Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 µ g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

[Construction and transduction of hNUDC shRNA interference lentiviral vector].

AIM: To construct a lentiviral vector carrying human nuclear distribution protein C (hNUDC)-specific shRNA (sh-NUDC-F) and knock down hNUDC expression in Dami cells by infection of the lentivirus. METHODS: After labeling of green fluorescent protein (GFP), sh-NUDC-F was cloned into lentiviral vector pRRL-cPPT-CMV-X-PRE-SIN, and the high-quality plasmid was transfected into 293T cells to produce lentiviral particles by the calcium phosphate method. After high-speed centrifugation, lentiviral particles were collected and determined for its titer. The high-titer lentiviral particles were then transduced into Dami cells. By detecting the expression of GFP in lentiviral vector using a microscope, the transduction efficiency was readily figured out. And hNUDC protein level was detected by Western blotting. RESULTS: hNUDC was totally knocked down by the efficient transduction of Dami cells with the lentivirus carrying sh-NUDC-F. CONCLUSION: Lentiviral vector containing sh-NUDC-F can be successfully packaged in 293T cells and then efficiently transduced into Dami cells to silence hNUDC gene.

[The construction of NUDC shRNA interference vector with red fluorescence protein].

AIM: Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence. METHODS: NUDC and RFP genes were cloned into pDs vector separately, resulting in pDs-NUDC- RFP. as above, human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs- NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed. Construct- ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluorescence photographs were taken. RESULTS: Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector. fluorescence photographs showed that shNUDC-A is the best effective fragment. CONCLUSION: The NUDC gene targeted shRNA and its vector are successfully constructed.

[Analysis on genetic defects of patients with azoospermia and severe oligospermia--report of 2 novel abnormal karyotypes].

G banding karyotype analysis of peripheral lymphocytes in 217 patients with azoospermia or severe oligospermia were performed and the Y-chromosome AZFc region from 7 cases with Y chromosome abnormality was amplified by polymerase chain reaction (PCR). Out of 187 cases with azoospermia, 77 patients or 41.18% had chromosome abnormalities, including number and structural aberrations, heteromorphic chromosomes (Y chromosome heteromorphisms and pericentric inversion of chromosome 9) and 46, XX sex reversal. Two novel abnormal karyotypes were found: 46, XY, t(6; 14)(p21; p13) and 46, XY, t(8; 12)(p21; q24). Out of 30 patients with severe oligospermia, 4 had chromosome abnormalities, including structural aberration and 46, XX sex reversal. Therefore, aberration of the sex chromosome causes the most serious spermatogenic failure and certain breakpoints in the autosomes may also affect spermatogenesis. The deletion of AZFc also affects spermatogenesis.

Association between cholesteryl ester transfer protein gene polymorphisms and variations in lipid levels in patients with coronary heart disease.

BACKGROUND: The Taq/B, Msp/ and I405V polymorphisms of cholesteryl ester transfer protein (CETP), an important regulatory factor of lipid metabolism, have been attracted much more attention by the researchers. In this study, we investigated the associations between these 3 polymorphisms of CETP gene and variations in plasma lipid and lipoprotein levels in patients with coronary heart disease (CHD). METHODS: Genomic DNA was extracted from leukocytes of 203 CHD patients and 100 control subjects using the salting out method. Genotyping of the CETP gene was performed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques. Statistical analysis was conducted using the SPSS 10.0 software package. RESULTS: The distribution of allele and genotype frequencies of the Taq/B, MspI, and I405V polymorphisms was similar in the CHD patient group and the control group. The B1B1 genotype of the Taq/B polymorphism was associated with significantly higher TC (P=0.039) and LDL-C (P=0.044) levels than the B2B2 genotype in CHD patients, and with significantly higher LDL-C (P=0.034) levels than the B2B2 genotype in controls. Homozygotes of the I405V polymorphism exhibited significantly higher HDL-C levels than VV homozygotes among control subjects (P=0.023). In male CHD patients with unambiguously assigned haplotypes, B2-M2-V/B2-M2-I patients demonstrated significantly higher HDL-C concentrations than B1-M2-V/B1-M2-I (P=0.023) and B1-M2-V/B1-M2-V patients (P=0.047). CONCLUSIONS: Genetic variations in the CETP gene may account for a significant proportion of the differences in plasma lipid and lipoprotein concentrations among the general population. The B1B1 genotype of the Taq/B polymorphism is probably a genetic risk factor for CHD in the study population.

