Age-dependent dichotomous effect of superoxide dismutase Ala16Val polymorphism on oxidized LDL levels

We investigated the association between superoxide dismutase (SOD) Ala16Val polymorphism and the levels of oxidized LDL lipoprotein-C (ox-LDL-C) in two age-different Greek cohorts. Four hundred fifteen middle-aged (n=147 females: 43.2+/-13 years, n=268 males: 43.3+/-14 years) Caucasian Greek subjects consisted the middle aged cohort. One hundred seventy five elderly (n=88 females: 79.9+/-4 years; n=87 males: 80.6+/-4 years) were selected from the elderly cohort. Genotype data were obtained for all of them. Multiple linear regression analysis, stratified by gender and adjusted for age, smoking habits and body mass index as covariates, showed higher ox-LDL-C levels for the middle aged men with the Val/Val genotype, compared to the other allele (Ala/Ala and Ala/Val) carriers (65.9+/-25.7 vs. 55.7+/-20.5 mg/dl; standardized beta coefficient=0.192, P=0.012). On the contrary, elderly women with the Val/Val genotype occurred with lower ox-LDL-C levels compared to the Ala/Ala or Ala/Val genotype (74.2+/-22.1 vs. 86.5+/-26.6 mg/dl; standardized beta coefficient= -0.269, P=0.015). The same trend was also recorded in elderly men, however without reaching statistical significance (standardized beta coefficient= -0.187, P=0.077). Moreover, elderly men and women with the Ala/Ala or Ala/Val genotype presented higher triglycerides levels compared to Val/Val (women: 145.2+/-68.7 vs. 114.3+/- 34.3 mg/dl, P= 0.027; men: 147.8+/-72.4 vs. 103.7 +/-38.0 mg/dl, P=0.002). Additionally, middle aged men with the Val/Val genotype had higher HDL-C levels compared to the Ala allele carriers. The results suggest that SOD Ala16Val polymorphism is an age-dependent modulator of ox-LDL-C levels in middle-aged men and elderly women.     

Association of ADIPOR1 polymorphisms with bone mineral density in postmenopausal Korean women

Adiponectin may affect bone through interactions with two known receptors, adiponectin receptors (ADIPOR) 1 and 2. We examined the association between polymorphisms of ADIPOR1 and ADIPOR2 and bone mineral density (BMD) in postmenopausal Korean women. Six polymorphisms in ADIPOR1 and four polymorphisms in ADIPOR2 were selected and genotyped in all study participants (n = 1,329). BMD at the lumbar spine and femur neck were measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment and the occurrence of non-vertebral fractures examined using self-reported data. P values were adjusted for multiple testing using Bonferroni correction (P(corr)). ADIPOR1 rs16850799 and rs34010966 polymorphisms were significantly associated with femur neck BMD (P(corr) = 0.036 in the dominant model; P(corr) = 0.024 and P(corr) = 0.006 in the additive and dominant models, respectively). Subjects with the rare allele of each polymorphism had lower BMD, and association of rs34010966 with BMD showed a gene dosage effect. However, ADIPOR2 single nucleotide polymorphisms and haplotypes were not associated with BMD at any site. Our results suggest that ADIPOR1 polymorphisms present a useful genetic marker for BMD in postmenopausal Korean women.     

Lycopene effects on serum mineral elements and bone strength in rats

This study investigated the beneficial effect of lycopene on bone biomarkers in ovariectomized (OVX) rats. Female Wistar rats were either sham operated or surgically ovariectomized and then fed with lycopene for 8 weeks. Serum Ca, P, alkaline phosphatase (ALP), interleukin 6 (IL-6) and bone gla protein (BGP) concentration was significantly higher in the untreated OVX group compared with that of the sham group, whereas serum estrogen levels were lower. Bone mineral density (BMD), BMD/wt, bone mineral content (BMC), BMC/wt values, maximum load, stiffness, energy and maximum stress were significantly lower in the untreated OVX group compared with that of the sham group. Administration of lycopene (20, 30 and 40 mg/kg b.w.) for 8 weeks significantly decreased serum Ca, P, ALP, and IL-6 concentration, and enhanced serum estrogen level, BMD, BMD/wt, BMC, BMC/wt values, maximum load, stiffness, energy and maximum stress in lycopene-treated OVX groups. In conclusion, the consumption of lycopene may have the most protective effect on bone in OVX rats.     

