Effects of chronic fluorosis on expressions of matrix metalloproteinase-9 mRNA and protein in the osteoclast of bone tissue of rats / 中华地方病学杂志

Objective To investigate the effects of chronic fluorosis on the expressions of matrix metalloproteinase-9 (MMP-9) mRNA and protein and the differentiation and maturation process of bone cell in the osteoclast of bone tissue of rats.Methods According to body weight,thirty-six healthy SD rats(body mass 100-120 g) were divided into three groups by random number table,twelve in each group,half male and half female.The rats of control group were given tap water(NaF ＜ 1 mg/L),and rats of low-fluorine and high-fluorine groups were fed with tap water containing 5 and 50 mg/L NaF to establish chronic fluorosis model.Rats were sacrificed after eight months; the contents of urinary fluoride in 24 hours and bone fluoride were analyzed by fluoride selective electrode.Serum content of tartrate resistant acid phosphatase 5b(TRACP5b)was detected by enzyme-linked immunosorbent assay (ELISA).The paraffin section of bone tissue was stained by hematoxylin-eosin (HE) and pathological morphometry was observed under optical microscope.The protein and mRNA levels of MMP-9 in the osteoclast of bones were detected by immunohistochemistry (IHC) and in situ hybridization (ISH),respectively.Results The differences of fluoride contents of urine and bone in rats were statistically significant between groups(F =400.612,48.229,all P ＜ 0.05).Fluoride contents of urine and bone were increased in lowfluorine and high-fluorine groups[(6.09 + 0.56),(7.69 + 0.64)mg/L,(12.65 ± 3.07),(26.53 + 5.88)mg/kg] compared to the control groups[(1.36 ± 0.51)mg/L,(0.67 ± 0.16)mg/kg,all P ＜ 0.05],and the fluoride contents of urine and bone were gradually increased with increasing fluoride doses(all P ＜ 0.05).The difference of TRACP5b content in serum was statistically significant between groups (F =9.607,P ＜ 0.05),in low-fluorine and high-fluorine groups,the TRACP5b contents[(1.86 ± 0.13),(1.92 ± 0.22)U/L] were higher than that of control group [(1.57 + 0.20)U/L,all P ＜ 0.05].The pathological examination showed osteosclerosis in fluoride exposed groups.The differences of MMP-9 mRNA and protein expressions were statistically significant between groups (F =365.727,331.382,all P ＜ 0.05).Compared to the control groups(97.22 ± 2.24,78.51 ± 1.16),the expressions of MMP-9 protein(108.18 ± 1.97,119.28 ± 1.76) and mRNA(89.44 ± 2.86,102.14 ± 2.39) were increased(all P ＜ 0.05),and the expressions of MMP-9 mRNA and protein were gradually increased with increasing fluoride doses (all P ＜ 0.05).Conclusions Chronic fluorosis might influence osteoclast differentiation and maturation process through regulating the expression levels of MMP-9 protein and mRNA.

Genetic diversity analyses reveal novel recombination events in Grapevine leafroll-associated virus 3 in China.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent causal agent of grapevine leafroll disease (GLD). Of the 75 grapevine samples collected from three regions in China, 46.7% and 94.7% of samples tested positive for GLRaV-3 in reverse transcription-PCR (RT-PCR) and reverse transcription nested PCR (RT-nPCR), respectively. The SSCP analysis for the clones of complete CP gene from 16 GLRaV-3 isolates showed that 15 isolates contained one predominant haplotype and one isolate had no predominant haplotype. The sequences of the CP genes showed 89.9-100% identities at the nucleotide level. Phylogenetic analysis of the CP gene sequences revealed the existence of four well defined variant groups, which corresponded to previously reported phylogenetic groups (1, 2, 3, and 5). Two new sub-groups designated as sub-group 1B and sub-group 3B in groups 1 and 3, respectively, were identified in the Chinese GLRaV-3 population. Recombination analyses illustrated that those two new sub-groups (1B and 3B) were emerged as a result of recombination events between variants in groups 1 and 2, and variants in groups 1 and 3, respectively. These results further indicated that the variants in those new sub-groups are viable and evolutionary successful. Recombinants with highly similar coat protein structure to variants of group 1 were abundantly found in the viral population. In addition, these analyses provided evidence about CP gene as one of the recombination hotspots in GLRaV-3 genome. The population genetic parameters of all available CP sequences suggested that the recombinants might have emerged due to population bottlenecks during transmission. The results provide new insights into the variability and evolution of GLRaV-3.

Probe binding to host proteins: a cause for false positive signals in viroid detection by tissue hybridization.

Molecular hybridization assay, especially involving the use of tissues directly, has been developed as a rapid, simple and important technique for plant pathogen detection and/or gene expression analysis on a large scale. In theory, this method relies on the specific binding of a labeled probe to a target nucleotide sequence. However, occasional false positive reactions can pose a problem in its application and the cause is often not well understood. Here, we show that in tissue-printing hybridization to detect Peach latent mosaic viroid (PLMVd) strong signals could arise by interactions between the viroid probe and plant proteins. Such probe-protein interactions made it difficult to show significant correlations between viroid infection and the level of hybridization signals. These results challenge the traditional view that proteins may hamper PCR reactions but have no influence on molecular hybridization. They further demonstrate that such probe-protein interactions in a plant could compromise the quality of molecular hybridization assays for viroid detection. Our results uncovered an important source of false positive reactions in tissue-printing hybridization and suggest that specificity can be improved by removing proteins.