Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells.

Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB) showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

Molecular analysis of thiopurine S-methyltransferase alleles in South-east Asian populations.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. In Caucasians, four variant TPMT alleles have been detected in over 80% of individuals with low or intermediate TPMT activity. The wild-type allele is designated as TPMT*1 and the mutant alleles are designated TPMT*2 through TPMT*8. The frequency of these alleles in different ethnic groups has not been well defined. In this study, one hundred individuals, from each of the Indonesian, Thai and Philippine populations, together with 249 Taiwanese, were analysed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing methods. The results showed that the allelic frequencies of TPMT*3C were 0.6% for Taiwanese and 1% for Filipino, Thai and Indonesian populations, respectively. One Filipino with a Caucasian parent was found to be heterozygous for the TPMT*2 allele. This study provides the first analysis of the allele frequency distribution of all known TPMT mutations in South-east Asian populations.

Interleukin-1beta and interleukin-1 receptor antagonist gene polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis.

OBJECTIVE: Osteoporosis is a common disorder with a strong genetic component. Our aim was to investigate the correlations of the interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms with bone mineral density (BMD) and their relationship to osteoporosis. METHODS: The IL-1beta (promoter and exon 5) and IL-1Ra (intron 2) gene polymorphisms were determined using polymerase chain reaction. BMD of the lumbar spine and proximal femur were measured using dual-energy X-ray absorptiometry. RESULTS: The prevalence of each genotype of the interleukin-1 related genes in the study population was: (1) 14% C/C, 71.5% C/T, and 14.5% T/T in IL-1beta promoter; (2) 95.3% E1/E1 and 4.7% E1/E2 in IL-1beta exon 5; (3) 92.4% I/I, 6.4% I/II, and 1.2% II/II in IL-1Ra intron 2. After adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake, subjects with genotype E1/E2 (n=8) in IL-1beta exon 5 had lower BMD values and a significantly greater risk for osteoporosis (OR 10.6, 95% CI 1.3-83.8) at the lumbar spine when compared with subjects with genotype E1/E1 (n=164) in IL-1beta exon 5. CONCLUSION: The Taq I IL-1beta exon 5 gene polymorphism is associated with reduced BMD and predisposes women to osteoporosis at the lumbar spine, but our results should be interpreted with caution because of the small number of subjects with the unfavorable E1/E2 genotype.

Association of TAP2 gene polymorphisms in Chinese patients with rheumatoid arthritis.

The aim of this study was to investigate the association between the polymorphism of transporters associated with antigen processing ( TAP1/TAP2) genes and rheumatoid arthritis in Chinese patients. A total of 100 RA patients and 99 healthy control subjects were enrolled. Analyses with polymerase chain reaction (PCR) based restrictions were used to identify the polymorphisms of the TAP1 and TAP2 genes, which were mapped on chromosome 6. There was a significant difference in the distribution of the TAP2 gene codon 565 polymorphism frequency between the RA patients and healthy control subjects ( p<0.001). The odds ratio for the risk of the 'A' allele in RA patients was 1.60 (95% CI: 0.82-2.92). No statistical associations in the distribution of the TAP1 gene polymorphism frequency were found between RA patients and controls. There were some physical links found between TAP1/TAP2 gene polymorphism loci. However, there was no linkage observed from TAP1/TAP2 gene polymorphisms and HLA-DRB1*04 between RA patients and healthy controls. We concluded that the TAP2 gene codon 565 'A' allele was associated with RA in Chinese patients in Taiwan. Individuals possessing the 'A' allele had a higher incidence of RA. A lack of association of TAP1 gene polymorphisms between RA patients and healthy individuals was noted. The results of this study provide genetic evidence that TAP2 gene codon 565 polymorphism may play a role in RA.

Interleukin-1 receptor antagonist gene polymorphism in chinese patients with systemic lupus erythematosus.

