Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Piericidin A Aggravates Tau Pathology in P301S Transgenic Mice

ObjectiveThe P301S mutation in exon 10 of the tau gene causes a hereditary tauopathy. While mitochondrial complex I inhibition has been linked to sporadic tauopathies. Piericidin A is a prototypical member of the group of the piericidins, a class of biologically active natural complex I inhibitors, isolated from streptomyces spp. with global distribution in marine and agricultural habitats. The aim of this study was to determine whether there is a pathogenic interaction of the environmental toxin piericidin A and the P301S mutation.MethodsTransgenic mice expressing human tau with the P301S-mutation (P301S^+/+) and wild-type mice at 12 weeks of age were treated subcutaneously with vehicle (N = 10 P301S^+/+, N = 7 wild-type) or piericidin A (N = 9 P301S^+/+, N = 9 wild-type mice) at a dose of 0.5 mg/kg/d for a period of 28 days via osmotic minipumps. Tau pathology was measured by stereological counts of cells immunoreative with antibodies against phosphorylated tau (AD2, AT8, AT180, and AT100) and corresponding Western blot analysis.ResultsPiericidin A significantly increased the number of phospho-tau immunoreactive cells in the cerebral cortex in P301S^+/+ mice, but only to a variable and mild extent in wild-type mice. Furthermore, piericidin A led to increased levels of pathologically phosphorylated tau only in P301S^+/+ mice. While we observed no apparent cell loss in the frontal cortex, the synaptic density was reduced by piericidin A treatment in P301S^+/+ mice.DiscussionThis study shows that exposure to piericidin A aggravates the course of genetically determined tau pathology, providing experimental support for the concept of gene-environment interaction in the etiology of tauopathies.     

Anti-Ulcerogenic Effect of Methanolic Extracts from Enicosanthellum pulchrum (King) Heusden against Ethanol-Induced Acute Gastric Lesion in Animal Models

A natural source of medicine, Enicosanthellum pulchrum is a tropical plant which belongs to the family Annonaceae. In this study, methanol extract from the leaves and stems of this species was evaluated for its gastroprotective potential against mucosal lesions induced by ethanol in rats. Seven groups of rats were assigned, groups 1 and 2 were given Tween 20 (10% v/v) orally. Group 3 was administered omeprazole 20 mg/kg (10% Tween 20) whilst the remaining groups received the leaf and stem extracts at doses of 150 and 300 mg/kg, respectively. After an additional hour, the rats in groups 2–7 received ethanol (95% v/v; 8 mL/kg) orally while group 1 received Tween 20 (10% v/v) instead. Rats were sacrificed after 1 h and their stomachs subjected to further studies. Macroscopically and histologically, group 2 rats showed extremely severe disruption of the gastric mucosa compared to rats pre-treated with the E. pulchrum extracts based on the ulcer index, where remarkable protection was noticed. Meanwhile, a significant percentage of inhibition was shown with the stem extract at 62% (150 mg/kg) and 65% (300 mg/kg), whilst the percentage with the leaf extract at doses of 150 and 300 mg/kg was 63% and 75%, respectively. An increase in mucus content, nitric oxide, glutathione, prostaglandin E2, superoxide dismutase, protein and catalase, and a decrease in malondialdehyde level compared to group 2 were also obtained. Furthermore, immunohistochemical staining of groups 4–7 exhibited down-regulation of Bax and up-regulation of Hsp70 proteins. The methanol extract from the leaves and the stems showed notable gastroprotective potential against ethanol.     

Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp. strain PT3

Multicomponent phenol hydroxylases (mPHs) are diiron enzymes that use molecular oxygen to hydroxylate a variety of phenolic compounds. The DNA sequence of the alpha subunit (large subunit) of mPH from 4-chlorophenol (4-CP)-degrading bacterial strain PT3 was determined. Strain PT3 was isolated from oil-contaminated soil samples adjacent to automobile workshops and oil stations after enrichment and establishment of a chlorophenol-degrading consortium. Strain PT3 was identified as a member of Pseudomonas sp. based on sequence analysis of the 16S rRNA gene fragment. The 4-CP catabolic pathway by strain PT3 was tentatively proposed to proceed via a meta-cleavage pathway after hydroxylation to the corresponding chlorocatechol. This hypothesis was supported by polymerase chain reaction (PCR) detection of the LmPH encoding sequence and UV/VIS spectrophotometric analysis of the culture filtrate showing accumulation of 5-chloro-2-hydroxymuconic semialdehyde (5-CHMS) with λ_max 380. The detection of catabolic genes involved in 4-CP degradation by PCR showed the presence of both mPH and catechol 2,3-dioxygenase (C23DO). Nucleotide sequence analysis of the alpha subunit of mPH from strain PT3 revealed specific phylogenetic grouping to known mPH. The metal coordination encoding regions from strain PT3 were found to be conserved with those from the homologous dinuclear oxo-iron bacterial monooxygenases. Two DE(D)XRH motifs was detected in LmPH of strain PT3 within an approximate 100 amino acid interval, a typical arrangement characteristic of most known PHs.     

A heparin-binding growth factor, midkine, binds to a chondroitin sulfate proteoglycan, PG-M/versican.

Midkine is a heparin-binding growth factor with survival-promoting and migration-enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine-binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG-M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG-M/versican isolated from day 13 mouse embryos bound midkine with a Kd of 1.0 nM. Pleiotrophin/heparin-binding growth-associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC-I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine-binding PG-M/versican released 4-sulfated, 6-sulfated, 2, 6-disulfated and 4,6-disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.     

Nanoemulsion as a Potential Ophthalmic Delivery System for Dorzolamide Hydrochloride

Dilutable nanoemulsions are potent drug delivery vehicles for ophthalmic use due to their numerous advantages as sustained effect and high ability of drug penetration into the deeper layers of the ocular structure and the aqueous humor. The aim of this article was to formulate the antiglaucoma drug dorzolamide hydrochloride as ocular nanoemulsion of high therapeutic efficacy and prolonged effect. Thirty-six systems consisting of different oils, surfactants, and cosurfactants were prepared and their pseudoternary-phase diagrams were constructed by water titration method. Seventeen dorzolamide hydrochloride nanoemulsions were prepared and evaluated for their physicochemical and drug release properties. These nanoemulsions showed acceptable physicochemical properties and exhibited slow drug release. Draize rabbit eye irritation test and histological examination were carried out for those preparations exhibiting superior properties and revealed that they were nonirritant. Biological evaluation of dorzolamide hydrochloride nanoemulsions on normotensive albino rabbits indicated that these products had higher therapeutic efficacy, faster onset of action, and prolonged effect relative to either drug solution or the market product. Formulation of dorzolamide hydrochloride in a nanoemulsion form offers, thus, a more intensive treatment of glaucoma, a decrease in the number of applications per day, and a better patient compliance compared to conventional eye drops.     

De la nature des vacuoles spermatiques aux résultats et indications de l’IMSI (intracytoplasmic morphologically selected sperm injection)

MSOME (high-magnification motile sperm organelle morphology examination) has shown that some spermatozoa, which appear morphologically normal when viewed at × 400 or × 200, present defects such as cephalic vacuoles when viewed under high magnification. The large vacuole is described as a nuclear thumbprint, but the nature may depend on the size, number, position and depth of the vacuole. This review summarizes the data available on the nature of vacuoles, analyzes the results of IMSI (intracytoplasmic morphologically selected sperm injection) and lists its potential indications.     

Genome map of Campylobacter fetus subsp. fetus ATCC 27374

Abstract A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by Sal I, Sma I and Not I. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4–14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA , rpoB , and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes ( flaAB ) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis , respectively, were used to identify the locations of flaAB , the gyrA and the gyrB genes on the ATCC 27374 chromosome.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).