Low Selenium and Low Protein Exacerbate Myocardial Damage in Keshan Disease by Affecting the PINK1/Parkin-mediated Mitochondrial Autophagy Pathway.

OBJECTIVE: Keshan disease (KD) is a myocardial mitochondrial disease closely related to insufficient selenium (Se) and protein intake. PTEN induced putative kinase 1 (PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body. This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury. METHODS: A low Se and low protein animal model was established. One hundred Wistar rats were randomly divided into 5 groups (control group, low Se group, low protein group, low Se + low protein group, and corn from KD area group). The JC-1 method was used to detect the mitochondrial membrane potential (MMP). ELISA was used to detect serum creatine kinase MB (CK-MB), cardiac troponin I (cTnI), and mitochondrial-glutamicoxalacetic transaminase (M-GOT) levels. RT-PCR and Western blot analysis were used to detect the expression of PINK1, Parkin, sequestome 1 (P62), and microtubule-associated proteins1A/1B light chain 3B (MAP1LC3B). RESULTS: The MMP was significantly decreased and the activity of CK-MB, cTnI, and M-GOT significantly increased in each experimental group (low Se group, low protein group, low Se + low protein group and corn from KD area group) compared with the control group (P<0.05 for all). The mRNA and protein expression levels of PINK1, Parkin and MAP1LC3B were profoundly increased, and those of P62 markedly decreased in the experimental groups compared with the control group (P<0.05 for all). CONCLUSION: Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.

Mild Anemia May Affect Thyroid Function in Pregnant Chinese Women During the First Trimester.

Background: Pregnant women are often susceptible to anemia, which can damage the thyroid gland. However, compared with moderate and severe anemia, less attention has been paid to mild anemia. The purpose of this study was to evaluate the effect of mild anemia on the thyroid function in pregnant women during the first trimester. Methods: A total of 1,761 women in the first trimester of their pregnancy were enrolled from Shenyang, China, and divided into mild anemia and normal control groups based on their hemoglobin levels. Thyroid-stimulating hormone (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) levels were compared between the two groups. Results: The TSH levels of pregnant women with mild anemia were higher than those of pregnant women without mild anemia (p < 0.05). Normal control women were selected to set new reference intervals for TSH, FT3, and FT4 levels during the first trimester, which were 0.11-4.13 mIU/l, 3.45-5.47 pmol/l, and 7.96-16.54 pmol/l, respectively. The upper limit of TSH 4.13 mU/l is close to the upper limit 4.0 mU/l recommended in the 2017 American Thyroid Association (ATA) guidelines, indicating that exclusion of mild anemia may reduce the difference in reference values from different regions. Mild anemia was related to 4.40 times odds of abnormally TSH levels (95% CI: 2.84, 6.76) and 5.87 increased odds of abnormal FT3 (95% CI: 3.89, 8.85). The proportion of hypothyroidism and subclinical hypothyroidism in patients with mild anemia was higher than that in those without anemia (0.6% vs. 0, p = 0.009; 12.1% vs. 1.9%, p < 0.001). Mild anemia was related to 7.61 times increased odds of subclinical hypothyroidism (95% CI: 4.53, 12.90). Conclusions: Mild anemia may affect thyroid function during the first trimester, which highlights the importance of excluding mild anemia confounding when establishing a locally derived specific reference interval for early pregnancy.

TGF-ß1/Smad3 Signaling Pathway Mediates T-2 Toxin-Induced Decrease of Type II Collagen in Cultured Rat Chondrocytes.

