The influence of chronic fluorosis on mitochondrial dynamics morphology and distribution in cortical neurons of the rat brain.

The present study was designed to evaluate the effects of chronic fluorosis on the dynamics (including fusion and fission proteins), fragmentation, and distribution of mitochondria in the cortical neurons of the rat brain in an attempt to elucidate molecular mechanisms underlying the brain damage associated with excess accumulation of fluoride. Sixty Sprague-Dawley rats were divided randomly into three groups of 20 each, that is, the untreated control group (drinking water naturally containing <0.5 mg fluoride/l, NaF), the low-fluoride group (whose drinking water was supplemented with 10 mg fluoride/l) and the high-fluoride group (50 mg fluoride/l). After 6 months of exposure, the expression of mitofusin-1 (Mfn1), fission-1 (Fis1), and dynamin-related protein-1 (Drp1) at both the protein and mRNA levels were detected by Western blotting, immunohistochemistry, and real-time PCR, respectively. Moreover, mitochondrial morphology and distribution in neurons were observed by transmission electron or fluorescence microscopy. In the cortices of the brains of rats with chronic fluorosis, the level of Mfn1 protein was clearly reduced, whereas the levels of Fis1 and Drp1 were elevated. The alternations of expression of the mRNAs encoding all three of these proteins were almost the same as the corresponding changes at the protein levels. The mitochondria were fragmented and the redistributed away from the axons of the cortical neurons. These findings indicate that chronic fluorosis induces abnormal mitochondrial dynamics, which might in turn result in a high level of oxidative stress.

[Influence of chronic fluorosis on the expression of mitochondrial fission protein dynamin-related 1 in the cortical neurons of rats].

OBJECTIVE: To explore the changes of protein expression of mitochondrial fission gene dynamin-related 1(Drp 1) in the cortical neurons of rats with chronic fluorosis. METHODS: A total of 120 one-month-old SD rats (each weighing approximately 100-120 g at the beginning of the experiment) were randomly divided into three groups, and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride & high-fluoride supplemented with 10 and 50 mg/L fluoride,respectively). After 3 or 6 months exposure, 20 rats from each group were killed. Then the protein expression of mitochondrial fission gene, Drp1, was detected by immunohistochemistry and western-blotting method. RESULTS: Dental fluorosis and urinary fluorosis were obviously found in the rats exposed to fluoride. At the experiment period of 3 months, the numbers of positive cells of Drp1 detected by immunohistochemistry changed. Compared with the control group (36.3 ± 5.8), the changes in low-fluoride group (34.7 ± 4.1) showed no significant difference (t = 1.5, P > 0.05),but the increase in high-fluoride group (45.0 ± 4.7) had statistical significance (t = 8.8, P < 0.05). The western-blotting method had consistent results. Compared with the control group (0.59 ± 0.03), a significant increase of the average topical density in low- fluoride (0.62 ± 0.03) and high-fluoride (0.71 ± 0.02) groups were found (t = 0.02,0.11, P < 0.05). At the experiment period of 6 months, the numbers of positive cells of Drp1 detected by immunohistochemistry significantly changed. Compared with the control group (33.2 ± 4.4), the number in low- fluoride and high-fluoride groups were separately (36.6 ± 3.8) and (39.4 ± 4.2),both increased significantly (t = 3.5,6.3, P < 0.05). Same results could be found in western-blotting method,compared with the control group (0.65 ± 0.06), the average topical density in low- fluoride (0.80 ± 0.09) and high-fluoride (0.76 ± 0.08) groups both increased significantly (t = 0.1,0.1, P < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the changes of expression of Drp1, and the neurons damage from the chronic fluorosis might be associated with the hyperfunction of mitochondrial fusion.