No association of prion protein gene polymorphisms with Alzheimer's disease in Korean population

The polymorphism at codon 129 (M129V) of the human prion protein gene (PRNP) is a known risk factor for Creutzfeldt-Jakob disease (CJD) in Caucasians. There are few reports of this polymorphism's effect on memory and on the risk of Alzheimer's disease (AD). The M129V genotype distributions among Asians are very different from Caucasians. Another polymorphism, codon 219 (E219K) is not found in Caucasians. We investigated two polymorphisms of PRNP, M129V (rs1799990) and E219K (rs1800014) in 297 Korean AD patients and 217 healthy subjects. The analysis of the genotype and allele distributions showed no significant difference between the AD patients and the controls in both polymorphisms (P=0.19 genotype, P=0.51 allele for M129V; P=0.64 genotype, P=0.50 allele for E219K). Also, the PRNP polymorphisms were not significantly associated with AD when the populations were stratified for the presence or absence of apolipoprotein E-epsilon4 (ApoE-epsilon4) allele. These results suggest that the PRNP genetic variants are not associated with the risk for AD in Korean population.     

Evaluation of a Apo-1/Fas promoter polymorphism in Korean stroke patients

Apoptosis has been implicated in the pathogenesis of neurodegenerative diseases such as stroke and Alzheimer's disease. Apo-1/Fas gene is one of the mediators of apoptosis in stroke. Mval polymorphism is the first polymorphic marker identified in the Apo-1/Fas gene promoter, which was typed by PCR and followed by Mval digestion and gel electrophoresis. DNA isolated from peripheral blood collected from 91 stroke patients and 103 healthy blood donors was used for genotypes of GG, GA and AA by sequence specific primer PCR. Mval polymorphism was examined based on Fas gene promotor region by restriction fragment length polymorphism (RFLP). The Fas-GG genotype was the least frequent in patients with stroke and healthy controls (P = 0.57). In normal Korean controls the Mval polymorphism GA, AA and GG were 48.6%, 34.9% and 16.5%. In stroke patients were 56.2%, 29.6% and 14.2% respectively. And the allelic frequencies of Mval*2 (G) allele were less frequent than Mval*1 (A) allele in patients with stroke and healthy controls (P = 0.76). In normal Korean controls Mval*1 (A) and Mval*2 (G) alleles were 59.2% and 40.8%. In stroke patients were 57.6% and 42.4%, respectively. Our results, pending confirmation in a larger study, indicate that the Fas genotype may not appear to be a risk factor for stroke in Korean stroke patients.     

Association of Paraoxonase 1 (PON1) polymorphisms with osteoporotic fracture risk in postmenopausal Korean women

There is increasing evidence of a biochemical link between lipid oxidation and bone metabolism. Paraoxonase 1 (PON1) prevents the oxidation of low-density lipoprotein (LDL) and metabolizes biologically active phospholipids in oxidized LDLs. Here, we performed association analyses of genetic variation in PON1 to ascertain its contribution to osteoporotic fractures (OFs) and bone mineral density (BMD). We directly sequenced the PON1 gene in 24 Korean individuals and identified 26 sequence variants. A large population of Korean postmenopausal women (n=1,329) was then genotyped for eight selected PON1 polymorphisms. BMD at the lumbar spine and femoral neck was measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment, and the occurrence of non-vertebral fractures (i.e., wrist, hip, forearm, humerus, rib, and pelvis) was examined using self-reported data. Multivariate analyses showed that none of the polymorphisms was associated with BMD at either site. However, +5989A>G and +26080T>C polymorphisms were significantly associated with non-vertebral and vertebral fractures, respectively, after adjustment for covariates. Specifically, the minor allele of +5989A>G exerted a highly protective effect against non-vertebral fractures (OR=0.59, P=0.036), whereas the minor allele of +26080T>C was associated with increased susceptibility to vertebral fractures (OR=1.73, P=0.020). When the risk for any OFs (i.e., vertebral or non-vertebral) was considered, the statistical significance of both polymorphisms persisted (P=0.002-0.010). These results suggest that PON1 polymorphisms could be one of useful genetic markers for OF risk in postmenopausal women.     