[Association of APOA5 gene single nucleotide polymorphism with levels of lipids and coronary heart disease in Chinese].

OBJECTIVE: To investigate the single nucleotide polymorphism 4 (SNP4) of the apolipoprotein A5 (APOA5) gene possible association with coronary heart disease(CHD) and its distribution of in Chinese Han population. METHODS: APOA5 SNP4 genotyping was performed using polymerase chain reaction and Hae III restriction fragment length polymorphism analysis. RESULTS: APOA5 allelic frequencies of T, C were 0.435, 0.565 and 0.374, 0.626 in CHD group and control group, respectively. There is significant difference in allele and genotype frequencies between CHD group and control group (P<0.05). The levels of plasma high density lipoprotein in CHD patients with CC genotype were higher than those in CHD patients with other genotypes (P<0.01). The frequencies of T allele and C allele in Chinese was significantly different from those in Caucasians (0.374 vs 0.663, 0.626 vs 0.337, P<0.01). The C allele was much more common in Chinese population. CONCLUSION: The association is found between the Hae III polymorphism and CHD, There is a significant correlation between the CC genotype of the APOA5 and the levels of plasma high density lipoprotein-cholosteal in the CHD group.

A novel missense mutation (L296 Q) in cholesteryl ester transfer protein gene related to coronary heart disease.

Cholesteryl ester transfer protein (CETP) is a key participant in the reverse transport of cholesterol from the peripheral tissues to the liver. To understand further the role that CETP gene plays in the pathogenesis of coronary heart disease (CHD), the promoter region, all 16 exons and adjacent intronic regions of CETP gene were screened for single nucleotide polymorphisms (SNPs) in 203 CHD patients and 209 healthy volunteers by the combination of PCR, denaturing high performance liquid chromatography (DHPLC), molecular cloning, and DNA sequencing. A novel missense mutation in the CETP gene was identified. This mutation (L(296)Q) was caused by a T-to-A conversion at codon 296 of exon 10 which resulted in the replacing of the codon for leucine (CTG) with the codon for glutamine (CAG). Further studies found that there was a significant increase in the mutant allele frequency in the CHD patients compared with that in the controls (0.160 vs. 0.091,cgr;(2) = 9.014, P = 0.003), and the odds ratio to develop CHD was 1.83 for the (296)Q allele carriers vs. (296)LL homozygotes. Statistical analyses also demonstrated that the mutant (296)Q allele carrier patients displayed significantly higher total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) concentrations than noncarrier patients. All these results suggest that the Q(296) mutation in CETP gene was closely related to CHD, and the identification of new mutations in the CETP gene will afford the opportunity to investigate the relationship between CETP gene and CHD.

[Association of 7alpha-hydroxylase gene polymorphism with levels of plasma lipids].

