The significant role of ATG5 in the maintenance of normal functions of Mc3T3-E1 osteoblast.

OBJECTIVE: To observe the effects of autophagy-related gene 5 (ATG5) on the proliferation, differentiation, and apoptosis of Mc3T3-E1 osteoblast as well as the effects of ATG5 on apoptosis of osteoblasts under the conditions of non-oxidative stress and oxidative stress. MATERIALS AND METHODS: ATG5 overexpressing and silencing cell lines were established in this experiment with lentiviral vector and transcription activator-like effect or nuclease (Talen) technique, respectively, using Mc3T3-E1 cells. Cell counting kit-8 (CCK-8) was used to detect the proliferation rate of osteoblasts, and flow cytometry was applied to detect the impacts of overexpressed and silenced ATG5 on the cell cycle. Alizarin red staining was used to detect the mineralization capacity of osteoblasts after 4-week osteoinduction differentiation. Quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blot methods were adopted to detect the levels of gene and protein expressions of runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and collagen I (COL-I) correlated with osteoblast differentiation after 48 h of osteoinduction differentiation. The staining with Annexin V-phycoerythrin/7-amino-actinomycin D (Annexin V-PE/7AAD) and flow cytometry were performed to detect the influence of ATG5 on osteoblast apoptosis. RESULTS: Stable ATG5 overexpressing and silencing Mc3T3-E1 cell lines were established successfully. CCK-8 test results showed that ATG5 silence inhibited cell proliferation, but the overexpression of ATG5 did not result in an obvious change in cell proliferation. Cell cycle did not change when ATG5 was overexpressed, while was stagnated in S-phase when silenced. The number of mineralized nodules of cells was reduced notably when ATG5 was silenced, while the overexpression of ATG5 did not have an impact on mineralization capacity of the cell after 4-week of osteoinduction differentiation. The test results of qRT-PCR and Western blotting suggested that ATG5 silence inhibited the gene and protein expressions of Runx2, OCN, and COL-I, while the influence of overexpressed ATG5 on the expressions of genes related to osteoblastic differentiation was not obvious after 48 h of osteoinduction differentiation. ATG5 silence made the cells easier to be damaged by hydrogen peroxide, which resulted in the rise of apoptosis rate of osteoblasts, while the overexpressed ATG5 inhibited osteoblast apoptosis after treatment with hydrogen peroxide for 12 h. CONCLUSIONS: ATG5 silence can lead to inhibition of osteoblast proliferation and differentiation. Moreover, it makes the cells easier to be damaged by oxidative stress, and it causes an increase in apoptosis. However, the overexpression of ATG5 strengthens the anti-oxidative capacity of osteoblasts and reduces apoptosis. ATG5 may be an effective target of anti-oxidative therapy for osteoporosis, which brings a new direction for the treatment of osteoporosis.

Impact of body composition on estimated glomerular filtration rate in relatively healthy adults in Taiwan.

BACKGROUND/OBJECTIVES: Chronic kidney diseases are associated with changes in cardiometabolic risk (CMR) factors in which body composition parameters have been used as sensitive predictors. This study aimed to explore the associations of anthropometric indicators, body fat (BF), body mass index (BMI) and waist circumference (WC) with estimated glomerular filtration rate (eGFR) in an adult healthy Chinese population. SUBJECTS/METHODS: A cross-sectional study was conducted for the subjects undergoing annual health examinations. The associations of subjects with body composition parameters were analyzed using the cutoff values of BMI, BF and WC in accordance with the criteria for Asian or Taiwanese population by gender. RESULTS: A total of 3473 subjects, aged 30-45 years, who received physical examinations in 2007 were analyzed. The levels of CMR factors were significantly higher in males than in females. eGFR was negatively associated with BMI but positively related to BF. The additional roles of BMI and WC were observed in the subjects who were categorized according to BF. Females with normal weight obese were associated with increased eGFR, whereas a higher eGFR was found in males with low/normal BF and BMI or normal WC. CONCLUSIONS: Our data provided evidence that anthropometric parameters were associated with changes of eGFR in relatively healthy adults. Higher eGFR was observed in females with normal weight obese in whom hyperfiltration may be suspected, and this finding deserves further studies.

