Layered microcapsules for daunorubicin loading and release as well as in vitro and in vivo studies

Anti‐cancer drug daunorubicin (DNR) was encapsulated in preformed multilayer microcapsules and was applied in tumor treatment by in vitro cell culture and in vivo animal experiments. The microcapsules were fabricated by an alternate deposition of oppositely charged polysaccharides, i.e. chitosan and alginate onto carboxymethyl cellulose (CMC) doped CaCO₃ colloidal particles in a sequential assembly procedure, followed by crosslinking of the capsule shells with glutaraldehyde (GA) and removal of the templates by disodium ethylenediaminetetraacetic acid (EDTA). The as‐prepared microcapsules showed strong ability to induce the positively charged DNR to deposit into the microcapsule interiors. Confocal microscopy and transmission electron microscopy observed homogeneous distribution of the drug within microcapsules. The loaded DNR could be released again, following a diffusion‐controlled model at the initial stage. In vitro experiments demonstrated that the encapsulated DNR can effectively induce the apoptosis of BEL‐7402 tumor cells, as evidenced by various microscopy techniques after acridine orange (AO), Hoechst 33342, and osmium tetraoxide staining. By seeding the BEL‐7402 hepatoma cells into BALB/c/nu mice, tumors were created for the animal experiments. The results showed that the encapsulated DNR had better efficacy than that of the free drug in terms of tumor inhibition in a 4 week in vivo culture period. Copyright © 2007 John Wiley & Sons, Ltd.     

Biotin synthase is catalytic in vivo, but catalysis engenders destruction of the protein

Biotin synthase is responsible for the synthesis of biotin from dethiobiotin and sulfur. Although the name of the protein implies that it functions as an enzyme, it has been consistently reported that biotin synthase produces <1 molecule of biotin per molecule of protein in vitro. Moreover, the source of the biotin sulfur atom has been reported to be the [2Fe-2S] center of the protein. Biotin synthase has therefore been designated as a substrate or reactant rather than an enzyme. We report in vivo experiments demonstrating that biotin synthase is catalytic but that catalysis puts the protein at risk of proteolytic destruction.     

Induction of hepatic inducible nitric oxide synthase by cholesterol in vivo and in vitro

Cholesterol-rich diet impairs endothelial NO synthase (eNOS) and enhances inducible NOS (iNOS) expression. In this study, we investigated effects of cholesterol on iNOS expression in high-fat-fed rat models, HepG2 and RAW264.7 cells. The high-fat diet increased the plasma total cholesterol level 6-7 fold and low-density lipoprotein cholesterol level (LDL-C) approximately 70 fold and slightly increased the level of lipid peroxidation as determined by thiobarbituric acid-reactive substance assay. The high-fat diet also increased plasma nitric oxide (NO) concentrations up to 5 fold, and induced iNOS mRNA expression in liver. The contractile responses of the endothelium-denuded thoracic aortic rings to phenylephrine were significantly damaged in high-fat-fed rats when assessed by organ chamber study. Treatment with estrogen for 4 days failed to reduce iNOS expressions as well as aortic contractility, although it improved lipid profiles. In cultured HepG2 or murine macrophage RAW264.7 cells, 3 days treatment with either 25-hydroxycholesterol or 7-ketocholesterol induced iNOS mRNA expression, as determined by RT-PCR. Our data suggested that the chronic exposure of hepatocytes and macrophage cells to high concentration of cholesterol or oxysterols may induce iNOS expression and subsequent synthesis of NO, which may be important in the pathogenesis of atherosclerosis.     

80 kDa mouse sperm protein as a substrate of protein kinase C.

Calcium and phospholipid-dependent protein kinase (PKC) activity was detected mainly in the cytosol of the mouse sperm. The PKC in the cytosol fraction was partially purified by ion-exchange chromatography. Using the partially purified PKC, the phosphorylation of PKC substrates was examined in vitro. The phosphorylation of the 80 kDa protein was enhanced by phorbol ester treatment in vitro as well as in vivo. The partial amino acid sequence of this protein was homologous with that of guanosine 5'-cyclic monophosphate (cGMP)-dependent protein kinase and myosin light chain kinase, both of which are related to ligand-receptor-transduction. The present data suggest that the activation of PKC and subsequent specific protein phosphorylation might be involved in the regulation of the zona pellucida-induced acrosome reaction.     

