OBJECTIVETo study the effect of kallikrein-related peptidase 10 (KLK10) on the proliferation and invasiveness of human tongue cancer cell line Tca8113.

METHODSThe eukaryotic expression vector harboring KLK10 gene (pIRES2-EGFP-KLK10) was transfected in Tca8113 cells and the stable cell lines were selected by G418 screening. The mRNA and protein expression of KLK10 in transfected Tca8113 cells were assayed by RT-PCR and Western blotting, respectively, and the proliferation and invasiveness of the cells were evaluated by MTS cell growth assay and Transwell chamber invasion experiments.

RESULTSA stable Tca8113 cell line with high KLK10 expression was obtained, which showed significantly increased mRNA and protein expression levels of KLK10 and obviously attenuated proliferation and invasiveness compared with control and empty vector-transfected cells (P<0.05).

CONCLUSIONEnhancing KLK10 gene expression can decrease the proliferation and invasiveness of human tongue cancer cells in vitro.

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Kallikreins ; genetics ; Tongue Neoplasms ; genetics ; pathology

There is evidence that a substantial part of genetic predisposition to prostate cancer (PCa) may be due to lower penetrance genes which are found by genome-wide association studies. We have recently conducted such a study and seven new regions of the genome linked to PCa risk have been identified. Three of these loci contain candidate susceptibility genes: MSMB, LMTK2 and KLK2/3. The MSMB and KLK2/3 genes may be useful for PCa screening, and the LMTK2 gene might provide a potential therapeutic target. Together with results from other groups, there are now 23 germline genetic variants which have been reported. These results have the potential to be developed into a genetic test. However, we consider that marketing of tests to the public is premature, as PCa risk can not be evaluated fully at this stage and the appropriate screening protocols need to be developed. Follow-up validation studies, as well as studies to explore the psychological implications of genetic profile testing, will be vital prior to roll out into healthcare.
Genetic Predisposition to Disease ; genetics ; Genetic Testing ; Humans ; Kallikreins ; genetics ; Male ; Membrane Proteins ; genetics ; Prostatic Neoplasms ; diagnosis ; genetics ; Prostatic Secretory Proteins ; genetics ; Protein-Serine-Threonine Kinases ; genetics ; Risk Factors

OBJECTIVETo construct a Gpx1 and klk1 recombinant vector containing the kidney-specific promoter Ksp-cadherin.

METHODSHuman Gpx1, Klk1 and Ksp-cadherin cDNAs were amplified with PCR and inserted in a stepwise manner into the expressive vector pIRES-EGFP to construct the recombinant vector Ksp-cadherin-Gpx1-Klk1. The constructed vector was verified with restriction enzyme digestion and sequence analysis.

RESULTS AND CONCLUSIONThe recombinant expression vector Ksp-cadherin-Gpx1-Klk1 was constructed and identified successfully, which provides a potent tool for preparing transgenic animals to investigate gene therapy for ischemia-reperfusion injury in kidney transplantation.

Cadherins ; genetics ; Cloning, Molecular ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Glutathione Peroxidase ; genetics ; Humans ; Kallikreins ; genetics ; Kidney ; metabolism ; Promoter Regions, Genetic ; genetics

To prolong serum half-life of human kallikrein (hK) and enhance its secretion rate, we modified hK gene and constructed a new form of recombinant hK protein (hK'-Fc). We amplified hK gene and Fc sequence, replaced the signal peptide of hK gene with murine signal peptide, constructed native expression plasmid of pcDNA-hK-Fc and modified expression plasmid of pcDNA-hK'-Fc, then transfected to CHO cells respectively. After the stable cell lines were screened, we compared the secretion rate between native fusion protein and modified fusion protein, purified fusion protein through Protein A+G affinity chromatography column and investigated the bioactivity of fusion protein. The results showed that recombinant vectors encoding fusion protein hK-Fc and hK'-Fc were constructed successfully; CHO cell lines stably secreting fusion protein were obtained, the yield is higher than 11 mg/L; Secretion rate was enhanced by 5-10 times after the signal peptide of fusion protein was modified; Fusion protein has enzymatic activity in vitro. The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy.
Animals ; CHO Cells ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cricetinae ; Cricetulus ; Humans ; Mice ; Protein Sorting Signals ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; physiology ; Tissue Kallikreins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo study the expression of the kallikreins-kinins system in the corpus cavernosum of rats.

METHODSThe expression of tissue kallikrein I and kinin B2 receptor gene in the corpus cavernosum and heart of adult rats was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThe tissue kallikrein I and kinin B2 receptor were detected in the corpus cavernosum as well as in the heart of the rats and the contents were similar.

CONCLUSIONA kallikreins-kinins system exists in the corpus cavernosum of rats, and the content is rich, almost similar to that in the heart.