The purpose of this study was to determine whether IL-1 receptor antagonist (IL-1Ra) gene polymorphism is a marker of susceptibility to or severity of systemic lupus erythematosus (SLE) in Chinese patients. The study included 52 Chinese patients with SLE. One hundred and three unrelated, healthy individuals living in central Taiwan served as controls. From genomic DNA, the polymorphism of the gene for IL-1Ra was typed. Allelic frequencies and carriage rates were compared between SLE patients and controls. The relationship between allelic frequencies and clinical manifestations of SLE was evaluated. We found an increased frequency of IL1RN*2 in the SLE patients compared to normal controls (chi(2) = 4.15, P<0.05), with an odds ratio (of allele frequency) of 2.63 (95% confidence interval 1.00-6.96). The carriage rate of IL1RN*2 was also higher in the SLE patients (6.8% in the controls vs. 17.3% in the SLE patients). We observed increased frequencies of malar rash and photosensitivity among patients with IL1RN*2 (77.8%) compared to patients without the allele (48.8%). However, this difference did not reach statistical significance (chi(2) = 2.51, P = 0.11). This study indicated that the frequency of IL1RN*2 is higher in Chinese SLE patients than in Chinese normal controls in Taiwan. However, there was no association between the frequency of IL1RN*2 and clinical manifestations.

Androgen receptor (AR) gene microsatellite polymorphism in postmenopausal women: correlation to bone mineral density and susceptibility to osteoporosis.

OBJECTIVE: To investigate the correlation of the androgen receptor gene microsatellite polymorphism (CAG trinucleotide repeat polymorphism on exon 1) with bone mineral density and their relationship to osteoporosis in postmenopausal women. STUDY DESIGN: A number of 168 of 477 postmenopausal women were randomly recruited. The androgen receptor gene microsatellite polymorphism was determined using polymerase chain reaction-based microsatellite analysis. Bone mineral density of the lumbar spine and proximal femur was measured using dual-energy X-ray absorptiometry. RESULTS: The AR genotype was classified from "9" to "32" according to the number of CAG trinucleotide repeats they contained to represent "signposts". After adjustment for potential confounding factors such as age, height, weight, years since menopause, and daily calcium intake, subjects with genotype 20+ (n=64) had lower bone mineral density values and a significantly greater risk for osteoporosis (OR 4.2, 95% CI 1.0-17.2) when compared with subjects with genotype 20- (n=104) at the femoral neck. CONCLUSION: The present study suggests that the androgen receptor gene microsatellite polymorphism may be a candidate genetic marker for risk of osteoporosis in postmenopausal women.

No association of vitamin D receptor gene start codon fok 1 polymorphisms in Chinese patients with systemic lupus erythematosus.

OBJECTIVE: To examine whether the start codon Fok I of the vitamin D receptor (VDR) gene polymorphism is a marker of susceptibility to or severity of systemic lupus erythematosus (SLE) in Chinese patients in Taiwan. METHODS: A control group of 90 healthy people and 52 patients with SLE were examined. Using polymerase chain reaction (PCR) based restriction analysis, we evaluated the relationship between Fok I polymorphisms and SLE, where an unexcisable length was determined to be 265 bp (FF), and the 2 fragments measuring 169 bp and 96 bp were determined to be excisable lengths (ff). RESULTS: For the genotype of VDR Fok I start codon polymorphism, there were no statistically significant differences between the 2 groups (chi-squared test, p = 0.07). Additionally, we did not detect any association of VDR genotype with the clinical and laboratory profiles in SLE patients. CONCLUSION: Our results suggest that the vitamin D receptor Fok I start codon polymorphism is not related to patients with SLE in Taiwan.

Lack of association of interleukin-1beta gene polymorphisms in Chinese patients with systemic lupus erythematosus.

The purpose of this study was to examine whether interleukin-1beta (IL-1beta) (promoter and exon 5) gene polymorphisms were markers of susceptibility or clinical manifestation in Chinese patients with systemic lupus erythematosus (SLE). The study included 52 Chinese patients with SLE. In addition, 103 unrelated healthy individuals living in central Taiwan served as control subjects. From genomic DNA, polymorphisms of the gene for IL-1beta promoter and exon 5 were typed. Allelic frequencies were compared between SLE patients and control subjects. The relationship between allelic frequencies and clinical manifestations of SLE was evaluated. No significant differences were observed in the allelic frequencies of the IL-1beta promoter and IL-1beta exon 5 between patients with SLE and healthy control subjects. Additionally, we did not detect any association of IL-1beta promoter and the exon 5 genotype with the clinical and laboratory profiles in SLE patients. The results from the present study suggest that the polymorphisms of the IL-1beta promoter and IL-1beta exon 5 are not related to SLE patients in Taiwan.