Association of transforming growth factor-beta1 gene polymorphisms with a hepatocellular carcinoma risk in patients with chronic hepatitis B virus infection

Transforming growth factor-beta1 (TGF-beta1) can act as both a tumor suppressor and a stimulator of tumor progression. We have examined the relationship between polymorphisms of the TGF-beta1 gene and the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection. A total of 1,237 Korean subjects were prospectively enrolled; 1,046 patients with chronic HBV infection and 191 healthy controls with no evidence of recent or remote HBV infection. The patients were divided into two groups: those without (n = 809) and those with HCC (n = 237). Single nucleotide polymorphisms (SNPs) of TGF-beta1 were searched for and genotyped using the single base extension method. In Korean subjects, only two SNPs were found among the seven known polymorphisms of TGF-beta1, at position -509 and in codon 10. The risk of HCC was significantly lower in patients with the T/T or C/T genotypes than in those with the C/C genotypes at position -509 (P < 0.02), and also lower among those with the Pro/Pro or Leu/Pro genotypes than in those with the Leu/Leu genotypes in codon 10 (P < 0.007). Haplotype analysis revealed that the possession of [-509C > T; L10P] conferred a decreased likelihood of HCC (OR = 0.74; 95% CI, 0.59-0.93; P = 0.008). In conclusion, the presence of the TGF-beta1 -509C > T promoter or of the L10P polymorphism, and the combination of both [-509C > T; L10P] as a haplotype were strongly associated with a reduced risk of HCC in patients with chronic HBV infection.     

Methionine synthase reductase polymorphisms are associated with serum osteocalcin levels in postmenopausal women

Homocysteine (Hcy) is thought to play an important role in the development of osteoporosis and fracture. Methionine synthase reductase (MTRR) is an enzyme involved in the conversion of Hcy to methionine. We hypothesized that certain genetic polymorphisms of MTRR leading to reduced enzyme activity may cause hyperhomocysteinemia and affect bone metabolism. We therefore examined the associations of the A66G and C524T polymorphisms of the MTRR gene with bone mineral density (BMD) and serum osteocalcin levels in postmenopausal women. Although we did not detect any significant associations between MTRR polymorphisms and BMD or serum osteocalcin levels, we found that the 66G/524C haplotype, which has reduced enzyme activity, was significantly associated with serum osteocalcin levels in a gene-dose dependent manner (P = 0.002). That is, the highest osteocalcin levels (34.5 +/- 16.8 ng/ml) were observed in subjects bearing two copies, intermediate osteocalcin levels (32.6 +/- 14.4 ng/ml) were observed in subjects bearing one copy, and the lowest levels of osteocalcin (28.8 +/- 10.9 ng/ml) were observed in subjects bearing no copies. These results suggest that the 66G/524C haplotype of the MTRR gene affect bone turn over rate.     

Analysis of the variations in IL-28RA gene and their association with allergic rhinitis

IL-28RA is one of the important candidate genes for complex trait of genetic diseases, but there is no published information of the genetic variation in this gene. We scanned the seven exons and their boundary introns sequence of IL-28RA including the promoter regions to analyze genetic variation sites, and identified eighteen single nucleotide polymorphisms (SNPs) and two variation sites. We chose seven SNPs (g.-1193 A>C, g.-30 C>T, g.17654 C>T, g.27798 A>G, g.31265 C>T, g.31911 C>T and g.32349 G>A) of them for large sample size genotyping, and assessed the association of genotype and allele frequencies of these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. We also compared the genotype frequencies between Korean controls and Han Chinese control or Korean Chinese control. We investigated the frequencies of haplotype constructed by these SNPs between allergic rhinitis patients and non-allergic rhinitis controls. Our results suggested that the g.32349 G>A polymorphism of IL-28RA might be associated with susceptibility to allergic rhinitis (P=0.032), but seems to have no relationship with serum total IgE levels. The haplotype frequencies by these SNPs also show significant association between controls and allergic rhinitis patients.     

