[Study of methylation of mitochondrial MT-COI of benzene poisoning].

Objective: To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec µlar biomarkers in patients with occupational chronic benzene poisoning. Methods: 38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups. Results: The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant (P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC (P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets (r=0.254、0.280, P<0.05) . Conclusion: The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

[In vitro study of histocompatibility and clearance of hemoperfusion adsorbents].

Objective: To evaluate the histocompatibility and clearance of chlorpyrifos and its metabolite of activated charcoal and adsorption resin by in vitro study. Methods: Venous blood from volunteers were incubation with activated charcoal or adsorbent resins, cytometry parameters and plasma components were detected for evaluation the histocompatibility of adsorbents. Venous blood from volunteers mixed with chlorpyrifos and its metabolite were incubation with activated charcoal or adsorbent resins, plasma concentration of chlorpyrifos and its metabolite were detected for evaluation the efficacy of adsorbents. Results: Incubation tests show that the absorbents reduce the blood platelet (F=3.671, P<0.05) , serum glucose (F=10.564, P<0.05) , albumin (F=5.239, P<0.05) , uric acid (F=7.175, P<0.05) , creatinine (F=23.673, P<0.05) , T3 (F=11.161, P<0.05) and free T3 (F=10.256, P<0.05) . However, other cytometry parameters and plasma components were not influenced. Both activated charcoal and adsorbent resins could reduce the plasma concentration of chlorpyrifos (F=798.110, P<0.01) and its metabolite (F=1495.212, P<0.05) . Conclusion: In vitro test show that both activated charcoal and adsorbent resins could clear chlorpyrifos and its metabolite, however, could not influence main cytometry parameters and plasma components, the histocompatibility of adsorbents are satisfactory.

[Detection of HLA-B*13:01 gene by dual allele-specific real-time polymerase chain reaction in patients with trichlorethylene-induced dermatitis].

Objective: To investigate the detection of a human leukocyte antigen-B (HLA-B) allele HLA-B*13:01 by dual allele-specific real-time polymerase chain reaction (PCR) in patients with trichlorethylene-induced dermatitis. Methods: A total of 20 patients with trichlorethylene-induced dermatitis who were admitted and treated from January 2014 to October 2016 were enrolled as case group, and 20 persons who underwent physical examination from January to October, 2016 were enrolled as control group. Peripheral cubital venous blood samples were collected from all subjects, and dual allele-specific real-time PCR was used to detect the HLA-B*13:01 gene. The two groups were compared in terms of the proportion of subjects carrying HLA-B*13:01 gene. Results: There were no significant differences between the case group and the control group in median age (25.0 years vs 27.0 years, Z=0.30, P>0.05) and the proportion of male subjects (60.0% vs 70.0%, χ(2)=0.44, P>0.05) . The mean time of exposure to trichloroethylene was 30.8 days in the case group, while the subjects in the control group were not exposed to trichloroethylene. The case group had a significantly higher frequency of HLA-B*13:01 gene than the control group (80.0% vs 20.0%, χ(2)=14.40, P<0.01) with an odds ratio of 16.00. Conclusion: Dual allele-specific real-time PCR can be used for detection of the HLA-B*13:01 gene in patients with trichlorethylene-induced dermatitis.

Amino acid composition of royal jelly harvested at different times after larval transfer.

The amino acids in royal jelly (RJ) have a wide range of pharmacological and health-promoting functions in humans. Multiple studies on the amino acid quality and composition in RJ have investigated RJ harvested at 72 h after larval transfer. In contrast, the concentration of amino acids in RJ harvested before 72 h remains unknown. In this study, the concentration of free amino acids (FAAs) and total amino acids (TAAs) in RJ harvested at 13 time points between 24 and 72 h after transfer of ten Apis mellifera colonies were measured. Our results indicated that the most abundant FAAs were Pro, Phe, Lys, Glu, and Tyr, whereas the most abundant TAAs were Asp, Glu, Leu, Lys, and Val. The total FAA concentration in RJ increased with increasing harvest time, from 4.30 mg/g at 24 h to 9.48 mg/g at 72 h. In contrast, the variation in concentration of TAAs observed was a decrease-increase-decrease trend with 40 h (149.53 mg/g) and 52 h (169.62 mg/g) as inflection points. The highest and lowest concentrations of TAA were 197.96 and 121.32 mg/g at 24 and 72 h, respectively. To our knowledge, this is the first study to investigate the concentration changes of FAAs and TAAs prior to 72 h after transfer. Our results will provide theoretical support to guide production practices of beekeeping, as well as elucidate the relationship between the harvest time point and RJ content.

