Effects of diallyl trisulfide on induction of apoptotic death in murine leukemia WEHI-3 cells in vitro and alterations of the immune responses in normal and leukemic mice in vivo.

Diallyl trisulfide (DATS), a chemopreventive dietary constituent and extracted from garlic, has been shown to against cultured many types of human cancer cell liens but the fate of apoptosis in murine leukemia cells in vitro and immune responses in leukemic mice remain elusive. Herein, we clarified the actions of DATS on growth inhibition of murine leukemia WEHI-3 cells in vitro and used WEHI-3 cells to generate leukemic mice in vivo, following to investigate the effects of DATS in animal model. In in vitro study, DATS induced apoptosis of WEHI-3 cells through the G0/G1 phase arrest and induction of caspase-3 activation. In in vivo study DATS decreased the weight of spleen of leukemia mice but did not affect the spleen weight of normal mice. DATS promoted the immune responses such as promotions of the macrophage phagocytosis and NK cell activities in WEHI-3 leukemic and normal mice. However, DATS only promotes NK cell activities in normal mice. DATS increases the surface markers of CD11b and Mac-3 in leukemia mice but only promoted CD3 in normal mice. In conclusion, the present study indicates that DATS induces cell death through induction of apoptosis in mice leukemia WHEI-3 cells. DATS also promotes immune responses in leukemia and normal mice in vivo.

High prevalence of Mycoplasma pneumoniae in hepatitis C virus-infected hemodialysis patients.

BACKGROUND: This study investigates the seroprevalence of mycoplasma pneumonia (M. pneumoniae) in patients with hepatitis C viral (HCV) infection undergoing hemodialysis (HD). METHODS: One hundred and nine HD patients were randomly selected from a medical center in Taiwan. Among the subjects, 42 patients (aged 60.93 +/- 13.94 years) had HCV infection and 67 patients (aged 64.87 +/- 13.02 years) did not have HCV infection. All subjects were analyzed for serum antibodies to HCV and M. pneumonia. Biochemical testing of liver function included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and gamma-glutamyl transferase. RESULTS: Seroprevalence of M. pneumoniae (p = 0.023) and levels of AST (p = 0.006) and ALT (p < 0.001) were significantly higher for HCV seropositive HD patients than for HCV seronegative HD patients. HCV seropositive HD patients had a significantly increased risk of infection with M. pneumoniae (ORs, 2.475; 95% CIs, 1.099 - 5.571) and high ALT level (HAL) (ORs, 5.020; 95% CIs, 1.250 - 20.164, p = 0.018). The serum AST and ALT levels of HD patients with HCV and M. pneumoniae infection were significantly higher than those of non-HCV and non-M. pneumoniae infected HD patients (p = 0.017 and p = 0.001, respectively). HAL were observed significantly in HCV infection and M. pneumoniae co-infection HD patients (p = 0.023). CONCLUSIONS: This study demonstrates that high M. pneumoniae infection in HCV-infected HD patients and that it was a factor exacerbating liver dysfunction.

Bilirubin concentrations in thalassemia heterozygotes in university students.

OBJECTIVES: To investigate the difference of bilirubin concentrations between α- and ß-thalassemia carriers and the role of variation status in the UDP-glucuronosyltransferase (UGT) 1A1 gene on such a difference. METHODS: A total of 2713 university freshmen who attended a regular physical examination were enrolled in underwent screenings for thalassemias. Finally, 123 subjects whose mean corpuscular volume was ≤80 fL and who had no iron deficiency anemia were tested by PCR and PCR-restriction fragment length polymorphism (RFLP) for α- and ß-thalassemias, respectively, and tested by PCR-RFLP for the five known variations of the UGT1A1 gene. RESULTS: Among the 123 subjects, 76 and 47 were diagnosed with heterozygous α-thalassemia and with heterozygous ß-thalassemia, respectively. Between the α- and ß-thalassemia heterozygotes, variation status of the UGT1A1 gene was not statistically different (P = 0.898), while hemoglobin and bilirubin concentrations differed significantly (P = 0.005 and 0.001, respectively). Bilirubin concentrations were significantly higher among individuals with compound heterozygous variations/homozygous variation in the UGT1A1 gene than in those possessing the wild type and heterozygous variation (P < 0.001 for both α- and ß-thalassemia heterozygotes). Compound heterozygous variations/homozygous variation in the UGT1A1 gene and anemia were the main causes of hyperbilirubinemia in α- and ß-thalassemia heterozygotes, respectively. CONCLUSIONS: The difference in bilirubin concentrations between α- and ß-thalassemia heterozygotes may be attributable to more bilirubin being produced in ß-thalassemia heterozygotes than in α-thalassemia heterozygotes, while variation status of the UGT1A1 gene affects bilirubin concentrations in both α- and ß-thalassemia heterozygotes.