[Changed expression of mito-fusion 1 and mitochondrial fragmentation in the cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To observe the mitochondrial fragmentation and the expression of mito-fusion 1 gene in the cortical neurons of rats with chronic fluorosis, and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis. METHODS: SD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages), and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride and high supplemented with 10 and 50 mg/L fluoride, respectively). After 3 or 6 months exposure, the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM), then the expression of mitochondrial fusion gene, Mfn1, were detected by immunohistochemistry and western-blotting, respectively. RESULTS: Dental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water, that is, (16 rats out of 20) numbers of I° detal fluorosis in the low-fluoride group, and (11 rats out of 20) numbers of I° and (9 rats out of 20) numbers of II° detal fluorosis in the high-fluoride group were observed after 3 months exposure. Moreover, (14 rats out of 20) numbers of I° and (6 rats out of 20) numbers of II° detal fluorosis in the low-fluoride group and (6 rats out of 20) numbers of Io, (13 rats out of 20) numbers of II°, and (1 rats out of 20) numbers of III° detal fluorosis in the high-fluoride group were observed after 6 months exposure. And both of untreated controls without detal fluorosis were also observed. The urinary level of fluoride in the low-fluoride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ± 0.35) were observed after 3 months exposure (F = 3.18, P < 0.05). Moreover, the urinary level of fluoride in the low-fluoride group (4.16 ± 1.39) mg/L and in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F = 3.17, P < 0.05). The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months, while the abnormal mitochondrial morphology of neurons with fluorosis was shown, presenting mitochondrial fragmentation with swollen cristae and even the fragmented, shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM. As compared with controls (53.0 ± 4.54 and 1.21 ± 0.18) at the experiment period of 3 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 51.09 ± 6.25) and western-blotting (1.22 ± 0.26) were no significant difference for low fluoride group (t = 1.7, 1.1, P > 0.05); Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 59.71 ± 5.64) and western-blotting (1.66 ± 0.20) were significantly increasing for high fluoride group (t = 2.1, 2.1, P < 0.05). As compared with controls (36.43 ± 4.04 and 1.00 ± 0.13) at the experiment period of 6 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and western-blotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t = 2.1, 2.2; 2.2, 2.2 respectively, all P value were < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1, and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.

[Alteration of mitochondrial distribution and gene expression of fission 1 protein in cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 (Fis1) protein in the cortical neurons of rats with chronic fluorosis. METHODS: Sixty SD rats were divided into 3 groups (20 each) according to weight hierarchy and fed with different concentrations of fluoride in drinking water (0, 10 and 50 mg/L, respectively) for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COXIV and tubulin-α were captured with fluorescence microscope. Fis1 protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fis1 mRNA was detected with real-time PCR. RESULTS: Varying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COXIV(34.8 ± 4.7 and 39.3 ± 3.0, respectively), and the expression of Fis1 protein (immunohistochemistry: 54.0 ± 3.6 and 51.3 ± 4.1, respectively; Western blot: 2.9 ± 0.4 and 2.6 ± 0.6, respectively) and mRNA (3773 ± 1292 and 1274 ± 162, respectively) was markedly increased as compared with controls (4.4 ± 2.3, 25.2 ± 2.5, 1.8 ± 0.2 and 277 ± 73) over the experimental period of 6 months. CONCLUSIONS: Excessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons (i.e., the increase in soma and the decrease in axon), increased expression of Fis1 gene and enhanced mitochondrial fission. The altered mitochondrial distribution may be related to the high expression level of Fis1 and a functional disorder of mitochondria.

[Establishment and application of rapid dipstick of latex immunochromatographic assay for the diagnosis of schistosomiasis].

OBJECTIVE: To establish a simple and fast diagnostic assay for schistosomiasis. METHODS: Based on the immunochromatographic technique and the principle of indirect assay of ELISA, using soluble egg antigen (SEA) of Schistosoma japonicum and mouse anti-human monoclonal antibody labelled with red latex as color developing agents, a latex immunochromatographic assay (DLIA) was developed. Serum samples from 69 schistosomiasis patients were detected by DLIA. Tested were also 264 sera from healthy people, 15 sera from clonorchiasis patients, 8 sera from patients with angiostrongyliasis cantonensis, 11 sera from patients with intestinal nematode infection and 19 sera from paragonimiasis patients. ELISA was used as a parallel control. RESULTS: The sensitivity for detecting schistosomiasis antibodies with DLIA and ELISA was 94.2% (65/69) and 95.7% (66/69), respectively (chi2=0.15, P>0.05). The specificity in examining healthy persons was 97.4% (257/264) and 94.7% (250/264), respectively (chi2=2.43, P>0.05). No cross reaction was found with the sera of clonorchiasis, intestinal nematode infection and angiostrongyliasis. The cross reaction rate with paragonimiasis of the two assays was 42.1% (8/19) and 47.4% (9/19), respectively (chi2=0.11, P>0.05). CONCLUSION: DLIA is a simple, fast, sensitive and specific assay for the diagnosis of schistosomiasis.

[A reviewing for profiling of highly degraded remains].

It's always a challenge to type from highly degraded biological remains. PCR-based STR genotyping is helpful and valuable for such degraded samples like bones, teeth et al, but the typing results are sometimes unstable or wrong. Here the methods for solving the problems and improving reproducibility are reviewed.

The decreased expression of mitofusin-1 and increased fission-1 together with alterations in mitochondrial morphology in the kidney of rats with chronic fluorosis may involve elevated oxidative stress.