Determination of stimulants and narcotics as well as their in vitro metabolites by online CE-ESI-MS

A simple, rapid and sensitive CE-ESI-MS method for the simultaneous analysis of seven stimulants and narcotics (amphetamine, ephedrine, methadone, pethidine, tetracaine, codeine and heroin) was developed. The CE-ESI-MS experimental conditions were optimized as follows: 20?mmol/L ammonium acetate with pH 9.0 as running buffer, the separation voltage of 22?kV and the sheath liquid of isopropanol/water (1:1 v/v) containing 7.5?mmol/L acetic acid with 3.0?μL/min flow rate. Under the optimized conditions, the stimulants and narcotics were well separated within 4.6?min using a 70-cm length fused-silica capillary (50?μm id). The detection limits (S/N=3) of the CE-ESI-MS analysis were in the range of 0.40-1.0?ng/mL. Method repeatability of intra-day and inter-day was satisfactory. The recoveries obtained from the analysis of spiked urine samples were between 84.1 and 108%. The developed method was successfully applied for the simultaneous analysis of methadone, pethidine and codeine and their in vitro metabolites.     

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

Background  

Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions.     

Resveratrol stimulates glucose transport in C2C12 myotubes by activating AMP-activated protein kinase

trans-Resveratrol (t-RVT), a naturally occurring polyphenol found in Polygonum cuspidatum, grape, and red wine, has been reported to have anti-inflammatory, cardioprotective, and cancer chemopreventive properties. However antidiabetic effect of t-RVT has not yet been reported. In this study, we show that t-RVT increases glucose uptake in C2C12 myotubes by activating AMP-activated protein kinase (AMPK), uncovering an antidiabetic potential of t-RVT for the first time. AMPK plays a central role in the regulation of glucose and lipid metabolism, and hence it is considered a novel therapeutic target for metabolic syndrome such as type 2 diabetes. t-RVT significantly induced glucose uptake in C2C12 cells, via AMPK activation, but not a phosphatidylinositol-3 kinase (PI-3 kinase) signal pathway. The induced glucose uptake was attenuated by pretreatment with a pharmacological inhibitor for AMPK, indicating that the effect of t-RVT primarily depends on AMPK activation. However, in the presence of insulin, t-RVT also potentiated the effect of insulin on glucose uptake via AMPK activation, which led to further activation of PI-3 kinase/Akt signal pathway.     

Phosphorylation of glycogen synthase kinase-3beta at serine-9 by phospholipase Cgamma1 through protein kinase C in rat 3Y1 fibroblasts

Phospholipase Cgamma1 (PLCgamma1) plays an important role in controlling cellular proliferation and differentiation. PLCgamma1 is overexpressed in some tumors, and its overexpression induces solid tumors in nude mice. However, the regulatory mechanisms underlying PLCgamma1-induced cell proliferation are not fully understood. Here we show that overexpression of PLCgamma1 highly phosphorylated glycogen synthase kinase-3beta (GSK-3beta) at serine-9 in 3Y1 fibroblasts. Inhibition of protein kinase C (PKC)s with GF109203X abrogated GSK-3beta phosphorylation by PLCgamma1. We also found that steady-state level of cyclin D1 protein, but not cyclin D1 mRNA, was highly elevated in response to serum stimulation in PLCgamma1-transfected cells as compared with vector-transfected cells. Since GSK-3beta is involved in cyclin D1 proteolysis in response to mitogenic stimulation, PLCgamma1-mediated GSK-3beta phosphorylation may function as a regulation of cyclin D1 accumulation in PLCgamma1-overexpressing cells.     

Organization of polyhydroxyalkanoate synthase for in vitro polymerization as revealed by atomic force microscopy

Individual polyhydroxyalkanoate synthase molecules from Ralstonia eutropha (PhaCRe) were directly visualized on highly oriented pyrolytic graphite (HOPG) by atomic force microscopy (AFM). PhaCRe molecule was observed as a spherical particle of 2.9 +/- 0.4 nm in height and 28 +/- 4 nm in width. In vitro polymerization reaction on HOPG was carried out for 5 min by reacting the PhaCRe molecules with (R)-3-hydroxybutyryl-CoA monomers. The reaction product was then observed after the removal of water solution. Several PhaCRe molecules associated with each other to form an assembly, which was attached to a fibrillar structure of ca. 0.2-0.3 nm in height. The fibrillar structure that elongated from the PhaCRe assembly was interpreted as the poly[(R)-3-hydroxybutyrate] polymer chain. High resolution AFM suggested that the PhaCRe assembly was composed of 3-4 subunits of PhaCRe molecules. This was further supported by SDS-PAGE analysis of the cross-linked PhaCRe enzyme. These results suggest that more than two subunits of PhaCRe are necessary for the in vitro polymerization of PHB molecular chains.     