Danthron induces DNA damage and inhibits DNA repair gene expressions in GBM 8401 human brain glioblastoma multiforms cells.

Our primary studies had shown that danthron induced cytotoxic effects, including apoptosis and inhibition of migration and invasion. However, danthron-affected DNA damage and repair gene expressions are not clear. In this study, we investigated to examine whether or not danthron induced DNA damage and inhibited DNA repair gene expression in human brain glioblastoma multiforms (GBM 8401) cells. The results from Comet assay indicated that incubation of GBM 8401 cells with 0, 50, 100 and 150 microM of danthron led to a longer DNA migration smear based on the single cell electrophoresis (Comet tail). The results from real-time PCR assay demonstrated that 100 microM of danthron for 24 h treatment in GBM 8401 cells led to decrease all examined ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA-1), 14-3-3 proteins sigma (14-3-3sigma), DNA-dependent serine/threonine protein kinase (DNA-PK) and O ( 6 )-methylguanine-DNA methyltransferase (MGMT) mRNA expressions. Taken together, the present study showed that danthron caused DNA damage and inhibited DNA repair genes, which may be the factors for danthron-inhibited cell growth in vitro.

Danthron induced apoptosis through mitochondria- and caspase-3-dependent pathways in human brain glioblastoma multiforms GBM 8401 cells.

Danthron (1,8-dihydroxyanthraquinone), is one of component from Rheum palmatum L. (Polygonaceae), has been shown several biological activities but did not show to induce apoptosis in human brain tumor cells. The aim of this study is to investigate the mechanisms by danthron for the induction of apoptotic potential on human brain glioblastoma multiforms GBM 8401 cell line. Danthron showed a marked concentration- and time-dependent inhibition of GBM 8401 cell viability and induced apoptosis in a dose-and time-dependent manner. There was an attenuation of mitochondrial membrane potential (DeltaPsi(m)) with the alterations of Bcl-2/Bax protein ratio in GBM 8401 cells, indicating the participation of a mitochondria-related mechanism. Pretreatment of a caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVE-FMK) significantly increased the viable of GBM 8401 cells implied that the participations of caspases. Western blotting analysis also showed the activation of initiator caspase-8 and caspase-9, and executor caspase-3 in GBM 8401 cells. Meanwhile, danthron also promoted the generation of reactive oxygen species (ROS) and cytosolic Ca(2+) in GBM 8401 cells. Taken together, our data showed that danthron induced apoptosis in GBM 8401 cells through mitochondria-related and caspase-related pathways, and it may be further evaluated as a chemotherapeutic agent for human brain cancer.

Evidence that highly conserved residues of Delonix regia trypsin inhibitor are important for activity.

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44-Cys89 and Cys139-Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4-5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139-Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44-Cys89) is not required.

Cigarette smoking exacerbates health problems in young men.

PURPOSE: To examines the hypothesis that smoking exacerbates health problems in young male smokers (age range, 18.6-22.8 yr; mean, 19.4 yr). METHODS: 1169 subjects were recruited, 25.41 % were smokers (2-15 cigarettes daily). All subjects were examined for body mass index, blood pressure, exhaled carbon monoxide content (carboxyl hemoglobin), blood hematology and biochemistry. RESULTS: Data for WBC (P < 0.001), hemoglobin (P=0.001), hematocrit (P=0.004), MCV (P=0.001), MCH (P=0.003), COHB% (P < 0.001), albumin/globulin (P < 0.001) and triglyceride (P < 0.001) were higher for smokers than non-smokers, while total-bilirubin (P < 0.001), total protein (P < 0.001) and globulin (P < 0.001) were markedly lower. The results of WBC (r=0.164, P < 0.004), COHB% (r=0.958, P < 0.001), gamma glutamyl transpeptidase (r=0.159, P=0.006), alkaline-phosphatase (r=-0.154, P=0.008) and triglyceride (r=0.144, P < 0.001) were closely correlated with number of cigarettes smoked daily. Investigation of associations with illness revealed that young smokers had an increased risk of hypertriglyceridemia to young non-smokers (adjusted ORs, 2.124; 95% CIs, 1.414-3.190), hyperglycemia (adjusted ORs, 1.980; 95% CIs, 0.803-4.901), neutrophilia (adjusted ORs, 1.947; 95% CIs, 1.248-3.037), RBC macrocytosis (adjusted ORs, 1.929; 95% CIs, 1.137-3.275), hyperchromia (adjusted ORs, 1.844; 95% CIs, 1.412-2.407) and polycythemia (adjusted ORs, 1.314; 95% CIs, 0.805-2.145) (all P < 0.05 for linear trends). CONCLUSION: The findings emphasize the importance of increasing surveillance of diseases exacerbated by smoking and reducing smoking in the young to prevent cardiovascular illnesses, metabolite disorders and other clinical diseases.

