv-CH3) on ovarian carcinoma bearing mice].

OBJECTIVE: An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer. METHODS: Using gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region. The fusion protein named minibody was induced with IPTG in E. coli and analyzed with Western blot and inhibition ELISA tests respectively. Twenty human-PBL-SCID mice bearing i.p. Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times. Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3). The latent period of ascites growth and the mean survival time were observed respectively. CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry. RESULTS: SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3). The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot. Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen. Ab3 could be detected in the sera of immunized mice with minibody by ELISA test. Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks. The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination. The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively. CONCLUSIONS: These results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function. Antigenicity is increased without adjuvants and partial humanization is realized. Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo. When ascites formation was delayed or prevented and the survival was prolonged in minibody group. We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.

Animals↗

[Pangenome].

A conception of general interaction between genetic information of different organisms is formulated. A new concept "Pangenom" is proposed for description of the total sum of genes of living organisms and viruses. Features of the Pangenom structure and processes controlling its saving and variability are discussed. It is demonstrated that this conception may be used in medicine for the aims of prognosis of epidemic situation, in particular, of newly spreading pathogen development of new methods of prophylaxis and diagnostics of oncological diseases, and also of the evaluation of possible consequences of molecular genetics and gene engineering interventions.

Animals↗

[Experimental study of human recombinant insulin].

The experimental study of recombinant human insulin revealed its high specific activity. The kinetics of the hypoglycemic effect of various dosage forms of the insulin developed at the National Research Centre of Antibiotics and the dosage forms manufactured by Eli Lilly was similar. The pharmacological investigation of the dosage forms showed that they had no side effects on the vitally important organs and systems. It was concluded that the dosage forms of the gene engineered human insulin developed at the National Research Centre of Antibiotics did not differ by their characteristics from the analogous dosage forms manufactured in other countries.

Animals↗

[The preparation of human anti-HBs Fab fragment by bioengineering technique].

AIM: To prepare human anti-HBs Fab by bioengineering technique. METHODS: The specific human anti-HBs Fab gene screened from combinatorial library was cloned into plasmid pBAD/g IIIA, then positive clone was transformed into E.coli Top10. After the fermentation and expression processes, the soluble Fab fragment in the periplasm were purified by Ni-NTA-agarose affinity chromatography,and the inclusion bodies were in turn denatured, solubilized, purified and renatured. The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot. RESULTS: The quantity of soluble Fab protein purified from periplasm with Ni-NTA-Agarose which possessed good specificity as well as excellent binding activity to antigens was up to 80 mg per liter, but the biologically active protein acquired after renaturation of the inclusion bodies was quite small. CONCLUSION: Using pBAD/g IIIA-Top10 expression system, the soluble Fab protein with biological activity could be produced from periplasm of the E.coli Top10, and the strategy is available to prepare human anti-HBsAg Fab fragments in large quantity by gene engineering technique.

Combinatorial Chemistry Techniques↗

[Expression and purification of mouse B lymphocyte chemoattractant].

A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR. The amplified fragment was inserted into prokaryotic expression vector PQE30. Recombinant protein was expressed in E. Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix. Then it was identified by sequence analysis and Western blot analysis. The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis. And a specfic band was shown by Western blot analysis. These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant.

Animals↗

[Preparation and characterization of anti-human IL-15 monoclonal antibody].

AIM: To prepare monoclonal antibody (mAb) against human interleukin-15 (hIL-15) and identify its characterization. METHODS: The GST-IL-15 was extracted from the gene-engineering bacteria E. coli and identified by SDS-PAGE. The gel strip containing GST-IL-15 was cut off to immunize BALB/c mice. The splenocytes of immunized mice were fused with Sp2/0 myeloma cells by a routine method and the hybridomas were selected in HAT medium. The hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution. The stability of the obtained hybridoma cells and the specificity of anti-hIL-15 mAb the hybridoma cells secreted were identified. In addition, the New Zealand rabbits were immunized with the rhIL-15 inclusion body protein (rhIL-15IBP) to prepare the polyclonal antibody (pAb) against hIL-15. A sandwich ELISA was established with the anti-IL-15 mAb and pAb as coating and sandwich antibodies, respectively, to detect hIL-15. RESULTS: One hybridoma cell line which could stably secrete specific mAb was obtained. A sandwich ELISA for detecting rhIL-15 protein was established and its sensitivity was as low as 10 microg/L. CONCLUSION: The anti-hIL-15 mAb was prepared successfully. A sandwich ELISA for the detection of hIL-15 was established.

Animals↗

[Research advance in nitrogen metabolism of plant and its environmental regulation].

Nitrogen metabolism is not only one of the basic processes of plant physiology, but also one of the important parts of global chemical cycle. Plant nitrogen assimilation directly takes part in the synthesis and conversion of amino acid through the reduction of nitrate. During this stage, some key enzymes, e.g., nitrate reductase (NR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), glutamine synthase (GOGAT), aspargine synthetase (AS), and asparate aminotransferase (AspAT) participate these processes. The protein is assimilated in plant cell through amino acid, and becomes a part of plant organism through modifying, classifying, transporting and storing processes, etc. The nitrogen metabolism is associated with carbonic metabolism through key enzyme regulations and the conversion of products, which consists of basic life process. Among these amino acids in plant cell, glutamic acid (Glu), glutamine (Gln), aspartic acid (Asp) and asparagines (Asn), etc., play a key role, which regulates their conversion each other and their contents in the plant cell through regulating formation and activity of those key enzymes. Environmental factors also affect the conversion and recycle of the key amino acids through regulating gene expression of the key enzymes and their activities. Nitrate and light intensity positively regulate the gene transcription of NR, but ammonium ions and Glu, Gln do the negative way. Water deficit is a very serious constraint on N2 fixation rate and soybean (Glycine max Merr.) grain yield, in which, ureide accumulation and degradation under water deficit appear to be the key issues of feedback mechanism on nitrogen fixation. Water stress decreases NR activity, but increases proteinase activity, and thus, they regulate plant nitrogen metabolism, although there are some different effects among species and cultivars. Water stress also decreases plant tissue protein content, ratio of protein and amino acid, and reduces the absorption of amino acid by plant. On the contrary, soil flooding decreases the content and accumulation amount of root nitrogen in winter wheat by 11.9% from booting to flowering stages and 39.1% during grain filling stage, and reduces the ratio of carbon and nitrogen by 79.6%. The results misadjust the metabolism between carbon and nitrogen, and result in the end of the root growth. Elevated CO2 level could decrease plant leaf nitrogen content under well-watered condition, but almost maintain stable under water deficit condition. The radiation of UV-B significantly reduces the partitioning coefficient and synthetic rate of Rubisco, which significantly decreases the photosynthetic rate. This paper reviewed the pathway of plant nitrogen assimilation, characteristics of key enzymes and their regulating mechanisms with picturing the regulating mode of NR, and described the signal sensing and conduct of plant nitrogen metabolism and the formation, transportation, storage and degradation of plant cell protein with picturing the schedule of protein transport of membrane system in plant cell. Seven key tasks are emphasized in this paper in terms of the review on the effects and mechanisms of key ecological factors including water stress on plant nitrogen metabolism. They are: 1) the absorption mechanism of plant based on different nitrogen sources and environmental regulations, 2) the localization and compartmentalization of the key enzymes of nitrogen mechanism in plant cell, 3) the gene and environmental regulating model and their relationships in various key enzymes of nitrogen metabolism, 4) the function of main cell organs and their responses to environmental factors in nitrogen metabolism process, 5) physiological and chemical mechanism of nitrogen and the relationship between the mechanism and protein formation during crop grain filling, 6) improving gene structure of special species or cultivars using gene engineering methods to enhance the resistance to environmental factor stress and the efficiency of absorption and transportation of nitrogen, and 7) the mechanism of natural nitrogen cycle and its response to human activity disturbance.

Environment↗

[Inhibitory effect and its kinetic analysis of baicalein on recombinant human protein kinase CK2 holoenzyme].

BACKGROUND & OBJECTIVE: Protein kinase CK2 is a ubiquitous and pleiotropic Ser/Thr protein kinase in eukaryotic cells. CK2 activity has been shown to be markedly elevated in solid tumors and leukemia cells. Its alpha or alpha' gene is a protooncogene. CK2 is attracting increasing attention as a potential target for anti-neoplastic. This study was to search specific CK2 inhibitors in tumor cells through observation of the inhibitory effects of baicalein on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro. METHODS: Recombinant human protein kinase CK2 alpha' and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. These 2 subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme. The CK2 activity was evaluated by detecting radioactivity of 32P of [gamma-32P]ATP which was incorporated into the substrate in various conditions. RESULTS: Baicalein was shown to strongly inhibit the holoenzyme activity of CK2 with IC50 of 2.54 micromol/L. Kinetic studies of baicalein on CK2 showed that baicalein acted as an inhibitor of noncompetitive with ATP(KI=7.73 micromol/L) and mixed types with casein(KI=3.07 micromol/L). CONCLUSION: Baicalein is an effective inhibitor of protein kinase CK2 in vitro.

Casein Kinase II↗

Humanized OKT3 antibodies: successful transfer of immune modulating properties and idiotype expression.

Antibodies that possess the Ag-binding regions of OKT3 within the context of a human framework (Hu-OKT3 Ab) offer distinct advantages for optimizing anti-CD3 mAb therapy. First, manipulation of Ab genes to produce humanized Ab that retain Ag-binding activity may circumvent antigenicity problems. Second, Ab gene engineering provides a means for modifying functional properties, including T cell activation and immune suppression. The purpose of this study was to determine the functional properties of Hu-OKT3 Ab and to compare the functional properties and idiotypes of Hu-OKT3 Ab to those of murine OKT3. Three Hu-OKT3 IgG4 Ab, a chimeric OKT3 antibody (cOKT3-1) (grafted sequences comprising all OKT3 VH and VL regions) and two complementarity determining region (CDR)-grafted antibodies, gOKT3-5 and gOKT3-6 (grafted sequences comprising only OKT3 VH and VL CDR and some framework amino acids, were analyzed. Initial studies demonstrated that the cOKT3 and gOKT3-5 Ab bound selectively to T cells and competitively inhibited OKT3-FITC binding with avidities similar to that of murine OKT3. Binding avidity of the gOKT3-6 Ab was markedly less than that of the other two Hu-OKT3 Ab. Serologic analysis suggested that cOKT3 and gOKT3-5 Ab possess idiotypes (combining sites) similar to murine OKT3. T cell activation potency of all three Hu-OKT3 Ab was assessed by proliferation, induction of activation marker expression (IL-2R and Leu 23), and lymphokine production (TNF-alpha and IFN-gamma). The cOKT3 and gOKT3-5 Ab demonstrated T cell activation potencies similar to murine OKT3 as assessed by each parameter. CD3 coating and modulation by these two Ab was effective but somewhat less potent than that observed with OKT3. Finally, cOKT3 and gOKT3-5 Ab both inhibited CTL activity comparably to murine OKT3. In conclusion, these studies indicate that gOKT3-5 and cOKT3 Ab possess immune modulating properties similar to murine OKT3 and thus offer attractive alternatives to murine OKT3 for in vivo therapy.

Amino Acid Sequence↗