Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Piper sarmentosum Roxb. Inhibits Angiotensin-Converting Enzyme Activity in Phorbol 12-Myristate-13-Acetate-Induced Endothelial Cells.

Angiotensin-converting enzyme (ACE) plays a crucial role in the pathogenesis of hypertension. Piper sarmentosum Roxb., an herb known for its antihypertensive effect, lacks a comprehensive understanding of the mechanism underlying its antihypertensive action. This study aimed to elucidate the antihypertensive mechanism of aqueous extract of P. sarmentosum leaves (AEPS) via its modulation of the ACE pathway in phorbol 12-myristate-13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were divided into five groups: control, treatment with 200 µg/mL AEPS, induction 200 nM PMA, concomitant treatment with 200 nM PMA and 200 µg/mL AEPS, and treatment with 200 nM PMA and 0.06 µM captopril. Subsequently, ACE mRNA expression, protein level and activity, angiotensin II (Ang II) levels, and angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression in HUVECs were determined. AEPS successfully inhibited ACE mRNA expression, protein and activity, and angiotensin II levels in PMA-induced HUVECs. Additionally, AT1R expression was downregulated, whereas AT2R expression was upregulated. In conclusion, AEPS reduces the levels of ACE mRNA, protein and activity, Ang II, and AT1R expression in PMA-induced HUVECs. Thus, AEPS has the potential to be developed as an ACE inhibitor in the future.

Exploring magnesium myristate for its dual functionality as a binder and lubricant in the formulation of tablet.

Purpose: To explore 'magnesium myristate' for its dual functionality as a lubricant and binder in the formulation of tablets. Methods: Using (DoE), tablet formulations using magnesium myristate and conventional excipients (magnesium stearate and PVP K30) were developed by wet granulation technique. The prepared granules and formulated tablets were evaluated for pre- and post-compression parameters, respectively. Results: Magnesium myristate exhibited excellent flow properties. The optimized formulations containing magnesium myristate exhibited increased hardness and in vitro drug release in comparison to conventional excipients. f2 similarity index for in vitro drug release showed no significant variations with optimized formulations and with the marketed formulations. Conclusion: Magnesium myristate shows a promising replacement for conventional excipients as both a lubricant and binder in tablet formulation.

Metabonomics and Transcriptomic Analysis of Free Fatty Acid Synthesis in Seedless and Tenera Oil Palm.

Oil palm, a tropical woody oil crop, is widely used in food, cosmetics, and pharmaceuticals due to its high production efficiency and economic value. Palm oil is rich in free fatty acids, polyphenols, vitamin E, and other nutrients, which are beneficial for human health when consumed appropriately. Therefore, investigating the dynamic changes in free fatty acid content at different stages of development and hypothesizing the influence of regulatory genes on free fatty acid metabolism is crucial for improving palm oil quality and accelerating industry growth. LC-MS/MS is used to analyze the composition and content of free fatty acids in the flesh after 95 days (MS1 and MT1), 125 days (MS2 and MT2), and 185 days (MS3 and MT3) of Seedless (MS) and Tenera (MT) oil palm species fruit pollination. RNA-Seq was used to analyze the expression of genes regulating free fatty acid synthesis and accumulation, with differences in genes and metabolites mapped to the KEGG pathway map using the KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis method. A metabolomics study identified 17 types of saturated and 13 types of unsaturated free fatty acids during the development of MS and MT. Transcriptomic research revealed that 10,804 significantly different expression genes were acquired in the set differential gene threshold between MS and MT. The results showed that FabB was positively correlated with the contents of three main free fatty acids (stearic acid, myristate acid, and palmitic acid) and negatively correlated with the contents of free palmitic acid in the flesh of MS and MT. ACSL and FATB were positively correlated with the contents of three main free fatty acids and negatively correlated with free myristate acid. The study reveals that the expression of key enzyme genes, FabB and FabF, may improve the synthesis of free myristate in oil palm flesh, while FabF, ACSL, and FATB genes may facilitate the production of free palmitoleic acid. These genes may also promote the synthesis of free stearic acid and palmitoleic acid in oil palm flesh. However, the FabB gene may inhibit stearic acid synthesis, while ACSL and FATB genes may hinder myristate acid production. This study provides a theoretical basis for improving palm oil quality.

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

BACKGROUND: Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. METHODS AND RESULTS: SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCß knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCß inhibitor LY333531. NADH (30-300 µmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCß small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCß expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCß inhibitor LY333531. CONCLUSIONS: NADH-induced inhibition of endothelial SK channel function is mediated via PKCß. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Water in oil microemulsions containing NaCl for transdermal delivery of fluorouracil / 药学学报