Effects of Tamm-Horsfall protein on the protection of MCDK cells from oxalate induced free radical injury.

Renal cell injury and fixed particle formation is one of the theories of urinary stone formation. The exposure of renal epithelial cells to oxalate ions and calcium oxalate monohydrate crystals can cause free radical generation and increase lipid peroxidation. Tamm-Horsfall protein (THP) has a protective effect on the production of free radicals in vitro. We aimed to show that THP (and its deglycosylated products, D-THP) could protect culture cells from free radical injury in vivo as well as the possible mechanism by which this is done. Exposure of Madin-Darby canine kidney (MDCK) cells to Ox resulted in a significant increase in the release LDH, NBT and MDA, as well as an increase in caspase 3 activity, all of which were further elevated when COM crystals were added. With the addition of THP at 500 nM, there was a significant decrease in the release of LDH and the production of MDA and NBT. A decrease in capase 3 activity was observed when 500 nM THP was added to the culture medium that reached 32.7% and 40.4% of inhibition in CaOx+THP and CaOx+COM+THP, respectively. THP decreased the adhesion of COM crystals to the MDCK cells but lost its effect when THP was deglycosylated. The results indicate that both Ox and COM crystals cause the release of LDH, MDA, NBT and increase the activity of capase 3 in MDCK cells. As a free radical scavenger, THP reduces the amount of free radicals and provides significant protection at a critical concentration of 500 nM. The deglycosylated THP decreased the effect of the protection of the MDCK cells from oxalate-induced injury and an increase of adhesion of the COM crystals to the MDCK cells. Therefore, the effects of THP on the protection of oxalate induced radical injury may be partly due to its intact glycosylation and its adhesion to the cell membrane.

Lack of association of tumor necrosis factor alpha gene polymorphism in patients with rheumatoid arthritis in central Taiwan.

The aim of this study was to investigate the association of tumor necrosis factor alpha (TNFalpha) gene promoter polymorphisms in Chinese patients with rheumatoid arthritis (RA) in central Taiwan. A total of 106 RA patients and 253 normal controls were studied. Polymerase chain reaction (PCR)-based restriction analysis was used to identify A/G polymorphism at position 308 in the promoter region of the TNFalpha, which is located at 6q21.3. For the genotype of TNFalpha-308 polymorphism, there was no statistically significant difference between RA patients and normal controls (Fisher's exact test, P=0.82). Additionally, no statistical association in the distribution of TNFalpha-308 polymorphism between rheumatoid factor (RF)-positive and -negative patients was noted. The lack of an association of TNFalpha-308 polymorphism with RA and RF in our study implies that TNFalpha-308 polymorphism cannot serve as a candidate gene marker for screening RA patients in Taiwan.

Vascular endothelial growth factor gene polymorphism is associated with calcium oxalate stone disease.

Growth factor-related genes regulate cell growth, differentiation and apoptosis in the kidney in response to cellular injury. One of the theories of stone formation is that cellular injury, and thus growth factors, play a role. We therefore investigated the association between growth factor genes and calcium oxalate stone disease. The most frequently seen polymorphism of the vascular endothelial growth factor (VEGF) gene is Bst U I C/T, which is located upstream at the -460th nucleotide. Other growth factor-related gene polymorphisms include the cytochrome P450c17alpha enzyme (CYP17) gene MspA I C/T polymorphism at the 5'-UTR promoter region, the epidermal growth factor receptor (EGFR) gene Bsr I polymorphism (A to T) at position 2,073, and the insulin-like growth factor-2 (IGF-2) gene Apa I A/G at exon 9. All four polymorphisms were used as genetic markers in this study in the search for an association between stone disease and growth factor related genes. A normal control group of 230 healthy people, and 230 patients with calcium oxalate stone, were examined. The polymorphism was seen following polymerase chain reaction based restriction analysis. The result revealed a significant difference between normal individuals and stone patients (P=0.0003, Fisher's exact test) in the distribution of the VEGF gene polymorphism as well as an odds ratio of 1.30 (95% confidence interval=0.993-1.715) per copy of the "T" allele. Whereas, the IGF-2, EGFR and CYP17 gene polymorphisms did not reveal a significant association with stone disease. We conclude that the VEGF gene Bst U I polymorphism is a suitable genetic marker of urolithiasis.