Effect of tones on vocal attack time in Cantonese speakers.

Vocal attack time (VAT) is the time lag between the growth of the sound pressure signal and the development of physical contact of vocal folds at vocal initiation. It can be derived by a cross-correlation of short-time amplitude changes occurring in the sound pressure and electroglottographic (EGG) signals. Cantonese is a tone language in which tone determines the lexical meaning of the syllable. Such linguistic function of tone has implications for the physiology of tone production. The aim of the present study was to investigate the possible effects of Cantonese tones on VAT. Sound pressure and EGG signals were simultaneously recorded from 59 native Cantonese speakers (31 females and 28 males). The subjects were asked to read aloud 12 disyllabic words comprising homophone pairs of the six Cantonese lexical tones. Results revealed a gender difference in VAT values, with the mean VAT significantly smaller in females than in males. There was also a significant difference in VAT values between the two tone categories, with the mean VAT values of the three level tones (tone 1, 3, and 6) significantly smaller than those of the three contour tones (tone 2, 4, and 5). The findings support the notion that norms and interpretations based on nontone European languages may not be directly applied to tone languages.

Synthesis and pharmacokinetic evaluation of novel N-acyl-cordycepin derivatives with a normal alkyl chain.

For slowing down the too fast metabolic velocity and increasing the bioavailability of cordycepin, four N-acyl-(propionyl-, octanoyl-, lauroyl- and stearoyl-) cordycepin derivatives were synthesized chemically and their pharmacokinetic profiles were investigated in this study. The results show that time of maximum concentration (T(max)) and half-life (t(1/2)) would be elongated with the increase of the alkyl chain length, but maximum concentration (C(max)) and area under concentration-time curve (AUC) increased initially, then decreased when the number of alkyl carbon exceeded eight. The T(max), C(max) and AUC of N-octanoyl-cordycepin were nearly 4, 30 and 68 times, respectively, higher than that of cordycepin. All derivatives could be transformed into cordycepin in vivo and the concentration of transformed cordycepin was proportional to that of derivatives. It indicated that N-octanoyl modification could decrease the metabolic velocity and increase the bioavailability of cordycepin to the maximum, thus it might be a promising prodrug of cordycepin.

Specific abnormalities in serotonin release in the prefrontal cortex of isolation-reared rats measured during behavioural performance of a task assessing visuospatial attention and impulsivity.

RATIONALE: Rats reared in social isolation exhibit hyperactivity and specific attentional disturbances in later adult life. These behavioural abnormalities may be relevant to impulsivity and other neuropsychiatric syndromes such as attention-deficit hyperactivity disorder and schizophrenia where disturbances in circuitry involving the prefrontal cortex have been identified. OBJECTIVE: To establish whether isolation-reared rats show a differential susceptibility to cognitive processes that depend on the prefrontal cortex and its monoaminergic innervation. METHODS: Rats were reared in isolation from postnatal day 28 or in social groups of four and trained on the five-choice serial reaction time task, which assesses spatially divided visual attention. Following a range of manipulations designed to tax visual attention and response control, in vivo microdialysis was used in conjunction with behavioural testing to assess dopamine (DA) and serotonin (5-HT) release in the prefrontal cortex, either under baseline conditions prior to task initiation, or during task performance. Subjects were challenged with amphetamine (0.125 mg/kg intravenously) every 15 min, commencing 15 min after the start of the task. RESULTS: Apart from being consistently slower to collect food rewards and showing more perseverative responses to an auditory distractor, isolates were unimpaired on accuracy, impulsivity and correct latency measures on the five-choice task. Basal levels of DA and 5-HT in the prefrontal cortex were also unaffected by isolation rearing. Amphetamine increased the speed of responding in control and isolation-reared animals and increased premature (impulsive) responding, but only in socially-reared animals. Cortical DA release increased to a similar extent in both groups following amphetamine challenge. By contrast, 5-HT release was attenuated in isolates under these conditions. CONCLUSIONS: These findings highlight a rather specific deficit in 5-HT release in the prefrontal cortex of isolation-reared rats, although this appears not to affect visual attentional function. Rather, these data may be relevant to reduced impulsiveness of isolation-reared rats on the five-choice task. These findings are important in the context of animal models of attentional disturbances in schizophrenia.

