Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

Metodologia para análise simultânea de ácido nicotínico, trigonelina, ácido clorogênico e cafeína em café torrado por cromatografia líquida de alta eficiência

A reverse phase liquid chromatography method was developed for simultaneous determination of trigonelline, caffeine, nicotinic and chlorogenic (5-CQA) acids in roasted coffee. A gradient of acetic acid/acetonitrile was used as mobile phase and detection was carried out in the UV. The samples were extracted with acetonitrile/water (5:95 v/v) at 80 ºC/10 min. Good recovery (89 to 104%), repeatability and linearity were obtained. Detection limits of 0.01, 0.15, 0.04 and 0.04 mg mL-1 were observed for nicotinic acid, trigonelline, 5-CQA and caffeine. The method, applied to arabica and robusta coffees with different degrees of roasting, was efficient and fast (~35 min) and also allowed identification of cinnamic acids.

Optimization of methodology to analyze ascorbic and dehydroascorbic acid in vegetables

In this study, different solutions to extract vitamin C were tested. High-performance liquid chromatography was chosen and the conditions were based on isocratic elution in reverse phase column. Dehydroascorbic acid was determined indirectly after its reduction using dithiothreitol. The use of metaphosphoric acid to stabilize the vitamin C was shown to be required and it was necessary to neutralize the pH of the extract to apply dithiothreitol. The average recovery was 90% in collard and tomato samples. The presence of oil did not interfere in extraction and the methodology can be used to analyze stir fried vegetables.

Uso de CLAE no controle de qualidade em produtos comerciais de Nim: reprodutibilidade da ação inseticida

The Neem tree, Azadirachta indica, provides many useful compounds that are used as pesticides. However, the efficiency in field of products like neem oil can be committed because they have not been observed reproductive content of secondary metabolic like azadirachtin. Based on reverse-phase high-performance liquid chromatography (HPLC) a new method was developed to permit the rapid quantitative analysis of azadirachtin from seeds, extracts and oil of Neem. In the present study it was evaluated the azadirachtin quantitative variation among various Neem's extracts and seeds showing the importance of quality control for reproduction of the insecticide efficiency, using S. frugiperda as target insect.

Development and validation of a novel stability indicating RP-UPLC method for simultaneous determination of nizatidine, methylparaben and propylparaben in oral liquid pharmaceutical formulation

A selective and accurate stability-indicating gradient reverse phase ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of nizatidine, methylparaben and propylparaben in pharmaceutical oral liquid formulation. The separation was achieved on Acquity UPLC TM HSS T3 1.8 µm column by using mobile phase containing a gradient mixture of solvent A (0.02 Mol L-1 KH2PO4, pH 7.5) and B (60:40 v/v mixture of methanol and acetonitrile) at flow rate of 0.4 mL min-1. Drug product was exposed to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. The developed method was validated as per international ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness.

Isolation and quantitative HPLC-PDA analysis of lupeol in phytopharmaceutical intermediate products from Vernonanthura ferruginea (Less.) H. Rob.

Prior to obtain a standardized dried extract from V. ferruginea, lupeol was first time isolated from leaves and used as chemical maker. An analytical method using HPLC-PDA for lupeol determination in V. ferruginea intermediate products was developed using a C8 reverse-phase column, acetonitrile-acetic acid (99.99:0.01, v/v) as mobile phase at 0.8 mL min-1, oven temperature at 23-25 ºC, sample injection volume at 30 µL and detection at 210 nm. The method presented linearity from 10 to 160 µg mL-1, accuracy, precision, robustness and suitable sensitivity proving to be a useful tool to the obtainment process of lupeol standardized dried extracts of V. ferruginea.

LC/ESI-MS method applied to characterization of flavonoids glycosides in B. forficata subsp. pruinosa

Bauhinia forficata is used in folk medicine for its hypoglycemiant effect. In the south of Brazil, the subspecies pruinosa is found in a region with the characteristic flora, pampa biome. This species has been consumed by the local population as a tea for diabetes treatment. We studied the chemical composition of hydroethanolic extracts using LC/ESI-MS. The leaf extracts were prepared by percolation with 50% (v/v) ethanol. The chromatographic analyses were performed using a reverse-phase system, gradient elution with acetonitrile:phosphoric acid 0.05%, and ESI-MS in the positive ion mode. The chemical profile of the flavonoids was suggested to involve four quercetin and kaempferol glycosides.

CROMATOGRAFIA POR INTERAÇÕES HIDROFILICAS (HILIC): ESTADO DA ARTE E APLICAÇÕES

Hydrophilic interaction liquid chromatography (HILIC) has been gaining increased attention for its effective separation of highly polar compounds, which include carbohydrates, amino acids, pharmaceutical compounds, proteins, glycoproteins, nucleosides, etc. Polar compounds are usually poorly retained on reverse-phase liquid chromatography (RP-HPLC) columns or have poor solubility in the apolar mobile phase of normal-phase high performance liquid chromatography (NP-HPLC). Since HILIC uses organic solvents such as ACN or MeOH ( > 70%), also used in RP-HPLC and polar stationary phases similar to NP-HPLC (bare silica, diol, amino, amide, saccharide, zwitterionic stationary phases, etc.), it represents a hybrid of the two separation modes. The high organic content in the MP leads to good compatibility with mass spectrometry (MS), increasing the detectivity. This review describes the fundamentals of HILIC and highlights some interesting applications.

