OBJECTIVETo explore the potential biochemical mechanisms of neuromuscular junction transmission (NMJT) dysfunction induced by organophosphorus insecticides.

METHODSTen rats were dosed with phoxim (1,144 mg/kg) and 5 of them developed myasthenia. The NMJT function was evaluated by the mean consecutive differences (MCD) measured by stimulation single fiber electromyography (SSFEMG) with the frequency of stimuli at 20 Hz. The activity of Ca(2+)-ATPase, Na(+), K(+)-ATPase, cAMP dependent protein kinase (PKA), Ca(2+)/phospholipid dependent protein kinase (PKC), and tyrosine protein kinase (TPK) were determined.

RESULTSIn comparison with the control and non-myasthenic rats, the results in myasthenic rats showed that: (1) the MCDs increased; (2) the activities of Ca(2+)-ATPase, Na(+), K(+)-ATPase and PKA decreased and were negatively correlated with MCD; (3) the activities of PKC and TPK increased, and were positively correlated with MCD; (4) the phosphorylation of serine residuals in sarcolemma was weaker and the phosphorylation of tyrosine residuals was stronger.

CONCLUSIONSThe NMJT dysfunction is likely associated with the decrease in Ca(2+)-ATPase and Na(+), K(+)-ATPase activity. The acceleration of desensitization and prolongation of resensitization of nicotinic acetylcholine receptors occur following the increase in tyrosine phosphorylation and the decrease in serine phosphorylation induced by OPs poisoning.

Animals ; Calcium-Transporting ATPases ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; Electromyography ; drug effects ; Insecticides ; pharmacokinetics ; toxicity ; Myasthenia Gravis ; chemically induced ; enzymology ; Organothiophosphorus Compounds ; pharmacokinetics ; toxicity ; Protein Kinase C ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Rats

Since there have been very few studies on nucleolar signaling, an attempt was made to establish nucleolar signal pathways which link the cell membrane to the nucleolus for the transfer of extracellular signals. Two pathways were studied. One was the G alpha s mediated cAMP pathway where two signal molecules were yielded, including RII and protein kinase A. The other was the G alpha q mediated DAG/IP3 pathway which yields two signals including protein kinase C and IP3/Ca2+. By the studying isolated nucleoli from resting liver, regenerating liver or weak carcinogen thioacetamide treated liver, it was possible to detect protein kinase A (PKA), protein kinase C (PKC) and RII subunits. In addition, CK2 was detected. It was found that external signals transmitted through G protein coupled receptors could reach into the nucleolus and that physical translocation of signal molecules was an integral step involved in membrane-nucleolus linked pathways. When an in vitro assay of the above signal molecules was carried out using [gamma-32P]-ATP, most kinase dependent phosphorylation was via the major CK2 (more than 95%). Therefore, it is suggested that the major CK2 dependent pathway is involved in 'house keeping' for nucleolar integrity and the minor pathways, dependent on PKA, PKC and others, are involved in subtle regulatory mechanisms such as 'extra-house-keeping' activities by nucleolar chromosomal remodeling.
Animal ; Blotting, Western ; Cell Membrane/metabolism ; Cell Nucleolus/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism ; GTP-Binding Proteins/metabolism ; Immunoblotting ; Liver/metabolism ; Liver Neoplasms, Experimental ; Male ; Models, Biological ; Nuclear Proteins/metabolism* ; Phosphoproteins/metabolism ; Protein Kinase C/metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Rats ; Rats, Sprague-Dawley ; Second Messenger Systems ; Signal Transduction* ; Thioacetamide/pharmacology ; Time Factors

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

This study aimed to further explore the relevant mechanism of action by network pharmacology integrated with animal experimental verification based on previous proven effective treatment of vertebral artery type of cervical spondylosis(CSA) by Panlongqi Tablets. Bionetwork analysis was performed to establish drug-disease interaction network, and it was found that the key candidate targets of Panlongqi Tablets were enriched in multiple signaling pathways related to CSA pathological links, among which phosphatidylinositol 3-kinase(PI3 K)/serine-threonine kinase(AKT/PKB) signaling pathway was the most significant. Further, mixed modeling method was used to build the CSA rat model, and the rats were divided into normal, model, Panlongqi Tablets low-, medium-and high-dose(0.16, 0.32, 0.64 g·kg~(-1)) and Jingfukang Granules(positive drug, 1.35 g·kg~(-1)) groups. After successful modeling, the rats were administered for 8 consecutive weeks. Pathological changes of rat cervical muscle tissues were detected by hematoxylin-eosin(HE) staining, and the content of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), vascular endothelial cell growth factor(VEGF) and chemokine(C-C motif) ligand 2(CCL2) in rat serum and/or cervical tissues was determined by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to detect the protein expression levels of chemokine(C-C motif) receptor 2(CCR2), PI3 K, AKT, phosphorylated AKT(p-AKT), I-kappa-B-kinase beta(IKK-beta/IKKβ), nuclear factor kappa B(NF-κB P65) and phosphorylated nuclear factor kappa B(NF-κB p-P65) in rat cervical tissues, and positive expression of p-NF-κB P65 in rat cervical muscle tissues was detected by immunofluorescence. The results showed that Panlongqi Tablets at different doses improved the degree of muscle fibrosis and inflammation in cervical muscle tissues of CSA rats, and reduced the content of inflammatory factors IL-1β, TNF-α, VEGF, CCL2 and CCR2 in serum and/or cervical tissues. The protein expression levels of PI3 K, p-AKT, IKKβ and p-NF-κB P65 as well as the nuclear entry of p-NF-κB P65 in cervical tissues were down-regulated. These findings suggest that Panlongqi Tablets can significantly inhibit the inflammatory response of CSA rats, and the mechanism of action may be related to the down-regulation of the activation of PI3 K/AKT signaling pathway.
Animals ; Drugs, Chinese Herbal ; I-kappa B Kinase/pharmacology* ; NF-kappa B/metabolism* ; Network Pharmacology ; Phosphatidylinositol 3-Kinases/metabolism* ; Protein Serine-Threonine Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; Spondylosis/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism* ; Vascular Endothelial Growth Factor A/genetics* ; Vertebral Artery/metabolism*