The effect of ionizing radiation on intraocular lenses.

BACKGROUND: The native crystalline lens is the principal shield against ultraviolet radiation (UV), damage to the human retina. Every year in the United States, more than one million patients undergo removal of the natural lens in the course of cataract surgery (phakectomy), at which time an intraocular lens (IOL) is placed in the lens capsule. The IOL thenceforth serves as the principal barrier to ultraviolet radiation over the life of the implant, potentially for decades. The synthetic organic molecules of which IOLs are composed offer little UV protection unless ultraviolet-absorbing chromophores are incorporated into the lens material during manufacture. However, chromophores are alkenes potentially subject to radiolytic degradation. It is unknown whether ionizing radiation at clinical doses (e.g., to the brain or in the head-and-neck region) affects the UV-absorbing capacity of chromophore-bearing IOLs and consequently exposes the retina to potentially chronic UV damage. In addition, the polymers of which IOLs are composed are themselves subject to radiation damage, which theoretically might result in optical distortion in the visible light range. OBJECTIVE: To determine whether megavoltage photon ionizing radiation alters the absorption spectra of ultraviolet-shielding polymethylmethacrylate (PMMA) and organopolysiloxane (silicone) intraocular lenses (IOLs) in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), and low-end near-infrared (700 nm < lambda < or = 830 nm) ranges. DESIGN: Prospective, nonrandomized trial of dose-paired IOL cohorts. METHODS: Fourteen IOLs, seven of PMMA (Chiron 6842B) and seven of silicone (IOLAB L141U), were paired and examined for absorption spectra in 1-nm intervals over the range lambda = 280-830 nm on a Cary 400 deuterium and quartz halogen source-lamp UV/visible spectrophotometer before and after undergoing megavoltage ionizing irradiation to doses of 2, 5, 10, 20, 40, 60, and 100 Gray, respectively. Because of artifactual aberrations inherent in analyzing convex lenses on a conventional flat-plate spectrophotometer, post-irradiation absorption spectra were subsequently reanalyzed on a Cary 300 spectrophotometer outfitted with a Labsphere Diffused Reflectance Accessory (DRA-CA-30-I) incorporating a Spectralon-coated integrating sphere. MAIN OUTCOME MEASURES: Primary: Changes in UV absorbance after irradiation. Secondary: Changes in visible and low-end near-infrared absorbance after irradiation. RESULTS: Photon ionizing radiation in the 2-Gy to 100-Gy range produced no detectable alterations in the UV (280 nm < or = lambda < 400 nm), visible (400 nm < or = lambda < or = 700 nm), or low-end near-infrared (700 nm < lambda < or = 830 nm) absorption spectra of any of the lenses irradiated. However, silicone IOLs as a group revealed peak post-irradiation UV absorption at a shorter wavelength than did PMMA IOLs, with marginally greater UV transmission at the uppermost extreme of the UV spectrum (lambda = 384.5-400 nm). CONCLUSIONS: At clinically relevant doses used in radiation therapy, megavoltage photon ionizing radiation produces no significant alterations in the absorption spectra of PMMA and silicone IOLs over the range lambda = 280- 830 nm. These findings indicate that, even at supraclinical doses, the UV-absorbing capacity of chromophore-bearing PMMA and silicone IOLs remains unimpaired. It is not clear whether the lower UV peak of silicone lenses represents a radiation effect or a peculiarity of the chromophore used in the lenses tested.

Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPases) have been implicated in the physiological regulation of the insulin signalling pathway. In cellular and molecular studies, the transmembrane, receptor-type PTPase LAR and the intracellular, non-receptor enzyme PTP1B have been shown to have a direct impact on insulin action in intact cell models. Since insulin signalling can be enhanced by reducing the abundance or activity of specific PTPases, pharmaceutical agents directed at blocking the interaction between individual PTPases and the insulin receptor may have potential clinical relevance to the treatment of insulin-resistant states such as obesity and Type II diabetes mellitus.

