Evaluation of stem aqueous extract and synthesized silver nanoparticles using Cissus quadrangularis against Hippobosca maculata and Rhipicephalus (Boophilus) microplus

The present study was to determine the efficacies of anti-parasitic activities of synthesized silver nanoparticles (Ag NPs) using stem aqueous extract of Cissus quadrangularis against the adult of hematophagous fly, Hippobosca maculata (Diptera: Hippoboscidae), and the larvae of cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Contact toxicity method was followed to determine the potential of parasitic activity. Twelve milliliters of stem aqueous extract of C. quadrangularis was treated with 88ml of 1mM silver nitrate (AgNO(3)) solution at room temperature for 30min and the resulting solution was yellow-brown color indicating the formation extracellular synthesis of Ag NPs. The synthesized Ag NPs were characterized with UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscope (FESEM) and energy dispersive X-ray (EDX) spectroscopy. The synthesized Ag NPs were recorded by UV-visible spectrum at 420nm and XRD patterns showed the nanoparticles crystalline in nature. FTIR analysis confirmed that the bioreduction of Ag((+)) ions to Ag NPs were due to the reduction by capping material of plant extract. FESEM image of Ag NPs showed spherical and oval in shape. By using the Bragg's Law and Scherrer's constant, the average mean size of synthesized Ag NPs was 42.46nm. The spot EDX analysis showed the complete chemical composition of the synthesized Ag NPs. The mortality obtained by the synthesized Ag NPs from the C. quadrangularis was more effective than the aqueous extract of C. quadrangularis and AgNO(3) solution (1mM). The adulticidal activity was observed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the adult of H. maculata with LC(50) values of 37.08, 40.35 and 6.30mg/L; LC(90) values of 175.46, 192.17 and 18.14mg/L and r(2) values of 0.970, 0.992 and 0.969, respectively. The maximum efficacy showed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the larvae of R. (B.) microplus with LC(50) values of 50.00, 21.72 and 7.61mg/L; LC(90) values of 205.12, 82.99 and 22.68mg/L and r(2) values of 0.968, 0.945and 0.994, respectively. The present study is the first report on antiparasitic activity of the experimental plant extract and synthesized Ag NPs. This is an ideal eco-friendly and inexpensive approach for the control of H. maculata and R. (B.) microplus.     

Heparin binding induces conformational changes in Adeno-associated virus serotype 2

Adeno-associated virus serotype 2 (AAV2) uses heparan sulfate proteoglycan as a cell surface-attachment receptor. In this study the structures of AAV2 alone and complexed with heparin were determined to 18 Å resolution using cryo-electron microscopy and three-dimensional image reconstruction. A difference map showed positive density, modeled as heparin, close to the icosahedral twofold axes and between the protrusions that surround the threefold axes of the capsid. Regions of the model near the threefold place the receptor in close proximity to basic residues previously identified as part of the heparin binding site. The region of the model near the twofold axes identifies a second contact site, not previously characterized but which is also possibly configured by heparin binding. The difference map also revealed two significant conformational changes: (I) at the tops of the threefold protrusions, which have become flattened in the complex, and (II) at the fivefold axes where the top of the channel is widened possibly in response to movement of the HI loops in the capsid proteins. Ordered density in the interior of the capsid in the AAV2–heparin complex was interpreted as nucleic acid, consistent with the presence of non-viral DNA in the expressed capsids.     

Heat shock-induced attenuation of hydroxyl radical generation and mitochondrial aconitase activity in cardiac H9c2 cells

A mild heat shock (hyperthermia) protects cells from apoptotic and necrotic deaths by inducing overexpression of various heat shock proteins (Hsps). These proteins, in combination with the activation of the nitric oxide synthase (NOS) enzyme, play important roles in the protection of the myocardium against a variety of diseases. In the present work we report that the generation of potent reactive oxygen species (ROS), namely ·OH in cardiac H9c2 cells, is attenuated by heat shock treatment (2 h at 42°C). Western blot analyses showed that heat shock treatment induced overexpression of Hsp70, Hsp60, and Hsp25. The observed ·OH was found to be derived from the superoxide (O₂^–·) generated by the mitochondria. Whereas the manganese superoxide dismutase (MnSOD) activity was increased in the heat-shocked cells, the mitochondrial aconitase activity was reduced. The mechanism of O₂^–· conversion into ·OH in mitochondria is proposed as follows. The O₂^–· leaked from the electron transport chain, oxidatively damages the mitochondrial aconitase, releasing a free Fe²⁺. The aconitase-released Fe²⁺ combines with H₂O₂ to generate ·OH via a Fenton reaction and the oxidized Fe³⁺ recombines with the inactivated enzyme after being reduced to Fe²⁺ by other cellular reductants, turning it over to be active. However, in heat-shocked cells, because of higher MnSOD activity, the excess H₂O₂ causes irreversible damage to the mitochondrial aconitase enzyme, thus inhibiting its activity. In conclusion, we propose that attenuation of ·OH generation after heat shock treatment might play an important role in reducing the myocardial ischemic injury, observed in heat shock-treated animals. proteins; free radicals; spin trapping; reactive oxygen species     

Molecular characterization of the heparin-dependent transduction domain on the capsid of a novel adeno-associated virus isolate, AAV(VR-942)

A new adeno-associated virus (AAV), referred to as AAV(VR-942), has been isolated as a contaminant of adenovirus strain simian virus 17. The sequence of the rep gene places it in the AAV serotype 2 (AAV2) complementation group, while the capsid is only 88% identical to that of AAV2. High-level AAV(VR-942) transduction activity requires cell surface heparan sulfate proteoglycans, although AAV(VR-942) lacks residues equivalent to the AAV2 R585 and R588 amino acid residues essential for mediating the interaction of AAV2 with the heparan sulfate proteoglycan receptor. Instead, AAV(VR-942) uses a distinct transduction region. This finding shows that distinct domains on different AAV isolates can be responsible for the same activities.     

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6(54/84) variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6(54/84) variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.     

Structural insights and functional implications of choline acetyltransferase

The biosynthetic enzyme for the neurotransmitter acetylcholine, choline acetyltransferase (ChAT) (E.C. 2.3.1.6), is essential for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. ChAT catalyzes the transfer of an acetyl group from acetyl-coenzyme A to choline to form the neurotransmitter acetylcholine. Since its discovery more than 60 years ago much research has been devoted to the kinetic studies of this enzyme. For the first time we report the crystal structure of rat ChAT (rChAT) to 1.55 A resolution. The structure of rChAT is a monomer and consists of two domains with an interfacial active site tunnel. This structure, with the modeled substrate binding, provides critical insights into the molecular basis for the production of acetylcholine and may further our understanding of disease causing mutations.     

Sodium molybdate improves the phagocytic function in alloxan-induced diabetic rats

Diabetes mellitus is a disease, which virtually affects all the systems in the body including the immune system. Bacterial infections are important causes of increased morbidity and mortality in diabetic patients. In the present study, we assessed the effect of molybdate on immune responses of diabetic rats. The phagocytic potency and nitroblue tetrazolium dye reduction capacity were found to be considerably lowered whereas soluble immune complex formation enhanced significantly in diabetic rats when compared with control rats. From our findings, it is suggested that dietary intake of molybdate may have a vital role in enhancing immune functions during diabetic mellitus.     

Human carbonic anhydrase III: structural and kinetic study of catalysis and proton transfer

The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.