Seroprevalence of Mycobacterium pneumoniae in healthy adolescents in Taiwan.

This study was designed to understand the seroprevalence of Mycoplasma pneumoniae infection in healthy Taiwanese adolescents. The study included 2,233 college freshmen (female:male = 1.29:1; mean age, 19.7 years). The percentages of subjects residing in northern, central, southern, and eastern Taiwan were 66.91, 15.89, 9.0, and 8.2%, respectively. All enrolled subjects underwent a serologic agglutination test to detect serum concentrations of antibodies to M. pneumoniae. The test results showed that 19.84% of the subjects were infected and, of those, 6.0% were estimated to have subsequently acquired a current or acute infection during this study period. Moreover, the percentage of seropositive females (22.77%) was significantly higher than that of seropositive males (16.07%) (odds ratio, 1.54; 95% confidence interval, 1.241-1.911). Subjects residing in eastern counties were more likely to contract M. pneumoniae than those residing in other areas of Taiwan.

The potential of at-home prediction of the formation of urolithiasis by simple multi-frequency electrical conductivity of the urine and the comparison of its performance with urine ion-related indices, color and specific gravity.

It is important to control daily diet, water intake and life style as well as monitor the quality of urine for urolithiasis prevention. For decades, many ion-related indices have been developed for predicting the formation of urinary stones or urolithiasis, such as EQUILs, relative supersaturation (RSS), Tiselius indices (TI), Robertson risk factor algorithms (RRFA) and more recently, the Bonn risk index. However, they mostly demand robust laboratory analysis, are work-intensive, and even require complex computational programs to get the concentration patterns of several urine analytes. A simple and fast platform for measuring multi-frequency electrical conductivity (MFEC) of morning spot urine (random urine) to predict the onset of urolithiasis was implemented in this study. The performance thereof was compared to ion-related indices, urine color and specific gravity. The concentrations of relevant ions, color, specific gravity (SG) and MFEC (MFEC tested at 1, 10, 100, 5001 KHz and 1 MHz) of 80 random urine samples were examined after collection. Then, the urine samples were stored at 4 °C for 24 h to determine whether sedimentation would occur or not. Ion-activity product index of calcium oxalate (AP(CaOx) EQ2) was calculated. The correlation between AP(CaOx) EQ2, urine color, SG and MFEC were analyzed. AP(CaOx) EQ2, urine color and MFEC (at 5 frequencies) all demonstrated good prediction (p = 0.01, 0.01, 0.01, respectively) for stone formation. The positive correlation between AP(CaOx) EQ2 and MFEC is also significant (p = 0.01). MFEC provides a good metric for predicting the onset of urolithiasis, which is comparable to conventional ion-related indices and urine color. This technology can be implemented with much ease for objectively monitoring the quality of urine at points-of-care or at home.

Efficacy of the nationwide hepatitis B infant vaccination program in Taiwan.