Association between waist-to-height ratio and chronic kidney disease in the Taiwanese population.

BACKGROUND: Obesity, metabolic syndrome (MS) and chronic kidney disease (CKD) are all becoming increasingly prevalent worldwide. Body mass index (BMI) has traditionally been employed to identify overweight or obese individuals, yet multiple studies have yielded conflicting results when BMI was used to evaluate the association between obesity and CKD. AIMS: The purpose of this large, population-based, multicentre study was to evaluate the associations of BMI and waist-to-height ratio (WHtR) with CKD. METHODS: A retrospective study of 41,600 subjects who had physical examinations from January 2010 to December 2011 was performed. Data such as life style and habits were collected by interviews, and systolic and diastolic blood pressure (SBP and DBP), height, body weight, waist circumference, total cholesterol (TC), high-density lipoproteins (HDL), triglycerides (TG), fasting blood glucose and creatinine levels were measured. The association of these factors with CKD was analysed by use of SPSS 15.0 software. RESULTS: The key findings of this study were that WHtR but not BMI was an independent predictor of CKD. Additionally, SBP was a predictor of CKD in males and females, and TG and TC were independent predictors of CKD in females. Such measures are components of MS, which may also be associated with the development of CKD. CONCLUSION: WHtR appears to be a better measure of central obesity than BMI, and is an easy-to-use, noninvasive tool for identifying individuals at risk of developing obesity-related CKD, and potentially also MS-related CKD.

Survival of staphylococcus aureus and Escherichia coli as affected by ethanol and NaCl.

Growth of three strains of Staphylococcus aureus and two strains of Escherichia coli on nutrient agar (NA) supplemented with ethanol and NaCl was investigated. S. aureus did not grow on NA containing > or =10% ethanol (wt/wt) combined with > or =0% NaCl (wt/wt), or 7.5% ethanol combined with 7.5% NaCl. Neither E. coli nor E. coli O157:H7 grew on NA containing > or =7.5% ethanol combined with > or =0% NaCl, 5% ethanol combined with > or =2.5% NaCl, or > or =5% NaCl combined with > or =0% ethanol. It is apparent that NaCl enhanced the inhibitory effect of ethanol on growth of S. aureus and E. coli When cells were suspended in nutrient broth containing 12.5, 20, or 40% ethanol combined with NaCl, viable cells decreased with an increase of ethanol concentration. Ethanol sensitivity among strains and between genera varied in a limited range. When the cells were exposed to 20% ethanol in combination with 5% NaCl, S. aureus and E. coli lost viability after 30 and 10 min, respectively. When treated with 40% ethanol combined with > or =0% NaCl, all test strains lost viability within 5 min.

Interaction of collagen alpha1(X) containing engineered NC1 mutations with normal alpha1(X) in vitro. Implications for the molecular basis of schmid metaphyseal chondrodysplasia.

Collagen X is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal nonhelical NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggests a critical role for NC1 in collagen X structure and function. In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that although alpha1(X) containing normal NC1 domains can form electrophoretically stable trimers, engineered SMCD NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal alpha1(X). To allow the detection of more subtle interactions that may interfere with assembly but not produce SDS-stable final products, we have developed a competition-based in vitro co-expression and assembly approach. Our studies show that alpha1(X) chains containing SMCD mutations reduce the efficiency of normal alpha1(X) trimer assembly, indicating that interactions do occur between mutant and normal NC1 domains, which can impact on the formation of normal trimers. This finding has important implications for the molecular pathology of collagen X mutations in SMCD. Although we have previously demonstrated haploinsufficiency as one in vivo mechanism (Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1998) J. Clin. Invest. 101, 1490-1499), the current study suggests dominant interference is also possible if the mutant protein is expressed in vivo. Furthermore, we establish that a conserved 13-amino acid aromatic motif (amino acids 589-601) is critical for the interaction between the NC1 domains, suggesting that this region may initiate assembly and the other NC1 mutations interfered with secondary interactions important in folding or in stabilizing the assembly process.

Direct antimicrobial susceptibility testing of gram-negative bacilli in blood cultures by an electrochemical method.

