To develop a method for the detection of surface-confined peptides containing cysteine residues or oligodeoxynucleotides (ODNs) whose 3' ends modified with thiol groups, and a thiol-specific fluorescent cross-linker, N-(9-acridinyl) maleimide (NAM) was used. The peptides studied herein include both the oxidized and reduced forms of glutathione, and a hexapeptide (FT). Peptides are first attached onto the activated 11-mercaptoundecanoic acid (MUA)-terminated alkanethiol self-assembled monolayers (SAMs) and then derivatized with NAM. The cysteine residues was determined by using electrochemical desorption and fluorescence detection. GSH concentration as low as 40 pmol x L(-1) can be measured. The fluorescence intensity in the case of FT is about 3 times as high as that for GSH, which is consistent with the molar ratio of cysteine residues in these two molecules. The analytical performance of gene analysis was also evaluated through the analyses of a complementary target and targets with varying numbers of mismatching bases. The method described here is simple, sensitive, reproducible, and does not require sophisticated analytical instrumentation and separation procedures.
Biosensing Techniques ; methods ; Cysteine ; analysis ; Electrochemistry ; methods ; Fluorescence ; Glutathione ; analysis ; chemistry ; Maleimides ; chemistry ; Oligodeoxyribonucleotides ; analysis ; Reproducibility of Results ; Sensitivity and Specificity

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus. Protein kinase C (PKC) inhibitor's has been thought to be a potential disease modifying drug's in DPN as it slows or reverse neuropathy's progression. To assesses the efficacy and safety of ruboxistaurin on the progression of symptoms, signs, or functional disability in DPN. METHODS: A systematic review of the literature databases like PubMed, ProQuest, EBSCO, EMBASE, and Cochrane Central was performed up to August 2012. We included randomized controlled trials (RCTs) comparing PKC inhibitor ruboxistaurin (RBX) with control and lasting at least 6 months. Our primary outcome measure was change in neurological examination, measured by neurological total symptom score (NTSS) and vibration detection threshold (VDT). Secondary outcome measures were total quality of life (QoL), skin microvascular blood flow and others. RESULTS: Six RCTs were included in review. Change in neurological function assessed by NTSS was reported in six studies, out of which significant difference between the RBX and placebo group seen in four studies favouring treatment group while remaining two studies reported no significant difference. VDT was assessed in only one study in which no significant difference seen between RBX and placebo group. Two studies reported significant improvement in QoL data. Safety data was reported in only two studies in which none of side effect was related to RBX. CONCLUSION: RBX had effects on DPN in some studies, but the evidence is not enough for meta-analysis and firm conclusion.
Diabetes Mellitus ; Indoles ; Maleimides ; NAD ; Neurologic Examination ; Outcome Assessment (Health Care) ; Peripheral Nervous System Diseases ; Protein Kinase C ; Quality of Life ; Skin ; Vibration

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus. Protein kinase C (PKC) inhibitor's has been thought to be a potential disease modifying drug's in DPN as it slows or reverse neuropathy's progression. To assesses the efficacy and safety of ruboxistaurin on the progression of symptoms, signs, or functional disability in DPN. METHODS: A systematic review of the literature databases like PubMed, ProQuest, EBSCO, EMBASE, and Cochrane Central was performed up to August 2012. We included randomized controlled trials (RCTs) comparing PKC inhibitor ruboxistaurin (RBX) with control and lasting at least 6 months. Our primary outcome measure was change in neurological examination, measured by neurological total symptom score (NTSS) and vibration detection threshold (VDT). Secondary outcome measures were total quality of life (QoL), skin microvascular blood flow and others. RESULTS: Six RCTs were included in review. Change in neurological function assessed by NTSS was reported in six studies, out of which significant difference between the RBX and placebo group seen in four studies favouring treatment group while remaining two studies reported no significant difference. VDT was assessed in only one study in which no significant difference seen between RBX and placebo group. Two studies reported significant improvement in QoL data. Safety data was reported in only two studies in which none of side effect was related to RBX. CONCLUSION: RBX had effects on DPN in some studies, but the evidence is not enough for meta-analysis and firm conclusion.
Diabetes Mellitus ; Indoles ; Maleimides ; NAD ; Neurologic Examination ; Outcome Assessment (Health Care) ; Peripheral Nervous System Diseases ; Protein Kinase C ; Quality of Life ; Skin ; Vibration

OBJECTIVETo investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells.

METHODSMC-3T3E1 cells were seeded in 96-well plates at the density of 1.5×10(4) cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-28), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34)+1 µmol/L Go6983, 1 µmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells.

RESULTSCCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05); this inhibitory effect was blocked by Go6983 (P<0.05).

CONCLUSIONs A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.

3T3 Cells ; Animals ; Apoptosis ; Cell Proliferation ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Mice ; Osteoblasts ; Parathyroid Hormone ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Signal Transduction

Myocardial fibrosis is the common results of the development of a variety of heart diseases which leads to extracellular matrix protein metabolic disorders and causes cardiac remodeling owing to cardiac fibroblasts proliferation, eventually results in malignant arrhythmia, heart failure, and even the occurrence of sudden cardiac death. Effective inhibition of myocardial remodeling could prevent the occurrence of sudden death. To know the protein kinase C (PKC) effective mechanism of regulation on myocardial fibrosis, a new therapeutic target for reversing myocardial remodeling might be provided.
Animals ; Cardiomegaly ; metabolism ; Fibrosis ; Humans ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Matrix Metalloproteinase 9 ; metabolism ; Myocardium ; pathology ; Protein Kinase C ; antagonists & inhibitors ; classification ; metabolism ; Ventricular Remodeling

AIMTo study the chemical constituents of the stems of Opuntia vulgaris Mill(Cactaceae).

METHODSThe compounds of Opuntia vulgaris were isolated by chromatography of Amberlite Dowex 50 and silica gel, and identified by means of UV, IR, MS, 1D and 2D NMR.

RESULTSThree compounds were isolated and identified as: opuntin B(I), 4-hydroxyproline(II) and tyrosine(III).

CONCLUSIONCompound I is a new alkaloid.

Hydroxyproline ; chemistry ; isolation & purification ; Maleimides ; chemistry ; isolation & purification ; Molecular Conformation ; Molecular Structure ; Opuntia ; chemistry ; Phenols ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Tyrosine ; chemistry ; isolation & purification

A sensitive, rapid method for determining reduced tiopronin concentration in rat plasma has been developed by using a high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl) maleimide (NPM). The analytes were separated on a Kromasil C18 column (250 mm x 4.6 mm, 5 microm) using 0.2% glacial acetic acid aqueous solution including 0.015 mol x L(-1) KH2PO4 and acetonitrile (56:44) as a mobile phase at a flow-rate of 0.8 mL x min(-1), and fluorescence detection wavelength were set at lamda(e x) = 340 nm and lamda(e m) = 375 nm, the column temperature was 30 degrees C. The calibration curve was found to be linear over a range of 0.1 - 10.0 microg x mL(-1), the limit of quantitation was 0. 1 mg x L(-1). The coefficients of the variation for the within-run and between-run precisions ranged from 5.3% to 10.8% and 7.0% to 10.8%, respectively. The percentage of absolute recovery ranged from 73.7% to 79.7%. The method was used to determine the concentration of tiopronin in rat plasma after a single intragastric administration of 25 mg x kg(-1) tiopronin to 6 healthy male Wistar rats. The pharmacokinetic process was fitted to a two-compartment model. The method has been successfully applied to the determination of tiopronin in rat plasma.
Animals ; Area Under Curve ; Chromatography, High Pressure Liquid ; methods ; Fluorescent Dyes ; chemistry ; Male ; Maleimides ; chemistry ; Rats ; Rats, Wistar ; Tiopronin ; blood ; pharmacokinetics

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.
Antibodies ; analysis ; chemistry ; Gas Chromatography-Mass Spectrometry ; methods ; Glycosylation ; Immunoconjugates ; analysis ; chemistry ; Maleimides ; analysis ; chemistry ; Molecular Weight ; Pharmaceutical Preparations ; analysis ; chemistry ; Spectrophotometry, Ultraviolet ; methods

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.
Adenosine Triphosphate ; Animals ; Calcium ; Constriction ; Extracellular Space ; Gallic Acid ; Genistein ; HEPES ; Indoles ; Maleimides ; Nimodipine ; PC12 Cells ; Phenol ; Protein Kinase C ; Protein-Tyrosine Kinases ; Thapsigargin

A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹⁰-1 × 10⁻⁵ mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻⁶ mol/L). Also it could be removed by intracellular dialysis of either GDP-β-S (5 × 10⁻⁴mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻⁶ mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.
Animals ; Arginine Vasopressin ; pharmacology ; Ganglia, Spinal ; cytology ; Guanosine Diphosphate ; analogs & derivatives ; pharmacology ; Indoles ; Maleimides ; Membrane Potentials ; Neurons ; drug effects ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; metabolism ; Signal Transduction ; Thionucleotides ; pharmacology ; gamma-Aminobutyric Acid ; pharmacology