BACKGROUND: Taiwan launched a nationwide infant vaccination program for hepatitis B (HB) in 1984. OBJECTIVES: This study evaluated the seroprevalence of hepatitis B virus (HBV) and the incidence of high alanine aminotransferase (ALT) level among young adults prior to, during, and since the introduction of the nationwide HBV vaccination program. STUDY DESIGN: Researchers recruited 101,584 freshmen (male:female=1.114:1; mean age, 18.5±0.5 years) from 21 universities between 1995 and 2009 (birth cohorts 1977-1991) in Taiwan, testing for serum hepatitis B surface antigens (HBsAg), hepatitis e antigens (HBeAg), antibodies against HBsAg (anti-HBs), and liver function tests, including ALT and aspartate aminotransferase (AST). RESULTS: The results showed that the prevalence of HBsAg decreased significantly from 14.3% in 1995 to 1.1% in 2009 and the seroprevalence of HBeAg decreased significantly from 5.9% in 1995 to 0.3% in 2009. Seroconversion to anti-HBs maintained a steady rate above 50% between 1995 and 2007, but declined considerably to 36.6% and 36.4% in 2008 and 2009, respectively. Subject with HBeAg seropositivity was in 43.94% of HBV carriers. Double seronegativity for HBsAg and anti-HBs was observed in 2007 (47.8%), 2008 (62.3%), and 2009 (62.5%). High ALT level was observed in 5.74% of the subjects, particular among HBV-carriers (16.5% of HBV carrier vs. 5.0% of non-HBV carrier; ORs, 3.733; 95% CIs, 3.463-4.023, p<0.0001). Subjects with high ALT level were significantly positively associated with HBeAg (10.5% of HBeAg seropositive vs. 1.9% of HBeAg seronegative; ORs, 6.195; 95%CI, 5.629-6.818; p<0.0001). Male subjects were more easily infected by HBV than female subjects were (HBsAg, ORs, 1.355, 95% CI, 1.283-1.431; HBeAg, ORs, 1.324, 95% CI, 1.218-1.439, p<0.0001), and significantly more male subjects had high ALT levels than female subjects did (ORs, 4.087; 95% CI, 3.819-4.375, p<0.0001). CONCLUSIONS: The mass vaccination program successfully reduced the HBV carrier rate and prevalence of chronic hepatitis B in Taiwan. However, the low percentage of anti-HBV in 2008 and 2009 remains unresolved.

Erythrocyte fragility increases with level of glycosylated hemoglobin in type 2 diabetic patients.

BACKGROUND: This study examines that erythrocyte was fragility-susceptible in diabetes. METHODS: Forty-five outpatients with type 2 diabetes mellitus (aged 46 +/- 13 years) and 20 healthy individuals with no history of diabetes disorders (aged 43 +/- 7 years) were randomly selected in this study. All subjects were analysis for blood glucose, glycosylated hemoglobin and erythrocyte osmotic fragility tests. RESULTS: The data for blood glucose (p<0.001), glycosylated hemoglobin (p<0.001), start hemolysis of erythrocyte (p<0.001), medium corpuscle hemolysis (p<0.001) and complete hemolysis (p=0.001) were significantly higher for diabetes than that of non-diabetes. The erythrocyte osmotic fragility in start hemolysis (r=0.479, p<0.001), medium corpuscle hemolysis (r=0.454, p<0.001) and complete hemolysis (r=0.277, p=0.025) were closely correlated with red blood cell glycosylated hemoglobin. Investigation of associations with red cell fragility-susceptible, diabetic patients had significantly increased risks of cell hemolysis on start hemolysis (ORs, 32.67; 95% CIs, 7.201-148.19), medium corpuscle hemolysis (ORs, 2.53; 95% CIs, 0.831-7.695) and complete hemolysis (ORs, 4.28; 95% CIs, 1.386-13.202) (all p<0.05 for linear trends) of erythrocyte to non-diabetes controls. CONCLUSIONS: This study demonstrated that osmotic fragility of erythrocyte was greater in type 2 diabetic subjects compared to non-diabetic controls and red blood cell fragility was positively correlated with glycosylated hemoglobin. Hence, it is necessary to emphasize increasing investigations of pathogenic mechanisms exacerbated by red cell fragility to prevent complications of diabetes mellitus.

Genomic and cDNA cloning, characterization of Delonix regia trypsin inhibitor (DrTI) gene, and expression of DrTI in Escherichia coli.

Degenerate primers were designed based on all possible sequences of the N-terminal and C-terminal regions of Delonix regia trypsin inhibitor (DrTI). Five hundred sixty-one bp of polymerase chain reaction (PCR) product was amplified using the above degenerate primers and genomic DNA and cDNA of Delonix regia as a template. The amplified PCR products were cloned and sequenced. DNA sequence analysis of cDNA and genomic clones of DrTI have the same nucleotide sequence in the coding region, and manifested a genomic clone without intervening sequences in the coding region. The amino acid sequence deduced from the DrTI genomic and cDNA clones agreed with that identified via amino acid sequencing analysis, except that two amino acid residues, Ser and Lys, existed between residues Lys141 and Ser142. DrTI open reading frame was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in Escherichia coli to yield a glutathione S-transferase (GST)-fusion protein with a calculated molecular mass of about 45 kDa. The recombinant DrTI (reDrTI) was derived by treating the GST-DrTI fusion protein with thrombin. Both the reDrTI and GST-DrTI fusion protein exhibited a strong identical inhibitory effect on trypsin activity.