T-2 toxin can cause damage to the articular cartilage, but the molecular mechanism remains unclear. By employing the culture of rat chondrocytes, we investigated the effect of the TGF-ß1/Smad3 signaling pathway on the damage to chondrocytes induced by T-2 toxin. It was found that T-2 toxin could reduce cell viability and increased the number of apoptotic cells when compared with the control group. After the addition of the T-2 toxin, the production of type II collagen was reduced at mRNA and protein levels, while the levels of TGF-ß1, Smad3, ALK5, and MMP13 were upregulated. The production of the P-Smad3 protein was also increased. Inhibitors of TGF-ß1 and Smad3 were able to reverse the effect of the T-2 toxin on the protein level of above-mentioned signaling molecules. The T-2 toxin could promote the level of MMP13 via the stimulation of TGF-ß1 signaling in chondrocytes, resulting in the downregulation of type II collagen and chondrocyte damage. Smad3 may be involved in the degradation of type II collagen, but the Smad3 has no connection with the regulation of MMP13 level. This study provides a new clue to elucidate the mechanism of T-2 toxin-induced chondrocyte damage.

Serum Thyroglobulin-A Sensitive Biomarker of Iodine Nutrition Status and Affected by Thyroid Abnormalities and Disease in Adult Populations.

OBJECTIVE: To evaluate the usefulness of the thyroglobulin (Tg) level in adults as a nutritional biomarker of iodine status and to identify the factors related to the serum Tg level. METHODS: A cross-sectional study was conducted in adult populations of areas differing in iodine nutrition from three provinces (Autonomous Region) in China. Serum levels of thyroid hormones and Tg as well as thyroid autoantibodies were measured. The thyroid volume and nodule were measured by ultrasound. A multivariate linear regression analysis was used to assess iodine intake and other indeterminate factors associated with the serum Tg level. RESULTS: A total of 573 adults were recruited for this study. The serum Tg levels differed significantly among the three groups (22.27 µg/L, 9.73 µg/L and 15.77 µg/L in the excess, more-than-adequate, and deficient groups, respectively). The results of multivariate linear regression analysis indicate that excess and deficient iodine intake, goiter, thyroid nodule, hypothyroidism are significantly related with higher Tg level, and TgAb positivity is significantly related with lower serum Tg. CONCLUSION: The serum Tg level reflects abnormal thyroid function and is a sensitive functional biomarker of iodine nutrition status.

Juglone reduces growth and migration of U251 glioblastoma cells and disrupts angiogenesis.

Accumulating data show that prolylisomerase (Pin1) is overexpressed in human glioblastoma multiforme (GBM) specimens. Therefore, Pin1 inhibitors should be investigated as a new chemotherapeutic drug that may enhance the clinical management of human gliomas. Recently, juglone, a Pin1 inhibitor, was shown to exhibit potent anticancer activity in various tumor cells, but its role in human glioma cells remains unknown. In the present study, we determined if juglone exerts antitumor effects in the U251 human glioma cell line and investigated its potential underlying molecular mechanisms. Cell survival, apoptosis, migration, angiogenesis and molecular targets were identified with multiple detection techniques including the MTT cell proliferation assay, dual acridine orange/ethidium bromide staining, electron microscopy, transwell migration assay, chick chorioallantoic membrane assay, quantitative real-time polymerase chain reaction and immunoblotting. The results showed that 5-20 µM juglone markedly suppressed cell proliferation, induced apoptosis, and enhanced caspase-3 activity in U251 cells in a dose- and time-dependent manner. Moreover, juglone inhibited cell migration and the formation of new blood vessels. At the molecular level, juglone markedly suppressed Pin1 levels in a time-dependent manner. TGF-ß1/Smad signaling, a critical upstream regulator of miR-21, was also suppressed by juglone. Moreover, the transient overexpression of Pin1 reversed its antitumor effects in U251 cells and inhibited juglone-mediated changes to the TGF-ß1/miR-21 signaling pathway. These findings suggest that juglone inhibits cell growth by causing apoptosis, thereby inhibiting the migration of U251 glioma cells and disrupting angiogenesis; and that Pin1 is a critical target for juglone's antitumor activity. The present study provides evidence that juglone has in vitro efficacy against glioma. Therefore, additional studies are warranted to examine the clinical potential of juglone in human gliomas.