Nonfastidious aerobic gram-negative bacilli (GNB) are commonly isolated from blood cultures. The feasibility of using an electrochemical method for direct antimicrobial susceptibility testing of GNB in positive blood cultures was evaluated. An aliquot (10 microliter) of 1:10-diluted positive blood cultures containing GNB was inoculated into the Bactometer module well (bioMérieux Vitek, Hazelwood, Mo.) containing 1 ml of Mueller-Hinton broth supplemented with an antibiotic. Susceptibility tests were performed in a breakpoint broth dilution format, with the results being categorized as resistant, intermediate, or susceptible. Seven antibiotics (ampicillin, cephalothin, gentamicin, amikacin, cefamandole, cefotaxime, and ciprofloxacin) were used in this study, with each agent being tested at the two interpretive breakpoint concentrations. The inoculated modules were incubated at 35 degreesC, and the change in impedance in each well was continuously monitored for 24 h by the Bactometer. The MICs of the seven antibiotics for each blood isolate were also determined by the standardized broth microdilution method. Of 146 positive blood cultures (1,022 microorganism-antibiotic combinations) containing GNB tested by the direct method, the rates of very major, major, and minor errors were 0, 1.1, and 2.5%, respectively. The impedance method was simple; no centrifugation, preincubation, or standardization of the inocula was required, and the susceptibility results were normally available within 3 to 6 h after inoculation. The rapid method may allow proper antimicrobial treatment almost 30 to 40 h before the results of the standard methods are available.

A nonsense mutation in the carboxyl-terminal domain of type X collagen causes haploinsufficiency in schmid metaphyseal chondrodysplasia.

Type X collagen is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggested a critical role for this type X collagen domain, but since no direct analysis of cartilage has been conducted in SMCD patients, the mechanisms of type X collagen dysfunction remain controversial. To resolve this problem, we obtained SMCD growth plate cartilage, determined the type X collagen mutation, and analyzed the expression of mutant and normal type X collagen mRNA and protein. The mutation was a single nucleotide substitution that changed the Tyr632 codon (TAC) to a stop codon (TAA). However, analysis of the expression of the normal and mutant allele transcripts in growth plate cartilage by reverse transcription PCR, restriction enzyme mapping, and a single nucleotide primer extension assay, demonstrated that only normal mRNA was present. The lack of mutant mRNA is most likely the result of nonsense-mediated mRNA decay, a common fate for transcripts carrying premature termination mutations. Furthermore, no mutant protein was detected by immunoblotting cartilage extracts. Our data indicates that a functionally null allele leading to type X collagen haploinsufficiency is the molecular basis of SMCD in this patient.

Multiexon deletions in the type I collagen COL1A2 gene in osteogenesis imperfecta type IB. Molecules containing the shortened alpha2(I) chains show differential incorporation into the bone and skin extracellular matrix.

Osteogenesis imperfecta (OI) type IB is a rare subset of the mildest form of OI, clinically characterized by moderate bone fragility, blue sclera, and dentinogenesis imperfecta. Cultured skin fibroblasts from two unrelated individuals (OI-197 and OI-165) with the typical features of OI type IB produced shortened alpha2(I) chains. Reverse transcription-polymerase chain reaction of the alpha2(I)-cDNA revealed deletions in the triple helical domain of 5 exons (exons 7-11) in OI-197, and 8 exons (exons 10-17) in OI-165. This exon skipping was caused by genomic deletions in one allele of COL1A2 with the breakpoints located in introns 6 and 11 in OI-197, and introns 9 and 17 in OI-165. The secretion and deposition of the mutant collagen into the matrix was measured in vitro in cultures of skin fibroblasts and bone osteoblasts, grown in the presence of ascorbic acid to induce collagen matrix formation and maturation, as well as in collagen extracts from skin and bone. The secretion of mutant collagen was impaired and long term cultures of fibroblasts showed that the mutant collagen was not incorporated into the mature collagenous matrix produced in vitro by skin fibroblasts from both patients. Likewise, the shortened alpha2(I) chain was not demonstrable in skin extracts. In contrast, bone extracts from OI-197 showed the presence of the mutant collagen. This incorporation of the abnormal collagen into the mature matrix was also demonstrated in long term cultures of the patient's osteoblasts. The deposition of the mutant collagen by bone osteoblasts but not by skin fibroblasts demonstrates a tissue specificity in the incorporation of mutant collagen into the matrix which may explain the primary involvement of bone and not skin in these patients.

