The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
Animals ; Apoptosis ; Diet ; Endothelial Cells ; Erectile Dysfunction/prevention & control* ; Fibrosis ; Humans ; Hyperhomocysteinemia/complications* ; Male ; Methionine ; Oxidative Stress ; Penis/pathology* ; Rats ; Rats, Sprague-Dawley ; Rats, Transgenic ; Signal Transduction/genetics* ; Tissue Kallikreins/genetics*

In 2011, a number of studies shed further light on the understanding of stroke etiologies and mechanism. Regarding eating habit and a risk of stroke, three studies from Swedish cohort showed independent relations of chocolate, red meat, and coffee consumption with a risk of stroke. In relation to biomarkers, plasma tissue Kallikrein and resistin levels have been shown to be associated with a risk of stroke. In addition, with regard to infection and stroke, one study reported an increasing incidence of HIV infection in stroke patients and another study demonstrated that new-onset atrial fibrillation among patients with severe sepsis is associated with an increased risk of stroke. Finally, European consortium of cervical artery dissection reported risk factors for cervical artery dissection and differences between carotid and verterbral artery dissections.
Arteries ; Atrial Fibrillation ; Biomarkers ; Cacao ; Coffee ; Cohort Studies ; Eating ; HIV Infections ; Humans ; Incidence ; Light ; Meat ; Plasma ; Resistin ; Risk Factors ; Sepsis ; Stroke ; Tissue Kallikreins

OBJECTIVE: To determine the predictive accuracy of the combined panels of serum human tissue kallikreins (hKs) and CA-125 for the detection of epithelial ovarian cancer. METHODS: Serum specimens collected from 5 Indonesian centers and 1 Vietnamese center were analyzed for CA-125, hK6, and hK10 levels. A total of 375 specimens from patients presenting with ovarian tumors, which include 156 benign cysts, 172 epithelial ovarian cancers (stage I/II, n=72; stage III/IV, n=100), 36 germ cell tumors and 11 borderline tumors, were included in the study analysis. Receiver operating characteristic analysis were performed to determine the cutoffs for age, CA-125, hK6, and hK10. Sensitivity, specificity, negative, and positive predictive values were determined for various combinations of the biomarkers. RESULTS: The levels of hK6 and hK10 were significantly elevated in ovarian cancer cases compared to benign cysts. Combination of 3 markers, age/CA-125/hk6 or CA-125/hk6/hk10, showed improved specificity (100%) and positive predictive value (100%) for prediction of ovarian cancer, when compared to the performance of single markers having 80-92% specificity and 74-87% positive predictive value. Four-marker combination, age/CA-125/hK6/hK10 also showed 100% specificity and 100% positive predictive value, although it demonstrated low sensitivity (11.9%) and negative predictive value (52.8%). CONCLUSION: The combination of human tissue kallikreins and CA-125 showed potential for improving prediction of epithelial ovarian cancer in patients presenting with ovarian tumors.
Asian Continental Ancestry Group ; Humans ; Neoplasms, Germ Cell and Embryonal ; Neoplasms, Glandular and Epithelial ; Ovarian Neoplasms ; ROC Curve ; Sensitivity and Specificity ; Tissue Kallikreins ; Biomarkers, Tumor

OBJECTIVETo investigate the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene transfer on platelet-derived growth factor-BB (PDGF-BB)-induced migration of vascular smooth muscle cells from spontaneously hypertensive rats (VSMC(SHR)).

METHODSA bicistronic recombinant adenovirus vector (Ad-hKLK1) carrying the target hKLK1 gene and the reporter gene EGFP was constructed. VSMCs isolated from the thoracic aorta of male SHR were passaged, and the quiescent VSMC(SHR) in passages 3-6 seeded in 6-well plates were treated with Ad-hKLK1 and control virus. Human PDGF-BB or icatibant Hoe140, a BK B2 antagonistat, was used as the chemoattractant and placed in the bottom chamber of the Boyden chamber. The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMC(SHR).

RESULTShKLK1 gene transfer significantly inhibited PDGF-BB-induced migration of VSMC(SHR), with the peak inhibition rate of 34.6% (P<0.001). PDGF-BB significantly increased the mRNA expression of B2 receptor but not B1 receptor in VSMC(SHR).

CONCLUSIONShKLK1 gene transfer can inhibit the migration of VSMC(SHR) induced by PDGF-BB, and the inhibitory effects may be not mediated by bradykinin B2 receptor.

Adenoviridae ; genetics ; metabolism ; Animals ; Aorta, Thoracic ; cytology ; Cell Movement ; drug effects ; genetics ; Cells, Cultured ; Gene Transfer Techniques ; Humans ; Hypertension ; pathology ; Male ; Muscle, Smooth, Vascular ; cytology ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Inbred SHR ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Tissue Kallikreins ; biosynthesis ; 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

OBJECTIVETo evaluate the effects of urinary kallidinogenase on subarachnoid hemorrhage (SAH) in rabbits.

METHODSRabbits symptomatic cerebral vasospasm model was built though Endo method, among the 40 rabbits, 8 died or had severe nervous system syndrome, the other 32 were randomly divided into 4 groups:group A, control group, injection of normal saline to the cisterna magna;group B, subarachnoid hemorrhage;group C, injection of human urinary tissue kallikreins;group D, treated with Nimodipine. The behavior scores, neurological scores and cerebral angiography changes were observed.

RESULTSFood intake obviously decreased and neurological deficit were seen in group B, while which were attenuated in group C and group D, and group A was normal. Comparing the diameter of basilar artery was (1.9 +/- 0.3) mm before SAH, the diameter of group B 4 d later was (1.5 +/- 0.3) mm, 7 d later (1.4 +/- 0.3) mm, the difference was significant (P < 0.05). Comparing with group C on the day 4th and 7th, the diameters of basilar artery were significantly different (P < 0.001). Comparing with group D on the day 4th, 7th and 14th, there was no obvious improvement.

CONCLUSIONUrinary kallidinogenase and Nimodipine can obviously alleviate symptomatic cerebral vasospasm in rabbits remarkably, but the former's effect of attenuating vasospasm is better than that of Nimodipine.

Animals ; Disease Models, Animal ; Female ; Humans ; Male ; Nimodipine ; therapeutic use ; Rabbits ; Random Allocation ; Tissue Kallikreins ; therapeutic use ; Vasodilator Agents ; therapeutic use ; Vasospasm, Intracranial ; drug therapy

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

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

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 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