Solution structure of the tissue-type plasminogen activator kringle 2 domain complexed to 6-aminohexanoic acid an antifibrinolytic drug.

The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-aminohexanoic acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure. All three sets of structures are well defined, with averaged atomic root-mean-square deviations between individual structures and mean set structures of 0.77, 0.99 and 0.70 A for backbone atoms, and 1.36, 1.55 and 1.41 A for all atoms, respectively. Kringle 2 is an oblate ellipsoid with overall dimensions of approximately 34 A x 30 A x 17 A. It exhibits a compact globular conformation characterized by a number of turns and loop elements as well as by one right-handed alpha-helix and five (1 extended and 4 rudimentary) antiparallel beta-sheets. The extended beta-sheet exhibits a right-handed twist. Close van der Waals' contacts between the Cys22-Cys63 and Cys51-Cys75 disulfide bridges and the central hydrophobic core composed of the Trp25, Leu46, His48a and Trp62 side-chains are among the distinguishing features of the kringle 2 fold. The binding site for 6-AHA appears as a rather exposed cleft with a negatively charged locus defined by the Asp55 and Asp57 side-chains, and with an aromatic pocket structured by the Tyr36, Trp62, His64 and Trp72 side-chains. The Trp62 and His64 rings line the back surface of the pocket, while the Tyr36 and Trp72 rings confine it from two sides. The Trp62 and Trp72 indole rings conform a V-shaped groove. The methyl groups of Val35 also contribute lipophilic character to the ligand-interacting surface. It is suggested that the positively charged side-chains of Lys34 and, potentially, Arg69 may favor interactions with the carboxylate group of the ligand. The Trp25 and Tyr74 aromatic rings, although conserved elements of the binding site structure, seem not to undergo direct contacts with the ligand.     

Therapeutic and pharmacokinetic characterizations of an anti-amyloidogenic bis-styrylbenzene derivative for Alzheimer’s disease treatment

Alzheimer’s disease drug discovery regarding exploration into the molecules and processes has focused on the intrinsic causes of the brain disorder correlated with the accumulation of amyloid-β. An anti-amyloidogenic bis-styrylbenzene derivative, KMS80013, showed excellent oral bioavailability (F = 46.2%), facilitated brain penetration (26%, iv) in mouse and target specific in vivo efficacy in acute AD mouse model attenuating the cognitive deficiency in Y-maze test. Acute toxicity (LD₅₀ >2000 mg/kg) and hERG channel inhibition (14% at 10 μM) results indicated safety of KMS80013.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.

     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

II. Structure and specificity of the interaction between the FHA2 domain of Rad53 and phosphotyrosyl peptides

The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the first structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr-containing peptide (826)EDI(pY)YLD(832) from Rad9, with a moderate affinity (K(d) ca. 100 microM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phosphate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the +2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence specificity of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes specific sequences C-terminal to pTyr with the following consensus: XX(pY)N(1)N(2)N(3), where N(1)=Leu, Met, Phe, or Ile, N(2)=Tyr, Phe, Leu, or Met, and N(3)=Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FYMIR, bind FHA2 with K(d) values of 1.1 and 5.0 microM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences.     

Association between Weight Misperception Patterns and Depressive Symptoms in Korean Young Adolescents: National Cross-Sectional Study

Purpose

Although distortion of weight perceptions has been known as a risk factor for adolescent depression, little has been known about the relationship between weight misperception patterns and depressive symptoms. This study explored the relationship between distortion of weight misperception patterns and depressive symptoms in Korean adolescents.

Methods

The subjects of this study were 109,373 middle school students who participated in the Korea Youth Risk Behavior Web-based Survey (KYRBWS) from 2011 to 2013. By comparing the difference between Body Mass Index (BMI) and subjective perceptions of body weight, misperceptions of weight were classified into two categories: misperception of underweight and misperception of overweight.

Results

When confounding variables were adjusted, the results of the logistic regression analysis revealed that male students who perceived themselves as underweight despite their normal weight were 110% more likely to have depressive symptoms (OR = 1.10, 95% CI: 1.03–1.18) than male students with accurate weight perceptions. On the contrary, for female students, misperceptions of underweight had no significant relationship with depression symptoms. Regarding misperceptions of overweight, female students who perceived themselves as overweight despite their normal weight were 107% (OR = 1.07, 95% CI: 1.02–1.11) more likely to have depressive symptoms than female students with accurate weight perceptions. Moreover, female students who perceived themselves as overweight when they were underweight were 137% (OR = 1.37, 95% CI: 1.18–1.58) more likely to have depressive symptoms.

Conclusion

Male students who underestimate their body weight and female students who overestimate their body weight were at a greater risk of depression emotions than students with accurate weight perceptions.     

Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2

Circoviruses are the smallest circular single-stranded DNA viruses able to replicate in mammalian cells. Essential to their replication is the replication initiator, or Rep protein that initiates the rolling circle replication (RCR) of the viral genome. Here we report the NMR solution three-dimensional structure of the endonuclease domain from the Rep protein of porcine circovirus type 2 (PCV2), the causative agent of postweaning multisystemic wasting syndrome in swine. The domain comprises residues 12-112 of the full-length protein and exhibits the fold described previously for the Rep protein of the representative geminivirus tomato yellow leaf curl Sardinia virus. The structure, however, differs significantly in some secondary structure elements that decorate the central five-stranded beta-sheet, including the replacement of a beta-hairpin by an alpha-helix in PCV2 Rep. The identification of the divalent metal binding site was accomplished by following the paramagnetic broadening of NMR amide signals upon Mn(2+) titration. The site comprises three conserved acidic residues on the exposed face of the central beta-sheet. For the 1:1 complex of the PCV2 Rep nuclease domain with a 22mer double-stranded DNA oligonucleotide chemical shift mapping allowed the identification of the DNA binding site on the protein and aided in constructing a model of the protein/DNA complex.     

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.