To study the distribution of Eco31I restriction polymorphism in nucleotide -204 of 7alpha-hydroxylase gene(CYP7A1)in Sichuan Han population of China and association of the polymorphism with coronary heart disease(CHD),CYP7A1 genotyping was performed by using PCR-RFLP approach in 183 CHD patients and 101 control subjects. 7alpha-hydroxylase gene allele frequencies of C,A were 0.840 and 0.160 in CHD group and 0.822 and 0.178 in control group,respectively. There was no significant difference in frequencies of allele and genotypes in A-204C polymorphism between CHD group and control group (P>0.05). However, in CHD patients there was significant difference in total cholesterol (TC) levels among CC,CA and AA genotypes (P<0.05) ,and the levels of high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in CHD patients with AA genotype were lower than those in CHD patients with CC and CA genotypes(P<0.05). In control group there was significant difference in TC levels between CC and CA genotypes (P<0.05) . The frequencies of C,A alleles at A-204C polymorphic site were significantly different from those reported in white people(P<0.05). The results indicating that no direct association was found between the A-204C polymorphism and CHD,but there was significant correlation between this polymorphism and the levels of TC ,and there was significant correlation in CHD patient group between this polymorphism and levels of HDL-C and LDL-C.

[Study on the association of cholesteryl ester transfer protein gene mutations with the susceptibility to coronary atherosclerotic heart disease].

OBJECTIVE: To determine the frequencies of 4 mutations of cholesteryl ester transfer protein (CETP) gene in Chinese population and to investigate the association of the mutations with lipid metabolism and the susceptibility to coronary atherosclerotic heart disease (CHD). METHODS: The target fragments of CETP gene were amplified and analyzed by PCR-restriction fragment length polymorphism technique in 209 unrelated control individuals and 203 CHD patients. The test for Hardy-Weinberg equilibrium was performed using HWE program and statistical analysis was implemented in statistical package SPSS. RESULTS: IVS14A and 451Q mutant genes were not found in either control group or patient group. The frequencies of 405V mutant allele were 0.443 and 0.413 in controls and patients, respectively, while 442G mutant gene frequencies were 0.007 and 0.025, respectively. The observed allele frequencies of I405V and D442G mutation were in accord with Hardy-Weinberg equilibrium. The frequency of 442G mutant gene in patients was significantly higher than that in controls (P=0.043). Compared with the CHD patients without D442G mutation, the 442G heterozygous CHD patients exhibited a significant increase in plasma TC and LDL-C concentration (P=0.017; P=0.041). CONCLUSION: IVS14A and 451Q mutants of CETP gene were rare in Chinese population and 442G mutant gene was possibly one of the susceptibility factors to CHD in Chinese.

[Cloning, sequencing, expression, and primary identification of recombinant mouse protein kinase CK2 alpha subunit].

BACKGROUND & OBJECTIVE: Protein kinase CK2 is a highly conserved and ubiquitous eukaryotic serine/threonine kinase that is elevated and can serve as an oncogene in many tumor cells. To further research the structure and function of CK2, this study was designed to construct, express, and preliminarily identify a recombinant expression plasmid which contains the cDNA encoding mouse protein kinase CK2 alpha subunit. METHODS: The aimed cDNA was obtained from NIH 3T3 mouse fibroblasts by RT-PCR. Nde I/BamH I-digested PCR product was directly cloned into pT7-7 expression vector which had been digested by Nde I/BamH I and dephosphorylated by calf intestinal alkaline phosphatase in advance. After E. coli DH5 alpha was transformed with the recombinant DNA by CaCl2 method, transformants were obtained. The positive clones were screened out primarily by gel electrophoresis, and then analyzed by digesting with restriction enzyme. Four positive clones were selected at random and sequenced respectively. The correct recombinant plasmid was transformed into E. coli BL21(DE3) and then expressed by inducing with IPTG. The products were identified with Western blotting. RESULTS: The positive rate of transformants was 100%. The results of restriction analysis indicated that DNA band size of the insert fragment and recombinant plasmid were consistent with theoretically predicated values. The sequencing results showed one of the four clones possessed the cDNA sequence which has no mutation in the processing of PCR, which was termed as pTMCKA. One protein with molecular mass of 42 kDa was overexpressed by inducing with IPTG. The Western blot results confirmed that the recombinant product could specially react with antibody against human CK2 alpha subunit. CONCLUSIONS: The authors have successfully cloned and expressed recombinant mouse protein kinase CK2 alpha subunit in this experiment.