Site-directed mutagenesis of human type X collagen. Expression of alpha1(X) NC1, NC2, and helical mutations in vitro and in transfected cells.

Type X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The alpha1(X) homotrimer consists of three protein domains, a short triple helix (COL1) flanked by nonhelical amino-terminal (NC2) and carboxyl-terminal (NC1) domains. While mutations of the NC1 domain result in Schmid metaphyseal chondrodysplasia, which suggests a critical role for this protein domain, little biochemical detail is known about type X collagen synthesis, secretion, and the mechanisms of molecular assembly. To study these processes, a range of mutations were produced in human alpha1(X) cDNA and the biochemical consequences determined by in vitro expression, using T7-driven coupled transcription and translation, and by transient transfection of cells. Three NC1 mutants, which were designed to be analogous to Schmid mutations (1952delC, 1963del10, and Y598D), were unable to assemble into type X collagen homotrimers in vitro, but the mutant chains did not associate with, or interfere with, the efficiency of normal chain assembly in co-translations with a normal construct. Expression in transiently transfected cells confirmed that mutant type X collagen assembly was also compromised in vivo. The mutant chains were not secreted from the cells but did not accumulate intracellularly, suggesting that the unassociated mutant chains were rapidly degraded. In-frame deletions within the helix (amino acid residues 72-354) and the NC2 domain (amino acid residues 21-54) were also produced. In contrast to the NC1 mutations, these mutations did not prevent assembly. Mutant homotrimers and mutant-normal heterotrimers were formed in vitro, and the mutant homotrimers formed in transiently transfected cells had assembled into pepsin-stable triple helical molecules which were secreted.

Identification of a human-specific epitope in a conserved region of the La/SS-B autoantigen.

Human anti-La/SS-B autoantibodies are known to react with highly conserved epitopes suggested to be functional or active sites on the La/SS-B polypeptide. This study was designed to determine whether the autoantibodies also react with poorly conserved regions of La/SS-B as predicted by an antigen-driven autoimmune response. Binding of human autoantibodies to purified human, mouse, and bovine recombinant fragments representing immunodominant regions of the La/SS-B polypeptide was compared using Western blotting and ELISA. A cross-reactive epitope was located in the highly conserved NH2-terminal region of La/SS-B. Significantly, human-specific epitopes were identified in both the conserved RNA-recognition motif and a poorly conserved COOH-terminal fragment, providing direct evidence for an autoantigen-driven response. The lack of autoantibody cross-reactivity with a conserved domain of mouse and bovine La/SS-B implies that a small number of residues in human autoepitopes may be critical for autoimmunogenicity.

N-nitrosamine and mutagenicity formation in Chinese salted fish after digestion.

Salted and dried fish (Nemipterus virgatus), acquired from Hong Kong, was treated with 0.43-110 mM nitrite during in vitro digestion using gastric enzymes and the volatile N-nitrosamine content and mutagenicity on Salmonella typhimurium TA100 assayed without concentration. N-Nitrosodimethylamine (NDMA; the only nitrosamine detected) formation was second order in nitrite concentration. When 10 g of fish was treated with 6.96 mM nitrite, 394 nM NDMA was formed. Thiocyanate was catalytic for NDMA formation at nitrite concentration greater than 0.87 mM and when the ratio of thiocyanate to nitrite was greater than 1. Approximately a 50% inhibition in NDMA formation by ascorbic acid was seen when the ratio of ascorbate to nitrite was approximately 2 or greater and the nitrite concentration was 1.74 mM. Mutagenicity increased with increasing nitrite concentration but the addition of thiocyanate did not increase mutagenicity over nitrite alone. Ascorbate increased mutagenicity even though NDMA formation was inhibited. Even at nitrite concentrations greater than 100-fold higher than expected in vivo, there was insufficient NDMA formed to account for the observed mutagenicity. These data do not exclude the possibility that the observed mutagenicity was due to non-volatile N-nitroso compounds, however, this possibility seems unlikely given the effects of ascorbate and thiocyanate which would be expected to inhibit and enhance non-volatile N-nitroso compound formation.