This study was designed to characterize changes in the expression of mitofusin-1 (Mfn1) and fission-1 (Fis1), as well as in mitochondrial morphology in the kidney of rats subjected to chronic fluorosis and to elucidate whether any mitochondrial injury observed is associated with increased oxidative stress. Sixty Sprague-Dawley (SD) rats were divided randomly into 3 groups of 20 each, i.e., the untreated control group (natural drinking water containing <0.5mg fluoride/L), the low-fluoride group (drinking water supplemented with 10mg fluoride/L, prepared with NaF) and the high-fluoride group (50mg fluoride/L), and treated for 6 months. Thereafter, renal expression of Mfn1 and Fis1 at both the protein and mRNA levels was determined by immunohistochemistry and real-time PCR, respectively. In addition, the malondiadehyde (MDA) was quantitated by the thiobarbituric acid procedure and the total antioxidative capability (T-AOC) by a colorimetric method. The morphology of renal mitochondria was observed under the transmission electron microscope. In the renal tissues of rats with chronic fluorosis, expression of both Mfn1 protein and mRNA was clearly reduced, whereas that of Fis1 was elevated. The level of MDA was increased and the T-AOC lowered. Swollen or fragmented mitochondria in renal cells were observed under the electronic microscope. These findings indicate that chronic fluorosis can lead to the abnormal mitochondrial dynamics and changed morphology in the rat kidney, which in mechanism might be induced by a high level of oxidative stress in the disease.

[Emission characteristics of a diesel car fueled with coal based Fischer-Tropsch (F-T) diesel and fossil diesel blends].

According to the first type test cycle of China national standard GB 18352.3-2005, the CO, NO(x), HC, PM and CO2 emission characteristics of a PASSAT diesel car fueled with Shanghai local IV diesel, coal based Fischer-Tropsch (F-T) diesel, and the blends of coal based F-T diesel and Shanghai local IV diesel up to 10% and 50% by volume were analyzed respectively. And the environmental impacts such as decreased air quality, health impact, photochemical ozone, global warming, and acidification that could be caused by CO, NO(x), HC, PM and CO2 emission of the diesel car were also assessed. The results showed that under GB 18352.3-2005 No. 1 test driving cycle, which consisted of four urban driving cycles and one extra urban driving cycle, the CO, HC, PM and CO2 emissions were released mainly in the urban driving cycles whereas the NO(x) emissions occurred mainly in the extra urban driving cycle. Compared with Shanghai local IV diesel, all of the CO, NO(x), HC, PM and CO2 emissions of the diesel car decreased to different extents when fueled with coal based F-T diesel blends. Moreover, the aerosol generation potential, global warming potential and acidification potential of F-T diesel fueled diesel car were also reduced. To sum up, coal based F-T diesel would be one of the alternative fuels to diesel in China.

Development of a rapid dipstick with latex immunochromatographic assay (DLIA) for diagnosis of schistosomiasis japonica.

BACKGROUND: Schistosomiasis japonica (schistosomiasis) is a zoonosis that can seriously affect human health. At present, the immunodiagnostic assays for schistosomiasis detection are time-consuming and require well-trained personnel and special instruments, which can limit their use in the field. Thus, there is a pressing need for a simple and rapid immunoassay to screen patients on a large scale. In this study, we developed a novel rapid dipstick with latex immunochromatographic assay (DLIA) to detect anti-Schisaosoma japonicum antibodies in human serum. RESULTS: Using latex microspheres as a color probe, DLIA was established to test standard positive and negative sera, in comparison with the classical enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of DLIA were 95.10% (97/102) and 94.91% (261/275), respectively. The cross-reaction rates with clonorchiosis, intestinal nematodes, Angiostrongylus cantonensis and paragonimiasis were 0, 0, 0 and 42.11% respectively. All the results showed no significant difference to the ELISA. In field tests, 333 human serum samples from an endemic area were tested with DLIA, and compared with ELISA and Kato-Katz method. There was no significant difference between DLIA and ELISA on positive and negative rates of detection; however, significant differences existed between DLIA and Kato-Katz method, and between ELISA and Kato-Katz method. The kappa value between DLIA and ELISA was 0.90. CONCLUSIONS: This is the first study in which DLIA was used to detect anti-Schistosoma japonicum antibody. The results show that DLIA is a simple, rapid, convenient, sensitive and specific assay for the diagnosis of schistosomiasis and is therefore very suitable for large-scale field applications and clinical detection.

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii.

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-gamma and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.