Effects of intramammary infection on whey proteinograms of sheep during lactation

The study aimed to identify potential biomarkers of mammary gland infection in Santa Inês sheep. Commercial flocks of sheep provided the same hygiene, sanitary, and nutritional management under semi-intensive production systems were monitored during the lactation stage-and assessed 15, 30, 60, and 90 days after delivery (through the end of lactation and weaning). The California Mastitis Test (CMT) was performed on the mammary glands. Milk was collected for bacterial examination and protein analysis. Bacterial culture and biochemical characterization of the samples were performed. Forty-two milk samples from healthy glands (negative CMT and bacterial testing) and 43 milk samples from infected glands (positive CMT and bacterial testing) taken at the predefined time points were assessed. A rennin solution was used to obtain the whey. The proteins analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for the quantification of nine whey proteins produced in healthy glands: serum albumin, lactoferrin, IgA, IgG heavy-chain (IgG HC), IgG light-chain (IgG LC), total IgG (IgG HC + IgG LC), α-lactalbumin, β-lactoglobulin, protein with MW 15.000 Da, protein with MW 29.000 Da and eleven whey proteins secreted by infected glands, including haptoglobin and α-1-acid glycoprotein. A comparison of whey proteins between healthy and infected glands showed increases (P<0.05) in the secreted and total contents of all proteins, except for IgG LC and α-lactoalbumin. The most significant changes were observed in α-1-acid glycoprotein, lactoferrin and haptoglobin, which showed three-, five-, and seven-fold increases in secretion, respectively. This study showed that haptoglobin, α-1-acid glycoprotein, lactoferrin, albumin, and the IgA and IgG immunoglobulins may serve as potential biomarkers for mammary gland infection in sheep.

A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias

R,S-sotalol, a ß-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 µl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lex = 235 nm and lem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: CMAX = 1230 ng/ml, TMAX = 1.8 h, AUCT = 10645 ng h-1 ml-1, Kab = 1.23 h-1, a = 0.95 h-1, ß = 0.09 h-1, t(1/2)ß = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.

A micromethod for quantitation of debrisoquine and 4-hydroxydebrisoquine in urine by liquid chromatography

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred µl of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 min (D) and 16.6 min for the internal standard, metoprolol (20 µg/ml). The 5-µm CN-reverse-phase column (Shimpack, 250 x 4.6 mm) was eluted with a mobile phase consisting of 0.25 M acetate buffer, pH 5.0, and acetonitrile (9:1, v/v) at 0.7 ml/min with detection at lexcitation = 210 nm and lemission = 290 nm. The method, validated on the basis of measurements of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 390-6240 ng/ml (D) and 750-12000 ng/ml (4-OHD) linearity, and 5.7/8.2% (D) and 5.3/8.2% (4-OHD) intra/interassay precision. The method was validated using urine of a healthy Caucasian volunteer who received one 10-mg tablet of Declinax®, po, in the morning after an overnight fast. Urine samples (diuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretion of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and excreted into urine, was 6.4% and 31.9%, respectively. The hydroxylation capacity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person investigated and can be compared to reference limits of >12.5 for poor metabolizers (PM) and <12.5 for extensive metabolizers (EM). In parallel, the recovery ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: SD + 4-OHD) versus reference limits of RR <0.12 for PM and RR >0.12 for EM. The healthy volunteer was considered to be an extensive metabolizer on the basis of the debrisoquine test.

Nitric oxide, cholesterol oxides and endothelium-dependent vasodilation in plasma of patients with essential hypertension

The objective of the present study was to identify disturbances of nitric oxide radical (·NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of·NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 µM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 µM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ·NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ·NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.

Effects of sample solvent composition and injection volume on chromatographic peak profiles of methyl w- benzimidazolecarbamate and 3-butyl1-2,4-dioxo[1,2-a]-s- triazinobenzimidazole in RP-HPLC

The effects of sample solvent composition and the injection volume, on the chromatographic peak profiles of two carbamate derivatives, methyl 2-benzimidazolecarbamate (MBC) and 3-butyl-2,4-dioxo[1,2-a]-s-triazinobenzimidazole (STB), were studied using reverse phase high performance liquid chromatograph. The study examined the effects of acetonitrile percentage in the sample solvent from 5 to 50%, effects of methanol percentage from 5 to 50%, effects of pH increase from 4.42 to 9.10, and effect of increasing buffer concentration from ° to 0.12M. The effects were studied at constant and increasing injection mass and at four injection volumes of 10, 50, 100 and 200 uL. The study demonstrated that the amount and the type of the organic solvents, the pH, and the buffer strength of the sample solution can have a pronounced effect on the peak heights, peak widths, and retention times of compounds analysed. MBC, which is capable of intramolecular hydrogen bonding and has no tendency to ionize, showed a predictable increase .in band broadening and a decrease in retention times at higher eluting strengths of the sample solvent. STB, which has a tendency to ionize or to strongly interact with the sample solvent, was influenced in various ways by the changes in ths sample solvent composition. The sample solvent effects became more pronounced as the injection volume increased and as the percentage of organic solvent in the sample solution became greater. The peak height increases for STB at increasing buffer concentrations became much more pronounced at higher analyte concentrations. It was shown that the widely accepted procedure of dissolving samples in the mobile phase does not yield the most efficient chromatograms. For that reason samples should be dissolved in the solutions with higher aqueous content than that of the mobile phase whenever possible. The results strongly recommend that all the samples and standards, regardless whether the standards are external or internal, be analysed at a constant sample composition and a constant injection volume.