Solution structures of two structural isoforms of CMrVIA chi/lambda-conotoxin

alpha-Conotoxins possess a conserved four-cysteine framework and disulfide linkages (C(1)(-)(3), C(2)(-)(4)) that fold toward the globular conformation with absolute fidelity. Despite the presence of a similar conserved set of cysteine framework, chi/lambda-conotoxins adopt an alternate disulfide-pairing (C(1)(-)(4), C(2)(-)(3)) and its consequent ribbon conformation, exhibiting distinct biological activities from alpha-conotoxins. chi/lambda-Conotoxin CMrVIA (VCCGYKLCHOC-COOH) isolated from the venom of Conus marmoreus natively exists in the ribbon conformation and induces seizures in mice at a potency that is of three orders higher than the non-native globular form. We have chemically synthesized two isoforms of CMrVIA conotoxin in the ribbon and globular conformation and determined their structures by (1)H NMR spectroscopy. The ribbon (PDB ID 2B5P) and globular conformations (PBD ID 2B5Q) were calculated to have paired-wise backbone RMSDs of 0.48 +/- 0.1 and 0.58 +/- 0.1 A respectively. Unlike the native globular alpha-conotoxins, the globular canonical form of CMrVIA chi/lambda-conotoxin exhibited heterogeneity in its solution structure as noted by the presence of minor conformers and poorer RMSD of structure calculation. Paired-wise backbone comparison between the native ribbon and the non-native globular form of CMrVIA conotoxin revealed an RMSD of 4.73 A, emphasizing their distinct conformational differences. These structural data are essential for the understanding of the structure-function activity of chi/lambda-conotoxins, as well as unraveling the folding propensities of these short peptide toxins.     

Activation of α-secretase by curcumin-aminoacid conjugates

The extracellular senile plaques observed in Alzheimer's disease (AD) patients are mainly composed of amyloid peptides produced from the β-amyloid precursor protein (βAPP) by β- and γ-secretases. A third non-amyloidogenic α-secretase activity performed by the disintegrins ADAM10 and ADAM17 occurs in the middle of the amyloid-β peptide Aβ and liberates the large sAPPα neuroprotective fragment. Since the activation of α-secretase recently emerged as a promising therapeutic approach to treat AD, the identification of natural compounds able to trigger this cleavage is highly required. Here we describe new curcumin-based modified compounds as α-secretase activators. We established that the aminoacid conjugates curcumin-isoleucine, curcumin-phenylalanine and curcumin-valine promote the constitutive α-secretase activity and increase ADAM10 immunoreactivity. Strickingly, experiments carried out under conditions mimicking the PKC/muscarinic receptor-regulated pathway display different patterns of activation by these compounds. Altogether, our data identified new lead natural compounds for the future development of powerful and stable α-secretase activators and established that some of these molecules are able to discriminate between the constitutive and regulated α-secretase pathways.     

Structural and Functional Characterization of a Novel Homodimeric Three-finger Neurotoxin from the Venom of Ophiophagus hannah (King Cobra)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (αβγδ) and neuronal (α₇, α₃β₂, and α₄β₂) nicotinic acetylcholine receptors (nAChRs) with highest affinity for α₇-nAChRs. The high resolution (1.5 Å) crystal structure revealed haditoxin to be a homodimer, like κ-neurotoxins, which target neuronal α₃β₂- and α₄β₂-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain α-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain α-neurotoxins and κ-neurotoxins notwithstanding, haditoxin exhibited unique blockade of α₇-nAChRs (IC₅₀ 180 nm), which is recognized by neither short-chain α-neurotoxins nor κ-neurotoxins. This is the first report of a dimeric short-chain α-neurotoxin interacting with neuronal α₇-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.     