Transgenic mice deficient in the LAR protein-tyrosine phosphatase exhibit profound defects in glucose homeostasis.

Protein-tyrosine phosphatases (PTPases) play an integral role in the regulation of cellular insulin action. LAR, a transmembrane PTPase expressed in insulin-sensitive tissues, acts as a negative regulator of insulin signaling in intact cell models. The physiological role of LAR was studied in mice in which LAR expression was eradicated by insertional mutagenesis. In the fasting state, adult male homozygous LAR (-/-) mice had significantly lower plasma levels of insulin and glucose, as well as a reduced rate of hepatic glucose production compared with wild-type controls, suggesting a heightened level of insulin sensitivity. In euglycemic clamp studies, the LAR (-/-) mice exhibited a significant resistance to insulin-stimulated glucose disposal and suppression of hepatic glucose output. Examination of hepatic insulin action demonstrated that the major alteration involved a 47% reduction in insulin-stimulated phosphatidylinositol 3'-kinase (PI 3-kinase) activity in the knockout mice, indicating a post-receptor signaling defect. Taken together with previous work on the cellular effects of LAR, the present results are consistent with a physiological role for LAR in the negative regulation of insulin action, with secondary abnormalities that contribute to the resistance to insulin-stimulated signaling in the knockout mice. Overall, these data provide further evidence for an important role for LAR in the regulation of insulin action and glucose homeostasis in intact animals.

Inhibition of the transmembrane protein tyrosine phosphatase lar by 3S-peptide-I enhances insulin receptor phosphorylation in intact cells.

3S-peptide-I, a tris-sulfotyrosyl dodecapeptide that corresponds to the major autophosphorylation domain within the insulin receptor beta-subunit, selectively enhances insulin signal transduction by specifically inhibiting dephosphorylation of the insulin receptor catalyzed by protein tyrosine phosphatases (PTPases). Because of the potential role of the transmembrane PTPase LAR in the regulation of insulin signaling, we assessed the effect of 3S-peptide-I on recombinant LAR PTPase activity and in McA-RH7777 rat hepatoma cells overexpressing full-length LAR protein (McA4B/LAR). 3S-peptide-I significantly reduced insulin receptor dephosphorylation by recombinant LAR (p < 0.001) while blocking dephosphorylation of the insulin receptor by approximately 72% in semi-permeabilized McA4B/LAR cells (p < 0.001). Increased LAR expression resulted in 40% reduction in ligand-mediated phosphorylation of the insulin receptor compared with null vector control (p < 0.001). However, treatment of intact McA4B/LAR cells with a fatty acid derivative of 3S-peptide-I (50 microM) led to an enhancement of insulin-stimulated receptor phosphorylation by 89% (p < 0.001). As a result, control and McA4B/LAR cells showed comparable steady-state levels of insulin receptor phosphorylation in the presence of insulin. These findings provide evidence that 3S-peptide-I may improve insulin responsiveness in intact cells by inhibiting LAR, an enzyme whose activity has been implicated in the pathogenesis of insulin resistance.

Suppression of insulin receptor activation by overexpression of the protein-tyrosine phosphatase LAR in hepatoma cells.

Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of reversible tyrosine phosphorylation of cellular proteins that mediate insulin action. In order to explore the potential role of the transmembrane PTPase (LAR) in insulin receptor signal transduction, we overexpressed the full-length LAR protein in McA-RH7777 rat hepatoma cells and found that modest increases in the abundance of LAR protein expression downregulated a number of insulin-stimulated cellular responses closely related to the activation of the receptor kinase. An increase in LAR protein of 2.4-fold over the level in control cells caused a 40% reduction in insulin receptor autophosphorylation in intact cells, without an alteration in insulin receptor mass or a change in the insulin-stimulated receptor kinase activity measured with partially purified receptors in vitro. In addition, insulin-stimulated tyrosine phosphorylation of the endogenous insulin receptor substrates IRS-1 and Shc were decreased to 57% and 73% of control, respectively, and IRS-1 associated phosphatidylinositol 3'-kinase activity was reduced to 47% of control of the cells overexpressing LAR. The present results, taken with our recent data demonstrating that reducing the abundance of LAR by expression of antisense mRNA enhances insulin receptor signal transduction (Kulas D. T., et al. J. Biol. Chem. 270:2435, 1995), supports the hypothesis that LAR acts as a physiological modulator of insulin action in insulin-sensitive hepatoma cells.