胰凝乳蛋白酶显色底物的合成及动力学

胰凝乳蛋白酶是一类重要的蛋白水解酶,在其纯化及应用时常常需要进行酶活性的测定;现在一般应用的底物存在溶解度不好、不稳定、灵敏度低或需要特殊的仪器等问题。本文设计合成了一类以对硝基苯胺为显色基团的底物,通过动力学测定研究了氨基酸结构对专一性的影响,该类显色底物可以较好地用于胰凝乳蛋白酶活性测定。     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Clinical evaluation of osteocalcin in sera of the patients with neoplasms of bone

The clinical significance of osteocalcin in sera of bone tumors were measured in sera of various kinds of bone tumors. The levels of osteocalcin were higher in bone tumors than those in normal subjects. Serum osteocalcin levels in hypercalcemic groups of bone metastasis and myeloma showed a remarkable increase in a parallelism with serum ALP and LDH levels. These results suggested to be directly or indirectly activated on osteoblast by bone tumors and to be synthesized osteocalcin in osteoblast.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression

The mechanism of inhibitory action of bisphosphonates on bone resorption is not fully elucidated. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-kappaB ligand (RANKL) and the inhibitor, osteoprotegerin (OPG). To investigate in vitro effects of bisphosphonates on mouse osteoblastic cells, we examined the expression levels of RANKL and OPG in the cells treated with alendronate or pamidronate (10(-8) approximately 10(-5) M) alone, or combined with 10 nM of 1,25-(OH)2VitD3 for 24 or 48 h. Various concentrations of alendronate and pamidronate did not change the mRNA expression of RANKL and OPG consistently irrespective of 1,25-(OH)2VitD3 presence. When added into cocultures of mouse osteoblastic cells and bone marrow cells, both alendronate and pamidronate inhibited osteoclast formation and bone resorption but failed to alter the RANKL and OPG mRNA expression. These results indicate that the inhibition of bone resorption by bisphosphonates is not mediated by the regulation of RANKL and OPG expression.     

UPLC–Q‐TOF/MS characterization of efficacy substances on osteoblasts differentiation and function in rat serum after administration of Wang‐Bi tablet

Wang‐Bi tablet (WB) is popularly used for the treatment of rheumatoid arthritis. However, few studies have been carried out on its active ingredients and mechanism. In this study, the effect of WB medicated serum on the changes in differentiation and function in osteoblast was investigated, the results showed that WB induced the production of ALP and mineralized nodules to promote the final maturation of osteoblasts and enhance the function of osteoblasts. The potential mechanism may that WB significantly inhibits gene expressions of RANKL and miR‐141, up‐regulates the gene expressions of RUNX2 and OPG, decreases expression of DKK‐1 and increases levels of β‐catenin protein to promote the activation of Wnt/β‐catenin signaling pathways, which enhances osteogenesis and bone repair function. To investigate which compounds contributed to the activity and mechanisms, a total of 138 compounds were characterized from WB, and 13 parent molecules and eight metabolites in rat serum were rapidly characterized by UPLC–Q‐TOF/MS. Total glycosides of paeony, loganin, α‐linolenic acid, linoleic acid and naringin from WB may contribute to the actions on osteoblasts according to our study and literature review. Our research provides a method to explore the bioactive ingredients and action mechanisms of WB.     

Genotyping the -6A/G functional polymorphism in the core promoter region of angiotensinogen gene by microchip electrophoresis

Angiotensinogen (AGT) gene has been regarded as one of the candidate genes for essential hypertension. In our study, the role of AGT gene as a putatively predisposing gene for hypertension was evaluated by genotyping a A (-6) G polymorphism in the core promoter region in 123 patients with essential hypertension and 103 healthy controls. A microchip electrophoresis method coupled with polymorphism chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used for genotyping the A (-6) G single-nucleotide polymorphism. The separation and detection of the digested PCR amplicons were completed just in 280 s or less. The genotype frequency fulfilled the criteria of the Hardy-Weinbery equilibrium (X2 = 3.067, P > 0.05). The results showed a higher frequency of the -6 A allele (0.70) in the normotensive subjects, which is higher than those reported in Germany (0.47) and Czech (0.40) populations, but similar to that found in Japanese populations (0.73). The frequencies of genotype AA, AG, and GG were 0.46, 0.49, and 0.05 in hypertensive subjects, and 0.44, 0.53, and 0.03 in control subjects. There is no significant difference in the distributions of the genotype and allele between the two groups (X2 = 0.88, P > 0.05; X2 = 0.024, P > 0.05). These findings differ from some of the results obtained in other ethnic groups, indicating the potential importance of ethnic origin in the assessment of genetic risk identifiers for a complex disease.     

Studies on Synthesis of a Cycloheptapeptide and Effects of Different Metal Ions on the Cyclization

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Single-Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single-molecule-analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.