This study is to prepare the W/O microemulsion containing NaCl and fluorouracil (5-Fu) as a model drug to investigate the transdermal characteristics and skin irritation of the microemulsion in vitro. Isopropylmyristate (IPM) acting as oil phase, Aerosol-OT (AOT) as surfactant, Tween 85 as cosurfactant, NaCl solution was added dropwise to the oil phase to prepare W/O microemulsion at room temperature using magnetic stirring, and then 5-Fu powder was added. According to the area of microemulsion based on the pseudo-tertiary phase diagrams, the optimum formulation was screened initially. And the permeation flux of fluorouracil across excised mice skin was determined in vitro using Franz diffusion cells to study the influence of the amount of water and the drug loading capacity and optimize the formulation further. Refer to 5-Fu cream, the irritation of microemulsion on the rat skin was studied. The optimum formulation was composed of 0.7% (w/v) 5-Fu, 50% NaCl solution (0.05 mol x L(-1)), 20% mix-surfactant (AOT/Tween 85, K(m) = 2) and 29.3% oil (IPM). The cumulative amount of fluorouracil permeated in 12 h was (2 013.4 +/- 41.6) microg x cm(-2), 20.23 folds and 10.38 folds more than 0.7% fluorouracil aqueous solution and 2.5% (w/w) fluorouracil cream, respectively. Microemulsion exhibited some irritation, but could be reversed after drug withdrawal. The addition of NaCl significantly increased the content of water and the drug loading in microemulsion systems. The NaCl/AOT-Tween 85/IPM microemulsion system promoted the permeation of fluorouracil greatly, which may be a promising vehicle for the transdermal delivery of fluorouracil and other hydrophilic drug.

Molecular Identification of the Vaccine Strain from the Inactivated Rabies Vaccine

Since 1994, several different inactivated rabies vaccines have been used to immunize domestic animals such as dogs, cats, and cattle in South Korea. The Korean Veterinary Authority has conducted safety and efficacy testes of inactivated vaccines using laboratory animals. In this study, we applied a molecular method to investigate the genetic characterization of the rabies virus (RABV) genes in six commercial inactivated rabies vaccines, and determined the efficiency of two extraction reagents (i.e., sodium citrate or isopropyl myristate) to separate the vaccine antigens from the antigen/adjuvant complexes. Six partial nucleocapsid (N: 181 bp) and five partial glycoprotein (G: 306 bp) genes were successfully amplified with specific primer sets, which demonstrated that sodium citrate is more efficient than isopropyl myristate in extracting viral RNA from inactivated gel vaccines. In addition, we identified the viral strain of the vaccine by analyzing the nucleotide sequences of the N and the G genes. The nucleotide similarity of the partial N and G genes ranged from 97.1 to 99.4% and from 91.8 to 100% among rabies vaccine strains, respectively, indicating that each manufacturer used different rabies virus strains to produce their vaccines. The molecular method used in this study could also be used to identify viral strains in other inactivated vaccines.

Microemulsion-based gel of fluorouracil for transdermal delivery / 药学学报

This study is to prepare the microemulsion-based gel based on the W/O microemulsion and fluorouracil (5-Fu) as a model drug to study the transdermal characterization and observe its skin irritation of the microemulsion-based gel in vitro. IPM acted as oil phase, AOT as surfactant, Tween 85 as cosurfactant, water was added dropwise to the oil phase to prepare W/O microemulsion at room temperature using magnetic stirring, then 5-Fu powder was added. The gelatin was used as substrate to prepare 5-Fu microemulsion-based gel. The permeation flux of 5-Fu from 5-Fu microemulsion-based gel across excised mice skin was determined in vitro using Franz diffusion cell to study the influence of the amount of gelatin and the drug loading capacity. Refer to 5-Fu cream, the irritation of microemulsion and microemulsion-based gel on the rat skin was studied. Based on the water/AOT/Tween 85/IPM microemulsion, only the gelatin can form the microemulsion-based gel. At 25 degrees C, 32 degrees C and 40 degrees C, the amount of gelatin required for the formation of microemulsion-based gel were 7%, 14% and more than 17%, respectively. The 12 h transdermal cumulated permeation amount of 5-Fu from microemulsion-based gel containing 14% gelatin and 0.5% drug loading were (876.5 +/- 29.1) microg x cm(-2), 12.3 folds and 4.5 folds more than 0.5% 5-Fu aqueous solution and 2.5% (w/w) 5-Fu cream, respectively. Microemulsion-based gel exhibited some irritation, but could be subsided after drug withdrawal. Microemulsion-based gel may be a promising vehicle for transdermal delivery of 5-Fu and other hydrophilic drug.

Water in oil microemulsions for transdermal delivery of fluorouracil / 药学学报

An Aersol-OT (AOT) included microemulsion containing fluorouracil was prepared by using appropriate proportion of oil, co-surfactant and water for increasing the drug transdermal delivery ability. According to the area of microemulsion basing on the pseudo-tertiary phase diagrams, the optimum formulation was screened initially. And the permeation flux of fluorouracil across excised mice skin was determined in vitro using Franz diffusion cell to optimize the formulation further. The effect of the kind of co-surfactant, the content of water, the content of mixed surfactant, the mass ratio of surfactant/cosurfactant (Km) and the drug load on skin permeation of fluorouracil were evaluated. The optimum formulation was composed of 0.5% (w/v) fluorouracil, 30% water, 20% mix-surfactant (AOT/Tween 85, Km = 2) and 49.5% oil (IPM). The cumulative amount permeated of fluorouracil in 12 hour was 1 355.5 microg x cm(-2), 19.1 folds and 7 folds more than 0.5% fluorouracil aqueous solution and 2.5% (w/w) fluorouracil cream, respectively. The permeation of this microemulsion accorded with first-order model. The water/AOT/Tween 85/IPM microemulsion system promoted the permeation of fluorouracil greatly, which may be a promising vehicle for the transdermal delivery of fluorouracil and other hydrophilic drug.