Differential regulation of insulin-stimulated tyrosine phosphorylation of IRS-1 and SHC by Wortmannin in intact cells.

Wortmannin is an inhibitor of phosphatidylinositol (PI) 3'-kinase, a cellular kinase activated by docking to phosphotyrosyl residues of insulin receptor substrate-1 (IRS-1) that can also phosphorylate serine residues on IRS-1 in vitro. After treatment of hepatoma cells with 100 nM wortmannin, the tyrosine phosphorylation of IRS-1 in response to insulin was increased by 38.3 +/- 3.3% while its phosphoserine/threonine content was reduced by 19%. Treatment with 1 microM wortmannin further increased IRS-1 tyrosine phosphorylation to 180% of control, while under these conditions, tyrosine phosphorylation of the IR substrate p52 Shc was reduced to less than 50% of control. Thus, alteration of the serine phosphorylation of IR substrates by a wortmannin-sensitive kinase may regulate post-insulin receptor signaling pathways by differential modulation of their insulin-stimulated tyrosine phosphorylation.

Modulation of insulin signal transduction by eutopic overexpression of the receptor-type protein-tyrosine phosphatase LAR.

Protein-tyrosine phosphatases (PTPases) regulate insulin signaling by catalyzing the tyrosine dephosphorylation of the insulin receptor and its substrate proteins. Previous studies have implicated a PTPase localized to a cell membrane fraction in the regulation of the insulin receptor in situ. LAR (leukoyte antigen related) is a transmembrane PTPase in insulin-sensitive tissues with in vitro catalytic specificity for the insulin receptor kinase domain. When transfected into Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-hlR), the LAR protein was processed as expected into an 85-kDa subunit containing the transmembrane and cytoplasmic domains. LAR was increased an average of 6-fold in clonal lines of stably transfected cells, and cell fractionation confirmed its localization in the cell membrane. After stimulation with 100 nM insulin, tyrosine phosphorylation of the insulin receptor was decreased by 31% at 1 min (P < 0.01) and by 42% at 10 min (P < 0.01), and that of IRS-1 was decreased by 34% (P < 0.01) at 1 min and by 56% (P < 0.01) at 10 min in the LAR-overexpressing cells compared with empty vector transfectants. LAR overexpression also blocked insulin-stimulated receptor kinase activation as well as thymidine incorporation into DNA. Quantitatively similar results were obtained in populations of CHO-hlR cells transfected transiently by electroporation. In contrast, overexpression of recombinant LAR cytoplasmic domain, detected as a 72-kDa protein in the cell cytosol, did not significantly affect the insulin-stimulated tyrosine phosphorylation of the insulin receptor or IRS-1 (99% and 93% of control at 10 min, respectively). These studies provide the first evidence that increased expression of LAR has negative regulatory effects at a proximal site in the insulin-signaling pathway. Since this effect occurs only when LAR is eutopically expressed at the cell membrane, these data further suggest that LAR requires a transmembrane localization to directly interact with the insulin receptor in situ.

Cell density-dependent changes in the insulin action pathway: evidence for involvement of protein-tyrosine phosphatases.

