Effect of homologous overexpression of nucleoside diphosphate kinase on the yield and molecular weight of hyaluronic acid / 中国药科大学学报

@#To investigate the effect of nucleoside diphosphate kinase (NDK) on the synthesis of hyaluronic acid, nucleoside diphosphate kinase gene (ndk) was overexpressed along with the hyaluronic acid-producing genes in recombinant B.subtilis. Two engineered strains named Hp8tg and Pn8tg were constructed.Uniform hyaluronic acid (HA) could be obtained from both engineered strains.HA produced by both recombinant strains was confirmed by monosaccharide composition analysis, Fourier transform infrared spectometry and nuclear magnetic resonance spectroscopy.Inducing conditions of HA fermentation were optimized by response surface methodology.Overexpression of ndk could increase the production and molecular weight of HA by 1.3-fold and 1.1-fold, respectively. This study revealed for the first time that overexpression of ndk could relieve the inhibition effect of uridine diphosphate (UDP) on Class II HA synthase and increase the production and molecular weight of HA, which proves to be an efficient strategy for the production of HA, and the preparation of other polysaccharides.

Revisiting trypanosomatid nucleoside diphosphate kinases

BACKGROUND An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses

BACKGROUND NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.

Nucleoside Diphosphate Kinase from Microorganisms

Nucleoside diphosphate kinase (Ndk) is ubiquitous and highly conserved multifunctional key enzyme in nucleotide metabolism. It generates nucleoside triphosphates (NTPs) by transfer of gamma-phosphates from nucleoside triphosphates such as ATP or GTP to nucleoside diphosphate. The formation of an autophosphorylated enzyme intermediate is involved in that mechanism. The phosphate is usually supplied by ATP and Ndk activity in different subcellular compartments. Ndk may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. Ndk is playing an important role in bacterial pathogenesis and emerging evidences recognize multiple roles of Ndk in host-microbe interaction. Here, I review some examples of the role of Ndk in intra- and extracellular microorganism.

nm23-H1 Protein Expression and Gene Mutation in 150 Patients with Non-Hodgkin's Lymphomas

The metastasis-suppressing role of the nm23 gene in the metastatic spread of malignant tumor is still debated. We examined the nm23-H1 protein expression and gene mutation in non-Hodgkin's lymphomas to compare with the clinicopathologic parameters. The expression of nm23-H1 protein was immunohistochemically examined in 150 cases of non-Hodgkin's lymphomas; 85 diffuse large B cell lymphomas (DL-BCL), 18 marginal zone B cell lymphomas (MZL), 3 mantle cell lymphomas, 25 peripheral T cell lymphomas, not otherwise specified (TCLNOS), and 19 NK/T cell lymphomas (NK/T). Eighty-one cases (58 DLBCL, 6 MZL, 4 TCLNOS, and 13 NK/T) were studied for nm23-H1 gene mutation in exon 1 to 5. The high expression of nm23-H1 protein was associated with the high IPI score (p=0.019) and the low survival rate of the patients (p=0.0039). The gene mutation of nm23-H1 was detected in 10.3% of DLBCL and 30.7% of NK/T; but none in MZL and TCLNOS. The mutation was found in exon 1 in 5 cases, exon 2 in two cases, exon 4 in one case and both exon 1 and 2 in two cases. Our results suggest that the expression of nm23-H1 protein can be used as a poor prognostic marker in non-Hodgkin's lymphomas, and the mutational change of gene may operate in the lymphomagenesis.

Protective Efficacy of Recombinant Proteins Adenylate Kinase, Nucleoside Diphosphate Kinase, and Heat-Shock Protein 70 against Mycobacterium tuberculosis Infection in Mice / 결핵및호흡기질환

BACKGROUND: Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. METHOD: M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and IFN-gamma after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. RESULT: Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and IFN-gamma responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. CONCLUSION: Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

The action of recombinant human nucleoside diphosphate kinase A(rhNDPK-A)on the growth of S_(180),H_(22),Lewis and H460 tumors in vivo / 中国病理生理杂志