Animals ; Male ; Myocardium ; metabolism ; Penis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptor, Bradykinin B2 ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Kallikreins ; biosynthesis ; genetics

OBJECTIVE: To explore the association between single nucleotide polymorphism (SNPs) of KLK1 gene and cerebral hemorrhage in Changsha Han population. METHODS: We enrolled 273 patients with cerebral hemorrhage and 140 normal people. The SNPs (including rs3212855 and rs5515) of KLK1 gene were analyzed by Snapshot method and direct sequencing. RESULTS: We found rs5515 was not a polymorphic site in Changsha Han population. Genotype and allele frequency in rs3212855 were not different between patients with cerebral hemorrhage and the controls (P>0.05). The blood pressure level was not different between the genotype subgroups. CONCLUSION Neither rs5515 nor rs3212855 is associated with cerebral hemorrhage.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cerebral Hemorrhage ; genetics ; China ; ethnology ; Female ; Genotype ; Humans ; Kallikreins ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Young Adult

We studied the influence of the first intron and 3'-regulatory region of ovalbumin gene (ov) on oviduct-specific transgene expression. The 3'-regulatory region in the oviduct-specific expression vector containing human tissue kallikrein (hK1) cDNA was replaced with bovine growth hormone (BGH) poly A, and the first intron was deleted by restriction enzyme digestion, resulting in five new vectors pOV2K, pOV3K, pOV4K, pOV5K and pOV6K. After mixing with polyethylenimine, we injected same copies of the five vectors via wing vein route into laying hens and compared their expression levels by quantitative assay for enzymatic activities in the egg whites. Among the five vectors tested, the pOV2K containing both the 5'- and 3'-regulatory regions expressed highest level of rhK1 activity, followed by pOV3K with the 3'-regulatory region replaced with BGH poly A, and then by the first intron-shortened vectors pOV4K, pOV5K and pOV6K. These data suggest that both the first intron and 3'-regulatory region of ov gene have enhancing effect on transgene expression in oviduct cells, which should be included in oviduct-specific expression vectors.
Animals ; Animals, Genetically Modified ; Cattle ; Chickens ; Cloning, Molecular ; Female ; Gene Transfer Techniques ; Growth Hormone ; genetics ; Humans ; Introns ; genetics ; Ovalbumin ; genetics ; Oviducts ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Regulatory Sequences, Nucleic Acid ; genetics ; Tissue Kallikreins ; genetics ; Transgenes ; genetics

OBJECTIVETo investigate the effects of human tissue kallikrein 1(Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs).

METHODSCarotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 microl, n = 8); Vector virus (control virus, 1 x 10(9) IU in 50 microl, n = 8) and Ad-hKLK1(Ad-hKLK1, 1 x 10(9) IU in 50 microl, n = 8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor (B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p2lCip1 were determined by Western blot analysis.

RESULTSWall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8%in Ad-hKLK1 treated SHRs compared to angioplasty group (all P < 0.001). The expression of p27Kip1 and p2lCip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P < 0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P < 0.05) while mRNA expression of B1R was similar between the 2 groups.

CONCLUSIONhKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p2lCip1 signaling pathways in carotid arteries of SHRs after balloon injury.

Angioplasty, Balloon ; adverse effects ; Animals ; Carotid Artery, Common ; pathology ; Gene Transfer Techniques ; Humans ; Male ; Neointima ; etiology ; Rats ; Rats, Inbred SHR ; Tissue Kallikreins ; genetics

OBJECTIVETo explore the association between single nucleotide polymorphisms (SNPs) of KLK1 gene and cerebral hemorrhage in Changsha Han Chinese.

METHODSTwo hundred and seventy-three cerebral hemorrhage (CH) patients and 140 healthy controls were collected. The SNPs of rs5516 and rs5517 loci of KLK1 gene were analyzed by SNaPshot methods and direct sequencing.

RESULTS(1)Genotype and allele frequencies in rs5516 locus had no difference between the CH patients and controls (P> 0.05). However, the A allele frequency of the rs5517 locus in CH patients was higher than that in the control group (0.419, 0.321 respectively, P< 0.05). (2)In the control group,the levels of diastolic blood pressure (DBP) of the GA and AA genotype carriers of the rs5517 locus were significantly higher than those of the GG genotype (P< 0.05), while the levels of blood pressure were not significantly different among different genotypes of the rs5516 polymorphism in both CH patients and the control group(P> 0.05).

CONCLUSIONAuthor's preliminary results suggested that the rs5517 polymorphism was associated with cerebral hemorrhage, while the rs5516 polymorphism was not in Changsha Han Chinese.

Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Cerebral Hemorrhage ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Tissue Kallikreins ; genetics

Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZalphaA, then the yeast expression vector pPICZalpha-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 microg/mL) of YPD plate and medium. When temperature was 30 degrees C, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L (1.25 g/L). The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1-H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu(2+)-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of > 96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.
Amino Acid Sequence ; Base Sequence ; Chromatography, Ion Exchange ; methods ; Genetic Vectors ; genetics ; Humans ; Kidney ; metabolism ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Tissue Kallikreins ; biosynthesis ; genetics