Synthesis of 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine Derivatives as Antimycobacterial and Antifungal Agents: In Vitro Evaluation and In Silico Study

A series of novel alkyl substituted purines were synthesized. 6‐[4‐(4‐Propoxyphenyl)piperazin‐1‐yl]‐9H‐purine was used as the key starting material, which was synthesized via a multistep protocol and finally subjected for N‐alkylation with various alkyl halides with an aim to get prospective antimicrobial agents. The structures of the novel compounds were established by substantiating them through spectral techniques like ¹H‐NMR, ¹³C‐NMR, FT‐IR and EI‐MS. They were explored for antitubercular activity against Mycobacterium tuberculosis H37RV. Furthermore, they were checked for their antimicrobial activity concerning bacterial and fungal strains. The title compounds exhibited considerable antimicrobial activity without any significant toxicity. In silico studies depicted their good binding profile against Mycobacterium tuberculosis enoyl reductase (InhA; PDB ID: 4TZK) and Candida albicans dihydrofolate reductase (PDB ID: 1AI9). The title compounds obeyed Lipinski's parameters and have exhibited good drug‐like properties.     

Donkey milk kefir induces apoptosis and suppresses proliferation of Ehrlich ascites carcinoma by decreasing iNOS in mice

Donkey milk and donkey milk kefir exhibit antiproliferative, antimutagenic and antibacterial effects. We investigated the effects of donkey milk and donkey milk kefir on oxidative stress, apoptosis and proliferation in Ehrlich ascites carcinoma (EAC) in mice. Thirty-four adult male Swiss albino mice were divided into four groups as follows: group 1, administered 0.5 ml water; group 2, administered 0.5 ml water + EAC cells; group 3, administered 0.5 ml donkey milk + EAC cells; group 4, administered 0.5 ml donkey milk kefir + EAC cells. We introduced 2.5 x 10⁶ EAC cells into each animal by subcutaneous injection. Tap water, donkey milk and donkey milk kefir were administered by gavage for 10 days. Animals were sacrificed on day 11. After measuring the short and long diameters of the tumors, tissues were processed for histology. To determine oxidative stress, cell death and proliferation iNOS and eNOS, active caspase-3 and proliferating cell nuclear antigen were assessed using immunohistochemistry. A TUNEL assay also was used to detect apoptosis. Tumor volume decreased in the donkey milk kefir group compared to the control and donkey milk groups. Tumor volume increased in the donkey milk group compared to the control group. Proliferating cell nuclear antigen levels were higher in the donkey milk kefir group compared to the control and donkey milk groups. The number of apoptotic cells was less in the donkey milk group, compared to the control, whereas it was highest in the donkey milk kefir group. Donkey milk administration increased eNOS levels and decreased iNOS levels, compared to the control group. In the donkey milk kefir group, iNOS levels were significantly lower than those of the control and donkey milk groups, while eNOS levels were similar to the control group. Donkey milk kefir induced apoptosis, suppressed proliferation and decreased co-expression of iNOS and eNOS. Donkey milk promoted development of the tumors. Therefore, donkey milk kefir appears to be more beneficial for treating breast cancer than donkey milk.     

The role of glycogen synthase kinase 3 in regulating IFN-β-mediated IL-10 production

The ability of IFN-β to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-β stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-β to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-β activity. IFN-β-mediated suppression of GSK3-β promoted IL-10, because IL-10 production by IFN-β-stimulated dendritic cells (DC) expressing an active GSK3-β knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-β augmented IL-10 production. IFN-β increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-β to induce IL-10 from DC. IL-10 production by IFN-β-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-β induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-β.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

Full-Genome Characterisation of Orungo,Lebombo and Changuinola Viruses Provides Evidence for Co-Evolution of Orbiviruses with Their Arthropod Vectors

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein ‘outer-capsid protein 1′ (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 ‘subcore-shell protein’ as the third largest protein ‘VP3(T2)’ in the first of these groups, but the second largest protein ‘VP3(T2)’ in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors.     

Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase,a Biomarker for Malaria,Using the Specific DNA Aptamer

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.