Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences

Hyperinsulinism-hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype-phenotype correlations

Background: Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives: To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods: Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results: Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 µmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion: Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations

Phytochemical and Pharmacological potential of Annona cherimola-A Review

Several plant remedies have been employed in various medicinal systems for the treatment and management of different diseases. During past several years, there has been growing interest among the usage of various medicinal plants from traditional system of medicine for the treatment of different ailments. Traditional system of medicine consists of large number of plants with various medicinal and pharmacological importances and hence represents a priceless tank of new bioactive molecules. Annona cherimola Miller is a multipurpose tree with edible fruits and is one of the sources of the medicinal products. This review attempts to encompass the available literature on Annona cherimola with respect to its pharmacognostic characters, phytochemical constituents, pharmacological activities and traditional uses

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5

Qualitative Analysis and Antibacterial Activity of Pelargonium graveolenL’Herit

Medicinal plants are an important source of phytochemicals that offer traditional medicinal treatment of various ailments and one of the plants is Pelargonium Graveolens which was grown in Hyderabad province. The preliminary screening of their aerial leaves showed best results the presence of different phytochemical like alkaloids, flavonoids, glycosides, phenol, sterol and lignin found in Methanolic and Ethyl acetate extract. However chloroform extract revealed the absence of alkaloids and sterols, where as in Water extract flavonoids, phenol, sterol and lignin. The best resulted extracts from preliminary screening test were subjected to Antimicrobial studies like some on gram positive and gram negative bacterial strains which exhibited a significant effect.  The both ethyl acetate  and methanolic extracts were showed the similar zone of inhibition on gram positive bacteria (S.aurea and B.Subtilus) whereas  ethyl acetate  extract positive inhibition on k.pneumonia  when compare to methanolic  extract  and which are more active suppression on gram negative  bacterial (E.coli) in comparison with the standard antibiotic.

Qualitative Analysis and Antibacterial Activity of Pelargonium graveolenL’Herit

Medicinal plants are an important source of phytochemicals that offer traditional medicinal treatment of various ailments and one of the plants is Pelargonium Graveolens which was grown in Hyderabad province. The preliminary screening of their aerial leaves showed best results the presence of different phytochemical like alkaloids, flavonoids, glycosides, phenol, sterol and lignin found in Methanolic and Ethyl acetate extract. However chloroform extract revealed the absence of alkaloids and sterols, where as in Water extract flavonoids, phenol, sterol and lignin. The best resulted extracts from preliminary screening test were subjected to Antimicrobial studies like some on gram positive and gram negative bacterial strains which exhibited a significant effect.  The both ethyl acetate  and methanolic extracts were showed the similar zone of inhibition on gram positive bacteria (S.aurea and B.Subtilus) whereas  ethyl acetate  extract positive inhibition on k.pneumonia  when compare to methanolic  extract  and which are more active suppression on gram negative  bacterial (E.coli) in comparison with the standard antibiotic.

Direct replacement of oral sodium benzoate with glycerol phenylbutyrate in children with urea cycle disorders

Long-term management of urea cycle disorders (UCDs) often involves unlicensed oral sodium benzoate (NaBz) which has a high volume and unpleasant taste. A more palatable treatment is licenced and available (glycerol phenylbutyrate [GPB], Ravicti) but guidance on how to transition patients from NaBz is lacking. A retrospective analysis of clinical and biochemical data was performed for eight children who transitioned from treatment with a single ammonia scavenger, NaBz, to GPB at a single metabolic centre; UCDs included arginosuccinic aciduria (ASA) (n = 5), citrullinaemia type 1 (n = 2) and carbamoyl phosphate synthetase I deficiency (CPS1) (n = 1). Patients transitioned either by gradual transition over 1–2 weeks (n = 3) or direct replacement of NaBz with GPB (n = 5). Median initial dose of GPB was 8.5 mL/m2/day based on published product information; doses were revisited subsequently in clinic and titrated individually (range 4.5–11 mL/m2/day). Pre-transition and post-transition mean ammonia levels were 37 μmol/L (SD 28 μmol/L) and 29 μmol/L (SD 22 μmol/L), respectively (p = 0.09), and mean glutamine levels were 664 μmol/L (SD 225 μmol/L) and 598 μmol/L (SD 185 μmol/L), respectively (p = 0.24). There were no reductions in levels of branched chain amino acids. No related adverse drug reactions were reported. Patients preferred GPB because of its lower volume and greater palatability. Direct replacement of NaBz with GPB maintained metabolic control and was simple for the health service and patients to manage. A more cautious approach with additional monitoring would be warranted in brittle patients and patients whose ammonia levels are difficult to control

Analgesic activity of Cissus quadrangularis linn with Zingiber officinale rosc in male wistar rats

Background: The global scenario, human beings are using various forms of treatment for relief of pain; among them, medicinal plant products have gained popularity because of its wide range of use and less side effects.Methods: Adult Albino rats of either sex were selected and divided into 5 groups. The Eddy’s hot plate was used and maintained temperature (55±0.5ºC), the animals were placed on the hot plate and the time taken for paw licking or jumping was recorded and considered as nociceptive response. The reaction time was observed on 0, 30, 60, and 90 minute.Results: The hot plate reaction time in sec was collected in the intervals of 0, 30, 60 and 90 minutes in all groups. Reaction times as follows: group III (Cissus quadrangularis Linn.) were 2.18±0.04, 3.13±0.05**, 5.83±0.05**, 5.39±0.04**;  Group IV (Zingiber officinale Rosc.) were 2.12±0.03, 4.13±0.04**, 7.43±0.07**, 7.16±0.06**; Group V Cissus quadrangularis Linn+ Zingiber officinale Rosc.) were 2.21±0.75, 4.67±0.98**, 8.15±0.89**, 9.02±0.75**; Group II (Dexamethasone) were 2.14±0.05, 5.53±0.05**, 8.14±0.06**, 10.08±0.10** respectively, Results are presented as mean±SEM, (n=6), *p<0.01, **p<0.05 dunnet test used as compared to control.Conclusions: Present study reveals that, the combination treatment of Zingiber officinale Rosc. + Cissus quadrangularis Linn. has been shown significant analgesic effect. High analgesic effect was observed with combination therapy; the effect was shown same as standard drug dexamethasone

Urea Cycle Related Amino Acids Measured in Dried Bloodspots Enable Long-Term In Vivo Monitoring and Therapeutic Adjustment

BACKGROUND: Dried bloodspots are easy to collect and to transport to assess various metabolites, such as amino acids. Dried bloodspots are routinely used for diagnosis and monitoring of some inherited metabolic diseases. METHODS: Measurement of amino acids from dried blood spots by liquid chromatography-tandem mass spectrometry. RESULTS: We describe a novel rapid method to measure underivatised urea cycle related amino acids. Application of this method enabled accurate monitoring of these amino acids to assess the efficacy of therapies in argininosuccinate lyase deficient mice and monitoring of these metabolites in patients with urea cycle defects. CONCLUSION: Measuring urea cycle related amino acids in urea cycle defects from dried blood spots is a reliable tool in animal research and will be of benefit in the clinic, facilitating optimisation of protein-restricted diet and preventing amino acid deprivation