Modification of fullerene C60 by phosphorylated diazo compounds

New representatives of phosphorylated methanofullerenes were prepared by the reactions of [60]fullerene with O,O-diethyl -diazoethyl- and O,O-diethyl -diazobenzylphosphonates. Electrochemical reduction of the above-mentioned products proceeded stepwise and reversibly.     

Ruthenium complexes as protein kinase inhibitors

[reaction: see text] Replacing complex natural products with simple metal complexes could lead to a new class of metallopharmaceuticals in which the metal center plays mainly a structural role. A strategy is introduced for the creation of ruthenium complex-based protein kinase inhibitors 1 (X = CO or CH(2)), morphed out of the class of indolocarbazole inhibitors with the alkaloid staurosporine as its most prominent member.     

吴茱萸碱的合成及其结构表征和体内抗肿瘤作用

以色胺和N-甲基靛红酸酐作为起始原料合成了吴茱萸碱;利用元素分析仪、红外光谱仪、核磁共振谱仪、质谱仪等对合成产物进行了结构表征,并研究了其对小鼠移植性肉瘤S180和小鼠肝癌H22的体内抗肿瘤作用.结果表明,所合成的吴茱萸碱在高、中、低剂量(20 mg·kg-1、10 mg·kg-1、5 mg·kg-1)下均对S180和...     

Some characteristics of cotton plant protein kinase C

The change in the activity of the protein kinase C of cotton plant shoots as functions of the temperature, the time of incubation, and the concentration of Mg²⁺ ions has been investigated. It has been established that the maximum activity is shown at a temperature of 25–40°C an incubation time of 30 min, pH 7.0, and a Mg²⁺ concentration of 10 mM.Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 141–145, January–February, 1995. Original article submitted October 17, 1994.     

A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride

Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C₃N₄) conjugates triggered by Zr⁴⁺ ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC₅₀ value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.     

Focal adhesion kinase: from in vitro studies to functional analyses in vivo

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to have key roles in cell migration, proliferation and survival. FAK activity can be stimulated in response to several types of extracellular ligands, including components of the extracellular matrix and growth factors, suggesting that FAK is an important integrator of multiple cues in the extracellular milieu. Recently, major progress has been made in understanding the molecular mechanisms regulating FAK activity. In particular, several novel proteins have been identified that can bind to FAK and inhibit its activity and associated cellular functions, including cell motility and invasion. Consistent with its critical functions in signal transduction, FAK also plays a pivotal role in mouse development. The inactivation of FAK in mice results in embryonic lethality around E8.5; this early embryonic lethal phenotype limits the use of the FAK total knockout mouse model for studying FAK function in later embryonic development stages and in adult mice. To overcome this problem, three independent groups created FAK/flox mice and generated several different FAK tissue-specific knockout mice models. Here we summarize the progress that has been made regarding the regulation of FAK-mediated signaling events in cell-based systems and also highlight the in vivo functions of FAK in a number of terminally differentiated cell lineages, including vascular endothelial cells, cardiomyocytes, neuronal cells, keratinocytes and several cancerous cell types.     

Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS.     

A small-molecule catalyst of protein folding in vitro and in vivo

BACKGROUND: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo'). RESULTS: A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo. CONCLUSIONS: A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.     

Modulation of the substrate specificity of purified human protein kinase C by its activators

The substrate specificity of purified human protein kinase C was modulated by 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), dioleoylglycerol, arachidonic acid and lipid A when histone type III-S and myelin basic protein were used as phosphate acceptors. Each activator also showed a distinct pattern in the stimulation of phosphorylation of the kinase itself and of cytosolic placental proteins. The nature of the substrate and the presence of calcium and phospholipid determined the magnitude of the effect observed upon addition of all activators and also the dose dependency of kinase activation by TPA. The apparent Km value for phosphorylation of histone type III-S by the kinase activated by phorbol ester alone and with calcium was 20-30 fold higher than that observed for the enzyme activated by calcium and phospholipid. These observations indicate that the nature and extent of cellular response induced by the activation of C-kinase(s) may be determined by the type of cellular stimulus.