ClC-7/Ostm1 contribute to the ability of tea polyphenols to maintain bone homeostasis in C57BL/6 mice, protecting against fluorosis.

Epidemiological investigations indicate that certain ingredients in tea bricks can antagonize the adverse effects of fluoride. Tea polyphenols (TPs), the most bioactive ingredient in tea bricks, have been demonstrated to be potent bone-supporting agents. ClC­7 is known to be crucial for osteoclast (OC) bone resorption. Thus, in this study, we investigated the potential protective effects of TPs against fluorosis using a mouse model and explored the underlying mechanisms with particular focus on ClC­7. A total of 40, healthy, 3­week­old male C57BL/6 mice were randomly divided into 4 groups (n=10/group) by weight as follows: distilled water (control group), 100 mg/l fluoridated water (F group), water containing 10 g/l TPs (TP group) and water containing 100 mg/l fluoride and 10 g/l TPs (F + TP group). After 15 weeks, and after the mice were sacrificed, the long bones were removed and bone marrow-derived macrophages were cultured ex vivo in order to perform several experiments. OCs were identified and counted by tartrate­resistant acid phosphatase (TRAP) staining. The consumption of fluoride resulted in severe fluorosis and in an impaired OC function [impaired bone resorption, and a low mRNA expression of nuclear factor of activated T-cells 1 (NFATc1), ATPase H+ transporting V0 subunit D2 (ATP6v0d2) and osteopetrosis­associated transmembrane protein 1 (Ostm1)]. In the F + TP group, fluorosis was attenuated and OC function was restored, but not the high bone fluoride content. Compared with the F group, mature OCs in the F + TP group expressed higher mRNA levels of ClC­7 and Ostm1; the transportation and retaining of Cl­ was improved, as shown by the fluorescence intensity experiment. On the whole, our findings indicate that TPs mitigate fluorosis in C57BL/6 mice by regulating OC bone resorption. Fluoride inhibits OC resorption by inhibiting ClC­7 and Ostm1, whereas TPs attenuate this inhibitory effect of fluoride.

Prolactin rs1341239 T allele may have protective role against the brick tea type skeletal fluorosis.

OBJECTIVE: Prolactin (PRL) has been reported to be associated with increased bone turnover, and increased bone turnover is also a feature of skeletal fluorosis (SF). Autocrine/paracrine production of PRL is regulated by the extrapituitary promoter and a polymorphism in the extrapituitary PRL promoter at -1149 (rs1341239) is associated with disturbances of bone metabolism in other diseases. Here, we have investigated the possibility that the rs1341239 polymorphism is associated with SF, which results from the consumption of brick tea. DESIGN: We conducted a cross-sectional study in Sinkiang, Qinghai, Inner Mongolia in China. Demography survey questionnaires were completed and physical examination and X-ray diagnoses were used to diagnose SF. Brick tea water fluoride intake (IF) and urinary fluoride (UF) were tested by an F-ion selective electrode method. A Sequenom MassARRAY system was used to determine PRL gene polymorphisms. RESULTS: Subjects who were younger than 45 years of age and carried the T allele had a significantly decreased risk of SF [OR = 0.279 (95%CI, 0.094-0.824)] compared to those carrying the homozygous G allele. This phenomenon was only observed in Kazakh subjects [OR = 0.127 (95%CI, 0.025-0.646)]. Kazakh females who carried T alleles has a decreased risk of SF [OR = 0.410 (95%CI, 0.199-0.847)]. For Kazakh subjects which IF is less than 3.5 mg/d, a decreased risk of SF was observed among the participants who carried T alleles [OR = 0.118 (95%CI, 0.029-0.472)]. Overall, subjects with 1.6-3.2 mg/L UF and carried T alleles had a significantly decreased risk of SF [OR = 0.476 (95%CI, 0.237-0.955)] compared to homozygous G allele carriers. This phenomenon was only observed in Kazakh subjects [OR = 0.324 (95%CI, 0.114-0.923)]. CONCLUSIONS: Our results suggested that the PRL rs1341239 T allele decreases the risk of brick tea SF.