AIM:To study the action of nucleoside diphosphate kinase A(NDPK-A)on the growth of S_ 180,H_ 22,Lewis and H460.METHODS:S_ 180 or H_ 22 cell(5?106)were inoculate subcutaneously into the right armpit of 85 Kunming mice,which were randomized into 8 groups.Lewis lung carcinoma cells(2?105)were inoculate subcutaneously into the right armpit of 85 C57BL/6 mice,which were randomized as Kunming mice.From the 2nd day,the treated groups were given different dose of rhNDPK-A once a day for 8 days(for S_ 180 or H_ 22 by iv)or for 10 days(for Lewis by ip),and the control group was given physiological saline only.H460 tissue pieces about 1.5 mm?1.5 mm?1.5 mm each were inoculated subcutaneously into the armpit of 38 Balb/c/neu mice.After the volume of xenograft become 100 mm?100 mm?100 mm,the nude mice were randomized into 5 groups and given different dose of rhNDPK-A once a day for 17 days.2 days after above treatments,the mice were killed and dissected.The knubs were peeled off and weighted.RESULTS:The growth of S_ 180,H_ 22 and H460 were inhibited by rhNDPK and the growth of H_ 22 was inhibited by rhNDPK at dose of 20 mg/kg combined with cisplatin(0.5 mg/kg).But the growth of Lewis lung cancer was not inhibited.CONCLUSION:rhNDPK-A inhibited the growth of S_ 180,H_ 22 and H460.rhNDPK-A(20 mg/kg)potentiated the antitumor action of cisplatin on H_ 22.

Expression of nm23 Protein in Human Gastric Carcinoma: correlation between nm23 expression with the development and metastasis of gastric carcinoma

Gene expression of nm23 has been investigated in many kinds of tumors, including breast cancers, colon cancers, hepatocellular carcinomas, papillary carcinomas of the thyroid and malignant melanomas since the nm23 was dislovered by Dr. Steeg as a tumor metastatic suppressor gene. Reduced expression of nm23 gene implicated in high metastatic potential in a variety of malignancies. However, there have been only a few reports on genetic alteration and expression of nm23 in human gastric carcinomas even though gastric carcinoma is a leading malignancy in Korea. In this study, we examined the expression of nm23 protein by immunohistochemistry in advanced and early gastric carcinomas, adenomas, matching normal mucosa to elucidate the role of nm23 in the development, progression and metastasis of human gastric carcinomas. The results are as follows; 1) Nm23 was expressed in 39 cases(69.6%) of 56 advanced gastric carcinomas. Among them, strong positive cases(grade 3) were 26(46.4%) and weak positive cases(grade 2) were 13(23.2%). 2) Nm23 expression was significantly different (P<0.05) depending on the site of the neoplasm. Antral carcinomas showed grade3 positivity in the 22/37 cases(60%), but carcinomas of the body showed negative (grade 1) result in about half(42.1%). 3) Nm23 expression was more intense in the neoplasm than normal mucosa.(67.9%) 4) Nm23 expression was not significantly related to the lymph node metastasis, invasion of lymphatics or veins and depth of invasion. 5) In the well differentiated carcinomas, grade3 were more common(64.0%). But in cases of signet ring cell carcinoma, many cases were negative(50.0%). 6) Nm23 expression rate and intensity was significantly increased from the normal mucosa to the gastric adenomas, early gastric carcinomas and advanced gastric carcinomas.

Expression of nm23 Protein in Breast Carcinoma: An immunohistochemical study

To elucidate a possible prognostic factor, we studied 91 cases of breast carcinoma for the expression of n-tn23 protein using an immunohistochemical method, and compared these results with the known prognostic parameters of the breast carcinoma. The mn23 protein was intensely stained in the cytoplasm and/or the nucleus of carcinoma cells in 82 cases(90.1%). There were two patterns of cytoplasmic staining; heterogeneous pattern and homogeneous pattern. Among the positive cases, 43 cases(47.2%) were heterogeneous while 39 cases(42.8%) were homogeneous. Axillary lymph node metastases(p<0.005) was found more frequently in the heterogeneous pattern group(79.0%) than in the homogeneous pattern group(41.0%). There was no significant correlation between nm23 protein expression and other parameters such as patient age, tumor size, estrogen receptor, histopathologic grade, and p53 overexpression. Although axillary lymph node metastasis was correlated with the disease free status(p<0.0005) and patient survival (p<0.05), they showed no correlation with nin23 expression. Multivariate analysis showed that axillary lymph node metastasis was the only prognostic indicator(p<0.05), and the expression of nm23 protein was of borderline significance. The results suggest that the homogeneous and/or granular cytoplasmic expression of mn23 protein plays a role in the suppression of nodal metastasis in breast carcinoma and might contribute in predicting patient survival.

Expression of recombinant human tumor suppressor NDPK-A in E. coli / 中国病理生理杂志

AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6?His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6?His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni+-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49 6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6?His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.