In order to examine alterations in the phosphorylation state of proteins involved in insulin action that might accompany the reduced growth state of density-arrested cells, we measured the insulin-stimulated phosphorylation of the receptor and high M(r) cellular substrates of the receptor kinase in rat hepatoma cells at different cell densities. As cell density increased from 2 x 10(5) to 3.2 x 10(6) per 35-mm well, the rate of DNA synthesis fell to 22% of control, while insulin-stimulated tyrosine phosphorylation of high M(r) receptor substrates ("pp185") was enhanced to 198% of control, without a change in the abundance of insulin receptor substrate (IRS)-1 protein. In anti-IRS-1 immunoprecipitates, tyrosine phosphorylation was increased by only 30%, suggesting that increased tyrosine phosphorylation of additional high M(r) proteins (e.g., IRS-2) accounted for much of the observed increase in tyrosine phosphorylation of the receptor substrates. In spite of increased tyrosine phosphorylation of IRS-1 and total pp185-related proteins, however, cells studied at high growth density exhibited a 25% decrease in IRS-1-associated phosphatidylinositol 3'-kinase activity and only a 39% increase in phosphatidylinositol 3'-kinase activity in antiphosphotyrosine immunoprecipitates. To explore the potential role of hepatic protein-tyrosine phosphatases (PTPases) in the hyperphosphorylation of pp185 proteins, we found by immunoblotting that at high cell density the intracellular PTPase PTP1B and the transmembrane PTPase LAR were reduced in abundance by 49% and 55%, respectively, while the abundance of the SH2-domain containing PTPase SH-PTP2 was increased by 48%. These data demonstrate that the attenuation of post-receptor signaling by insulin in hepatoma cells at increasing growth density involves changes in endogenous substrate phosphorylation which may result from alterations in specific PTPases implicated in the regulation of the insulin action pathway.

Osmotic loading of neutralizing antibodies demonstrates a role for protein-tyrosine phosphatase 1B in negative regulation of the insulin action pathway.

Protein-tyrosine phosphatases (PTPases) have been postulated to balance the steady-state phosphorylation and the activation state of the insulin receptor and its substrate proteins. To explore whether PTP1B, a widely expressed, non-receptor-type PTPase, regulates insulin signaling, we used osmotic shock to load rat KRC-7 hepatoma cells with affinity-purified neutralizing antibodies that immunoprecipitate and inactivate the enzymatic activity of recombinant rat PTP1B in vitro. In cells loaded with PTP1B antibody, insulin-stimulated DNA synthesis and phosphatidylinositol 3'-kinase activity were increased by 42% and 38%, respectively, compared with control cells loaded with preimmune IgG (p < 0.005). In order to characterize the potential site(s) of action of PTP1B in insulin signaling, we also determined that insulin-stimulated receptor autophosphorylation and insulin receptor substrate 1 tyrosine phosphorylation were increased 2.2- and 2.0-fold, respectively, and that insulin-stimulated receptor kinase activity toward an exogenous peptide substrate was increased by 57% in the PTP1B antibody-loaded cells. Osmotic loading did not alter the cellular content of PTP1B protein, suggesting that the antibody acts in the cell by sterically blocking catalytic interactions between PTP1B and its physiological substrates. These studies demonstrate that PTP1B has a role in the negative regulation of insulin signaling and acts, at least in part, directly at the level of the insulin receptor. These results also show that insulin signaling can be enhanced by the inhibition of specific PTPases, a maneuver that has potential clinical relevance in the treatment of insulin resistance and Type II diabetes mellitus.

Evaluation of protein kinase inhibitors in an assay system containing multiple protein kinase activities.

Specificities of several known protein kinase inhibitors were evaluated using an assay system in which activities of protein kinase A, protein kinase C, protein tyrosine kinase and calmodulin-dependent protein kinase III were simultaneously detected. Inhibitory spectra of H-89, K252a, H-7, staurosporine, tyrphostin and herbimycin A observed in the assay system were similar to those reported in the purified protein kinase assay systems. It was also found that KN-62 selectively inhibited calmodulin-dependent protein kinase III activity. These data suggest that the assay system may be effective and practicable in evaluating specificities and screening of new protein kinase inhibitors.

Method for simultaneous detection of protein kinase A, protein kinase C, protein tyrosine kinase, and calmodulin-dependent protein kinase activities.

We report a simple method that permits simultaneous detection of multiple protein kinase activities using postnuclear supernatant of v-src transformed NIH3T3 cells. A supernatant is incubated with activators of protein kinases and [gamma-32P]ATP, and the phosphorylated proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The method enables detection of activities of at least four protein kinases (protein kinase A, protein kinase C, protein tyrosine kinase(s), and calmodulin-dependent protein kinase III) on a single gel. Experiments using various specific activators and inhibitors of protein kinases indicated that this method can, in crude preparations, reliably detect the protein kinase activities intended for measurement. Protein kinase C activity disappeared when membranes were solubilized, demonstrating the importance of membrane environment for its function. This method should be particularly useful for evaluating and screening protein kinase inhibitors.