The plasma levels of CST and BCKDK in patients with sepsis.

OBJECTIVES: CST has been recently identified as a mediator of various beneficial effects in animal models of sepsis. At present, no data are available concerning the levels of CST in sepsis patients. In sepsis the plasma amino acid pattern is characterized by decreased branced chain amino acids (BCAAs). We investigated the levels of plasma CST or branched-chain α-ketoacid dehydrogenase kinase (BCKDK) and their relationship to component traits in patients with sepsis. DESIGN AND METHODS: We studied 228 patients and divided them into two groups based on severity of infection. Blood samples were taken at study entry, and CST, BCKDK were measured. RESULTS: CST and BCKDK levels were significantly higher in patients with sepsis than in controls: the median plasma CST concentration was 103.1ng/ml (range, <83.13-189.7ng/ml) in patients with sepsis and 49.69ng/ml (range, <19.38-100.8ng/ml) in controls (p=0.0022); the median plasma BCKDK concentration was 801.7ng/ml in sepsis group and 745ng/ml in controls (p=0.0292). Additionally, there was correlation between the plasma concentrations of CST and BCKDK in sepsis patients (r2=0.6357, p<0.01). CONCLUSIONS: We conclude that the plasma levels of CST in sepsis patients were higher than in controls, and there is a relationship between CST and BCKDK in sepsis patients. Future experimental and clinical studies are needed to evaluate CST as a novel prognostic tool in sepsis patients and its potential therapeutic use in sepsis.

Variations in the Obesity Gene "LEPR" Contribute to Risk of Type 2 Diabetes Mellitus: Evidence from a Meta-Analysis.

Leptin is a hormone protein regulating food intake and energy expenditure. A number of studies have evaluated the genetic effect of leptin (LEP) and leptin receptor (LEPR) genes on T2DM. This study aimed to investigate the association between these gene polymorphisms and T2DM by a systematic review and meta-analysis. Published studies were identified through extensive search in PubMed and EMBASE. A total of 5143 T2DM cases and 5021 controls from 14 articles were included in this study. Five functional variants in LEPR were well evaluated. Meta-analysis showed that rs1137101 (p.R223Q) was significantly associated with T2DM in all genetic models: allele model (OR = 1.27, 95% confidence interval (CI) = 1.13-1.42), dominant model (OR = 1.19, 95% CI = 1.05-1.35), homozygote model (OR = 1.82, 95% CI = 1.38-2.39), and recessive model (OR = 1.75, 95% CI = 1.35-2.28), with minimal heterogeneity and no indication of publication bias. Similar associations with T2DM were also found for rs62589000 (p.P1019P) and 3'UTR ins/del, although the data was obtained from a small number of studies. For the other two polymorphisms rs1137100 (p.R109K) and rs8179183 (p.K656N), they were not significantly associated with T2DM. Our results provide robust evidences for the genetic association of rs1137101 (p.R223Q) in LEPR with T2DM susceptibility.

Differential expression of serum proteins in rats subchronically exposed to arsenic identified by iTRAQ-based proteomic technology-14-3-3 ζ protein to serve as a potential biomarker.

Arsenic is a multi-system toxicant. However, the mechanism of arsenic toxicity is not fully clarified and few effective protein biomarkers could be used for arsenic poisoning. This study was to investigate the differentially expressed proteins in the serum of rats subchronically exposed to arsenic. Sixty male rats were randomly divided into four groups, and the dose of sodium arsenite in drinking water for each group was 0, 2, 10, and 50 mg L-1, respectively. The exposure lasted for 12 weeks. An Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic approach was used to identify the differentially expressed proteins in serum between control and 50 mg L-1 groups. A total of 201 serum proteins were identified by iTRAQ, of which 12 were significantly changed by arsenic exposure with two up-regulated and ten down-regulated proteins. One down-regulated protein 14-3-3 ζ, an abundant protein expressed in the brain, was verified by ELISA using serum samples and by immunohistochemical, real time PCR, and western blot methods using brain tissues in four groups. Our work provided valuable insight into the serum protein changes in rats exposed to arsenic, and indicated that 14-3-3 ζ may serve as a useful biomarker for nervous damage caused by arsenic poisoning.

The metabolomic profiling of serum in rats exposed to arsenic using UPLC/Q-TOF MS.

Chronic arsenicosis induced by excessive arsenic intake can cause damages to multi-organ systems, skin cancer and various internal cancers. However, the key metabolic changes and biomarkers which can reflect these changes remain unclear resulting in a lack of effective prevention and treatments. The aim of this study is to determine the impact of chronic arsenic exposure on the metabolism of organism, and find the metabolites changes by using metabolomic techniques. Thirty male Wistar rats were randomly divided into three groups. The arsenite was administered in water, and the doses were 0, 10, and 50mg/L, respectively. The exposure lasted for 6 months. The endogenous metabolite profile of serum was investigated by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Partial least squares discriminant analysis (PLS-DA) enabled clusters to be visualized. Nine serum principal metabolites contributing to the clusters were identified, which were CPA (18:2(9Z,12Z)/0:0), LysoPC (14:0), LysoPC (18:4 (6Z,9Z,12Z,15Z)), LysoPC (P-18:0), l-palmitoylcarnitine, LysoPC (20:2(11Z,14Z)) in positive ESI mode and deoxygcholylglycine, LysoPE (0:0/20:2(11Z,14Z)), 15(S)-hydroxyeicosatrienoic acid in negative ESI. These changes of metabolites in rats suggested the changed metabolism in rats exposed to arsenic. These findings may further aid diagnose and serve as targets for therapeutic intervention of arsenicosis.

Associations between XRCC1 Arg399Gln, Arg194Trp, and Arg280His polymorphisms and risk of differentiated thyroid carcinoma: a meta-analysis.

BACKGROUND: Associations between Arg399Gln, Arg194Trp and Arg280His polymorphisms of the XRCC1 gene and risk of differentiated thyroid carcinoma (DTC) have been widely studied but the findings are contradictory. METHODS: We performed a meta-analysis in the present study using STATA 11.0 software to clarify any associations. Electronic literature databases and reference lists of relevant articles revealed a total of 10, 6 and 6 published studies for the Arg399Gln, Arg194Trp and Arg280His polymorphisms, respectively. RESULTS: No significant associations were observed between Arg399Gln and DTC risk in all genetic models within the overall and subgroup meta-analyses, while the Trp/Trp vs Arg/Arg and recessive model of the Arg194Trp polymorphism was associated with DTC susceptibility, and the dominant model of Arg280His polymorphism contributed to DTC susceptibility in Caucasians. CONCLUSIONS: Our meta-analysis suggests that XRCC1 Arg194Trp may be a risk factor for DTC development.

Oral exposure to the herbicide simazine induces mouse spleen immunotoxicity and immune cell apoptosis.

The authors investigated the toxic effects of simazine on mice spleen immune cells and the underlying mechanisms. Mice were given simazine at 0, 90, 200, or 400 mg/kg by gastric gavage for 3 weeks. The authors then measured immune cell proliferation and the expressions of apoptosis-related proteins (Bcl-2, Bax, Fas, and caspase-3), spleen cell intracellular [Ca(2+)], cellular oxidative stress level, and immune functions. After 3 weeks, mice exposed to simazine had reduced proliferation of both spleen T and B cells. The number of spleen CD4(+) T lymphocytes decreased with simazine exposure, while CD8(+) T cells remained unchanged. Exposure to simazine resulted in reduced immune function, higher intracellular [Ca(2+)], and oxidative stress. Finally, simazine induced spleen immune cells apoptosis by reducing Bcl-2, while increasing Fas and Caspase-3 level. Overall, the immunotoxicity of simazine may involve the induction of immune cell apoptosis and alterations in the immune and physiological functions of spleen cells.

Curcuminoids exert glucose-lowering effect in type 2 diabetes by decreasing serum free fatty acids: a double-blind, placebo-controlled trial.

SCOPE: We previously found that curcuminoids decreased blood glucose and improved insulin resistance by reducing serum free fatty acids (FFAs) and increasing fatty acid oxidation in skeletal muscle of diabetic rats. This study was to investigate whether curcuminoids have beneficial effects on type 2 diabetic patients, and its possible mechanisms. METHODS AND RESULTS: Overweight/obese type 2 diabetic patients (BMI ≥ 24.0; fasting blood glucose ≥ 7.0 mmol/L or postprandial blood glucose ≥11.1 mmol/L) were randomly assigned to curcuminoids (300 mg/day) or placebo for 3 months. Bodyweight, glycosylated hemoglobin A1c (HbA1c ,% ), serum fasting glucose, FFAs, lipids, and lipoprotein lipase (LPL) were determined. A total of 100 patients (curcuminoids, n = 50; placebo, n = 50) completed the trial. Curcuminoids supplementation significantly decreased fasting blood glucose (p < 0.01), HbA1c (p = 0.031), and insulin resistance index (HOMA-IR) (p < 0.01) in type 2 diabetic patients. Curcuminoids also led to a significant decrease in serum total FFAs (p < 0.01), triglycerides (P = 0.018), an increase in LPL activity (p < 0.01). CONCLUSION: These findings suggest a glucose-lowering effect of curcuminoids in type 2 diabetes, which is partially due to decrease in serum FFAs, which may result from promoting fatty acid oxidation and utilization.

The Raf-1 inhibitor GW5074 and the ERK1/2 pathway inhibitor U0126 ameliorate PC12 cells apoptosis induced by 6-hydroxydopamine.

6-Hydroxydopamine (6-OHDA) is a widely used dopaminergic neurotoxin that leads to cell apoptosis in vivo and in vitro, and is a widely accepted experimental model of neurodegeneration in Parkinson's disease. However, the molecular mechanisms responsible for 6-OHDA-induced cell apoptosis are unclear. We found that the treatment of PC12 cells with 6-OHDA resulted in a significant decrease in cell viability and elevated apoptosis as detected by MTT assay, Hoechst 33258 staining, and flow cytometry. In addition, 6-OHDA induced a time-dependent phosphorylation of ERK1/2 at Thr-202/Tyr-204 and of Raf-1 at Ser-338, but a decreased level of Raf-1 phosphorylation at Ser-259. Phosphorylation of ERK1/2 at Thr-202/Tyr-204 and Raf-1 at Ser-338 were inhibited by the Raf-1 inhibitor GW5074, while the ERK1/2 pathway inhibitor U0126 decreased phosphorylation of ERK1/2. Furthermore, 6-OHDA-induced PC12 cells apoptosis was suppressed by GW5074 and U0126. Our results suggest that GW5074 and U0126 act as neuroprotants against 6-OHDA toxicity in PC12 cells by modulating Raf-1/ERK1/2 signaling systems.

Action mechanism of Zuo Gui Yin Decoction's promotion on estradiol production in rats during the peri-menopausal period.

AIM OF THE STUDY: Although Zuo Gui Yin Decoction has long been used in Traditional Chinese Medicine to treat menopausal symptoms, the underlying mechanism(s) by which these effects are induced remains to be defined. The aim of this study was to investigate the action mechanism of Zuo Gui Yin Decoction on estradiol production in the rat ovary during peri-menopause. MATERIALS AND METHODS: The peri-menopausal animal model was established by natural aging. Peri-menopausal rats were treated by intragastric administration (ig) with low (13.78gkg(-1)), middle (20.67gkg(-1)) or high (31gkg(-1)) dose of Zuo Gui Yin Decoction per day for 8 weeks. At the 8th weekend, the rats were sacrificed for sampling. Estradiol (E(2)) levels in rats' serum were evaluated by radioimmunoassay (RIA). RT-PCR, in situ hybridization and immunohistochemistry were used to determine mRNA and protein expression of relevant genes. RESULTS: Medium- and high-dose of Zuo Gui Yin Decoction could significantly increase serum estradiol concentration, ovarian CYP19 mRNA levels, and P450(arom) protein expression in rats during peri-menopause. Zuo Gui Yin Decoction at three different dosages all could promote FSHR expression and the effect of low-dose was the greatest. Zuo Gui Yin Decoction could elevate LRH-1 and ER(α) expression in a dose dependent manner. CONCLUSIONS: Taken collectively, these findings suggest that Zuo Gui Yin Decoction could promote estradiol production in rat serum during peri-menopausal period through ovarian ER(α)→LRH-1→CYP19 pathway as well as the ovarian classical FSHR→CYP19 mechanism.

Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCdelta phosphorylation.

OBJECTIVE: The present study aims to investigate the role of protein kinase C delta subtype (PKCdelta) phosphorylation in the process of 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson's disease. METHODS: The pheochromocytoma (PC12) cell line was employed in the present study. Cells were treated with 2 mumol/L PKCdelta inhibitor Rottlerin, 10 nmol/L protein kinase C delta subtype (PKCdelta) inhibitor bisindolylmaleimide I, or 5 nmol/L Gö6976 that could specifically inhibit the calcium-dependent PKCdelta isoforms, respectively. PKC activator phorbol-12-myristate-13-acetate (PMA, 100 nmol/L) was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotoxicity of 6-OHDA was determined by methyl thiazolyl tetrazolium (MTT) reduction assay and PKCdelta phosphorylation levels in various groups were measured by western blotting. RESULTS: Bisindolylmaleimide I and Gö6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCdelta phosphorylation in PC12 cells. However, Rottlerin could inhibit the phosphorylation of PKCdelta and attenuate 6-OHDA-induced cell death, and the cell viability was raised to (69.6+/-2.63)% of that in control group (P<0.05). In contrast, PMA induced a significant increase in PKCdelta phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to (49.8+/-5.06)% of that in control group (P<0.001). CONCLUSION: Rottlerin can protect PC12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKCdelta phosphorylation. The results suggest that PKCdelta may be a key regulator of neuron loss in Parkinson's disease.

Mutational analysis of proto-oncogene Dbl on Xq27 in testicular germ cell tumors reveals a rare SNP in a patient with bilateral undescended testis.

OBJECTIVES: An abundance of X chromosomes in testicular germ cell tumors (TGCTs), and a candidate TGCTs susceptibility gene (TGCT1) on Xq27 highlight the potential involvement of X chromosomes in TGCT pathogenesis. However, the TGCT1 on Xq27 has so far not been identified. We hypothesized that a somatic mutation of dbl oncogene on Xq27 may play a role for the development of TGCTs. METHODS: We have screened 41 TGCT tissues for dbl mutations using single-strand conformation polymorphism (SSCP) analysis. These tissues are composed of 25 seminomatous TGCTs tissues and 16 non-seminomatous TGCTs tissues, including two cases with a rhabdomyosarcoma component. RESULTS: Somatic mutations were not detected in the 25 exons of dbl in these TGCTs. However, we found a rare single nucleotide polymorphism (SNP) (T to C nucleotide change) within intron 22 in one out of the 41 TGCTs cases (2%). Furthermore, the sample with the rare SNP was identified as the sole TGCTs case associated with bilateral undescended testis in our series. CONCLUSIONS: Our results indicate that proto-oncogene dbl is not a major target for sporadic TGCTs. However, the rare SNP in dbl may affect the susceptibility to undescended testis. Determining the frequency of this SNP in patients with various types of undescended testis in different ethnic groups is a warranted study.