Vitamin D Prevents Hypoxia/Reoxygenation-Induced Blood-Brain Barrier Disruption via Vitamin D Receptor-Mediated NF-kB Signaling Pathways

Maintaining blood-brain barrier integrity and minimizing neuronal injury are critical components of any therapeutic intervention following ischemic stroke. However, a low level of vitamin D hormone is a risk factor for many vascular diseases including stroke. The neuroprotective effects of 1,25(OH)2D3 (vitamin D) after ischemic stroke have been studied, but it is not known whether it prevents ischemic injury to brain endothelial cells, a key component of the neurovascular unit. We analyzed the effect of 1,25(OH)₂D₃ on brain endothelial cell barrier integrity and tight junction proteins after hypoxia/reoxygenation in a mouse brain endothelial cell culture model that closely mimics many of the features of the blood-brain barrier in vitro. Following hypoxic injury in bEnd.3 cells, 1,25(OH)₂D₃ treatment prevented the decrease in barrier function as measured by transendothelial electrical resistance and permeability of FITC-dextran (40 kDa), the decrease in the expression of the tight junction proteins zonula occludin-1, claudin-5, and occludin, the activation of NF—kB, and the increase in matrix metalloproteinase-9 expression. These responses were blocked when the interaction of 1,25(OH) )₂D₃ with the vitamin D receptor (VDR) was inhibited by pyridoxal 5’-phosphate treatment. Our findings show a direct, VDR-mediated, protective effect of 1,25(OH) )₂D₃ against ischemic injury-induced blood-brain barrier dysfunction in cerebral endothelial cells.     

Association of Household and Community Socioeconomic Position and Urbanicity with Underweight and Overweight among Women in Pakistan

Background

Similar to other developing countries, Pakistan is going through a rapid nutrition transition where shift from underweight to overweight and obesity is occurring. In this paper, we report on the relationship of household socioeconomic position (SEP), community SEP and urbanicity with under- and over-weight categories of BMI among Pakistani women.

Methods

We analyzed data on 4,767 women ages 15-49 years enrolled in a nationally representative Pakistan Demographic Health Survey (PDHS) conducted in 2012-13 that employed a multistage, stratified cluster sampling design. We assessed the association of urbanicity, household and community SEP derived from household assets and utilities, with categories of body mass index (BMI) using multinomial regression analysis where normal weight (BMI 18.6-22.5) was the reference category.

Results

Thirteen percent of women were underweight (BMI <18.5), 15% pre-overweight (BMI: 22.6-24.9), 25% overweight (BMI: 25.0–29.9) and 14% were obese (BMI≥30). Pre-overweight, overweight and obesity among women increased across household wealth quintiles (HWQs) in a graded fashion whereas there was no significant difference in underweight by household wealth. Women in urban areas were more likely to be obese. There was a pronounced increase in adjusted odds ratios (aORs) for overweight/obesity across HWQs within urban areas compared to rural areas. There was a steeper gradient in aORs for obesity from 1st to 5th HWQs in high income communities compared to the middle- and low income communities. In community-level analyses, communities in urban areas were more likely to have higher levels of obesity while in rural areas, especially in Sindh, more communities were more likely to have a higher level of underweight.

Conclusion

A shift to higher overweight and obesity than underweight in Pakistan is associated with high household and community wealth as well as living in urban areas. Clustering of obesity and underweight in distinct communities afford opportunity for tailored intervention programs.     

Tinkering with heparan sulfate sulfation to steer development

Heparan sulfate (HS) proteoglycans, at the cell surface and extracellular matrix, facilitate ligand-receptor interactions crucial to many physiological processes. The distinct sulfation patterns of HS sugar chains presented by their protein core are key to HS proteoglycan activity. Tight regulation of several Golgi complex enzyme families is crucial to produce complex tissue-specific HS sequences. Several in vivo models deficient in HS biosynthesis enzymes demonstrate that developmental abnormalities result from modified HS structure. This review will discuss the plasticity of sulfation requirements on HS for activating protein ligands, which might reflect a flexible HS biosynthetic mechanism. In addition, the latest discovery of HS acting enzymes, the Sulfs, responsible for extracellular tweaking of HS sulfation levels subsequent to biosynthesis will be considered.     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution

This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at −30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (−30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.