The mammalian target of rapamycin (mTOR) critically regulates several essential biological functions, such as cell growth, metabolism, survival, and immune response by forming two important complexes, namely, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTOR signaling is often dysregulated in cancers and has been considered an attractive cancer therapeutic target. Great efforts have been made to develop efficacious mTOR inhibitors, particularly mTOR kinase inhibitors, which suppress mTORC1 and mTORC2; however, major success has not been achieved. With the strong scientific rationale, the intriguing question is why cancers are insensitive or not responsive to mTOR-targeted cancer therapy in clinics. Beyond early findings on induced activation of PI3K/Akt, MEK/ERK, and Mnk/eIF4E survival signaling pathways that compromise the efficacy of rapalog-based cancer therapy, recent findings on the essential role of GSK3 in mediating cancer cell response to mTOR inhibitors and mTORC1 inhibition-induced upregulation of PD-L1 in cancer cells may provide some explanations. These new findings may also offer us the opportunity to rationally utilize mTOR inhibitors in cancer therapy. Further elucidation of the biology of complicated mTOR networks may bring us the hope to develop effective therapeutic strategies with mTOR inhibitors against cancer.
Glycogen Synthase Kinase 3 ; Mechanistic Target of Rapamycin Complex 2 ; Neoplasms/drug therapy* ; Phosphatidylinositol 3-Kinases ; Protein Kinase Inhibitors ; Proto-Oncogene Proteins c-akt ; TOR Serine-Threonine Kinases

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.
3T3 Cells ; Animal ; Cell Transformation, Neoplastic* ; Genes, ras* ; Mice ; Mitogen-Activated Protein Kinase Kinases/analysis ; Mitogen-Activated Protein Kinases/analysis ; Phosphorylation ; Protein-Serine-Threonine Kinases/analysis* ; Proto-Oncogene Proteins c-raf/analysis ; p42 MAP Kinase/analysis

OBJECTIVETo investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree.

METHODSThe content and activity of MPF were assessed by immunobloting and Gollicano method.

RESULTSThe cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent.

CONCLUSIONSThe content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.

CDC2 Protein Kinase ; analysis ; Cyclin B ; analysis ; Humans ; Immunoblotting ; Maturation-Promoting Factor ; analysis ; Mouth ; chemistry ; Mouth Neoplasms ; chemistry ; Protein Kinase C ; physiology

No abstract available.
Humans* ; Keratinocytes* ; Protein-Serine-Threonine Kinases* ; Serine* ; Threonine*

The recent development of a prosaposin -/- mouse model has allowed the investigation of the role of prosaposin in the development of the male reproductive organs. A morphometric analysis of the male reproductive system of 37 days old mice revealed that prosaposin ablation produced a 30 % reduction in size and weight of the testes, 37 % of the epididymis, 75 % of the seminal vesicles and 60 % of the prostate glands. Light microscopy (LM) showed that smaller testis size from homozygous mutant mice was associated with reduced spermiogenesis. Both, dorsal and ventral lobules of the prostate glands were underdeveloped in the homozygous mutant. LM analysis also showed that prostatic alveoli were considerably smaller and lined by shorter epithelial cells in the homozygous mutant. Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice, the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes, which showed the presence of nonciliated cells. Radioimmunoassays demonstrated that testosterone levels were normal or higher in mice with the inactivated prosaposin gene. Immunostaining of prostate sections with an anti-androgen receptor antibody showed that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue. Similarly, sections immunostained with antibodies to the phosphorylated MAPKs and Akts strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse. On the other hand, the epithelial cells in the homozygous prostate remained unstained or weakly stained. These findings demonstrate that inactivation of the prosaposin gene affected the development of the prostate gland and some components of the MAPK pathway.
Animals ; Glycoproteins ; metabolism ; Humans ; MAP Kinase Signaling System ; physiology ; Male ; Prostate ; growth & development ; metabolism ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-akt ; Saposins

Type 1 diabetes mellitus (T1DM)-induced cognitive dysfunction is common, but its underlying mechanisms are still poorly understood. In this study, we found that knockout of conventional protein kinase C (cPKC)γ significantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles, but did not affect the activities of GSK-3β and PP2A in the hippocampal neurons of T1DM mice. cPKCγ deficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice. Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγ deficiency in T1DM mice. Moreover, cPKCγ deficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro. The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor. In conclusion, these results indicated that cPKCγ promotes autophagy through the AMPK/mTOR signaling pathway, thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.
AMP-Activated Protein Kinases/metabolism* ; Animals ; Autophagy ; Diabetes Mellitus, Type 1 ; Glucose ; Glycogen Synthase Kinase 3 beta/metabolism* ; Mice ; Phosphorylation ; Protein Kinase C/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; tau Proteins/metabolism*