A Case of Allergic Contact Dermatitis from Silmazine(R) Cream / 대한피부과학회지

Silmazine(R) cream is an antibiotic agent widely used in burn therapy. It consists of Propylene glycol, Stearyl alcohol, Isopropyl Myristate, Sorbitan mono-oleate, Methyl-p-hydroxybenzoate, Polyoxyl 40 stearate and varseline. A 24-year- old female presented with well-demarcated erythematous papules and vesicles with an itching sensation on the dorsal area of her right hand. She had applied Silmazine(R) cream on the dorsal area of her right handfor 4 days and the skin lesion became aggravated. A patch test with Silmazine(R) cream 'as is' showed a positive reaction and propylene glycol and stearyl alcohol, ingredients in Silmazine(R) cream, revealed a positive reaction. These two agents are known as weak sensitizers that can produce allergic contact dermatitis. There are some reports of allergic contact dermatitis from propylene glycol and stearyl alcohol used topically. As far as we know, there are no reports of allergic contact dermatitis from propylene glycol and stearyl alcohol in the Silmazine(R) cream (Silver sulfadiazine) that is commonly used as topical antibiotic medication for burns. We report this rare case of allergic contact dermatitis from propylene glycol and stearyl alcohol in Silmazine(R) cream (Silver sulfadiazine).

Study on alpha-asarone reservoir-type patch / 中国中药杂志

<p><b>OBJECTIVE</b>To prepare the alpha-asarone reservoir patch and investigate its release and transdermal absorption characteristics in vitro. The efficient enhancers were chosen to improve the drug's permeation rate.</p><p><b>METHOD</b>The alpha-asarone reservoir patch was prepared using 1% hydroxypropyl methylcellulose (HPMC) of ethanol solution as medium and ethylene vinyl acetate (EVA) membrane to control the release of drug. The Franz diffusion cells were used and several permeation enhancers were evaluated. High performance liquid chromatorgraphy (HPLC) was used to determine alpha-asarone's content and permeation rate.</p><p><b>RESULT</b>The release mechanisms of alpha K-asarone patch in vitro coincided with zero-order kinetic. 30% ethanol cooperates with 1% Isopropyl Myristate (IPM) have the best effect on permeation of the patch. The permeation rate reaches (20.67 +/- 1.33) microg x cm(-2) h(-1).</p><p><b>CONCLUSION</b>Ethanol combined with IPM is good permeation enhancer, which facilitated the permeation of alpha K-asarone to fit the clinical requirements. However, the further studies of the skin's stimulation and bioavailability are needed.</p>

Isopropyl myristate molecular gels and drug-loaded transdermal capability / 药学学报

<p><b>AIM</b>To prepare of isopropyl myristate (IPM) molecular gels and investigate of its transdermal capability.</p><p><b>METHODS</b>Microstructure of IPM gels was studied by scanning electron microscope (SEM) and optical microscope (OM). The rheology and thixotropy of IPM gels were investigated by viscosity. Triptolide was used as model drug to investigate its transdermal capability.</p><p><b>RESULTS</b>The microstructure of IPM gels was a three-dimension network formed by the aggregation of Span 60 in IPM, which was rod-like tubular aggregate. It has good rheology and thixotropy. There was a good linear correlation between the accumulative permeated amount per unit area and the time for triptolide-loaded IPM gels. The permeation process agreed with zero order pharmacokinetics. The average permeability through rat skin for triptolide was 19.26 ng x cm(-2) x h(-1), which was 2.92 times of triptolide unguents obtained commercially available.</p><p><b>CONCLUSION</b>Isopropyl myristate molercular gel can be formed by span 60 assemblies. Transdermal capability drug-loaded IPM gels was better than that of triptolide unguents.</p>

Research on components of Cornus officinalis extracted by supercritical carbon dioxide / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Cornus officinalis extracted by supercritical carbon dioxide fluid extraction (SFE).</p><p><b>METHOD</b>The process was performed at 40 centigrade with pressures of 15 MPa for 2 hours and with CO2 fluid and gas at the flow rate of 22.0 kg x h(-1) and 18.0 kg x h(-1) respectively. The chemical constituents of the SFE extractions were determined by GC-MS.</p><p><b>RESULT</b>The total amount of extractable substances or yields by SFE is 2.42% (mass). 31 Chemical constituents were identified and their relative contents were determined by normalization method of area.</p><p><b>CONCLUSION</b>The major components identified in the extractions are 1,2-benzenedicarboxylic acid, butyl 2-methylpropyl ester, isopropyl myristate etc.</p>

Myristate exchange in glycolipid A and VSG of African trypanosomes

The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet know, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange