Unsaturated N-acetyl- D-glucosaminuronic acid glycosides as inhibitors of influenza virus sialidase

The threat of pandemic influenza is a significant concern of governments worldwide. There is a very limited and relatively expensive armament to tackle such a pandemic should it occur. This fact provides much impetus to the scientific community for the discovery of new and less expensive anti-influenza drugs. Our longstanding interest in the inhibition of influenza virus sialidase, coupled with the development of simple carbohydrates that mimic an unsaturated derivative of the enzyme's naturally-occurring ligand, N-acetylneuraminic acid, has led us to investigate the development of influenza virus sialidase inhibitors based on these mimetics. We have successfully prepared a range of these compounds, in good yield, from the relatively inexpensive carbohydrate N-acetylglucosamine utilising a short synthetic procedure. We have employed a sialidase inhibition assay for biological evaluation of the target compounds and to our delight these mimetics have displayed significant inhibition of influenza virus sialidase.     

G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages

Lactobacillus rhamnosus is a human commensal with known immunomodulatory properties. To date the mechanism of these immunomodulatory effects is not well understood. To unravel the immunomodulatory signalling mechanism, we investigated the effects of two strains of L. rhamnosus, L. rhamnosus GG and GR-1, in modulating production of tumour necrosis factor-alpha (TNF) in human monocytic cell line THP-1 and mouse macrophages. Live L. rhamnosus GG and GR-1 or their spent culture supernatant induced minuscule amounts of TNF production but large quantities of granulocyte-colony stimulating factor (G-CSF) in macrophages compared with those induced by pathogenic Escherichia coli GR-12 and Enterococcus faecalis. By using neutralizing antibodies and G-CSF receptor knockout mice, we demonstrated that G-CSF secreted from L. rhamnosus GG- and GR-1-exposed macrophages suppressed TNF production induced by E. coli- or lipopolysaccharide-activated macrophages through a paracrine route. The suppression of TNF production by G-CSF was mediated through activation of STAT3 and subsequent inhibition of c-Jun-N-terminal kinases (JNKs). The inhibition of JNK activation required STAT3alpha-mediated de novo protein synthesis. This demonstrates a novel role of G-CSF in L. rhamnosus-triggered anti-inflammatory effects and its mechanism in the suppression of TNF production in macrophages.     

Influence of Catastrophic Climatic Events and Human Waste on Vibrio Distribution in the Karnaphuli Estuary, Bangladesh

Vibrios are bacteria of marine and estuarine origin that can cause human diseases, such as cholera, and also affect aquatic organisms. The impact of storm-driven changes in salinity and suspended particulate matter (SPM) on cultivable Vibrio counts (CVC) and distribution in Karnaphuli estuary, Bangladesh, was compared before and after a strong cyclone in mid May 2007 and after a monsoon landslide a month later. CVC were higher (~10³ colony forming units—cfu/ml) at estuary’s mouth (salinity 20–15 parts per thousand, ppt) and steeply declined landwards. CVC and their proportion of total aerobic bacteria were highest after the cyclone and also increased after the landslide, likely due to higher SPM loads. The cyclone did not significantly change previous fecal coliform abundance, contrasting with the ten times increase after the landslide. Sewage input enhanced CVC near the point sources. CVC and salinity correlated highly significantly at salinities <10 ppt; however, at higher values dispersion increased, probably due to the effect of sediment resuspension on CVC. Cyclone or heavy rainfall-mediated turbidity changes jointly with salinity gradients can significantly influence abundance and distribution of estuarine vibrios. Extended salt intrusion and higher turbidities in tropical estuaries by stronger and more frequent storms and deforestation-derived erosion could favor Vibrio growth, with increasing risks for aquatic resources and human health in the coastal zone.     

Sub-optimal dose of Sodium Antimony Gluconate (SAG)-diperoxovanadate combination clears organ parasites from BALB/c mice infected with antimony resistant Leishmania donovani by expanding antileishmanial T-cell repertoire and increasing IFN-γ to IL-10 ratio

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O₂)₂(H₂O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (Sb^R) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-γ and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-γ inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.     

Structural Evidence of Substrate Specificity in Mammalian Peroxidases: STRUCTURE OF THE THIOCYANATE COMPLEX WITH LACTOPEROXIDASE AND ITS INTERACTIONS AT 2.4 ? RESOLUTION*S?

The crystal structure of the complex of lactoperoxidase (LPO) with its physiological substrate thiocyanate (SCN^–) has been determined at 2.4Å resolution. It revealed that the SCN^– ion is bound to LPO in the distal heme cavity. The observed orientation of the SCN^– ion shows that the sulfur atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of SCN^– forms a hydrogen bond with a water (Wat) molecule at position 6′. This water molecule is stabilized by two hydrogen bonds with Gln⁴²³ N^ε2 and Phe⁴²² oxygen. In contrast, the placement of the SCN^– ion in the structure of myeloperoxidase (MPO) occurs with an opposite orientation, in which the nitrogen atom is closer to the heme iron than the sulfur atom. The site corresponding to the positions of Gln⁴²³, Phe⁴²² oxygen, and Wat⁶′ in LPO is occupied primarily by the side chain of Phe⁴⁰⁷ in MPO due to an entirely different conformation of the loop corresponding to the segment Arg⁴¹⁸–Phe⁴³¹ of LPO. This arrangement in MPO does not favor a similar orientation of the SCN^– ion. The orientation of the catalytic product OSCN^– as reported in the structure of LPO·OSCN^– is similar to the orientation of SCN^– in the structure of LPO·SCN^–. Similarly, in the structure of LPO·SCN^–·CN^–, in which CN^– binds at Wat¹, the position and orientation of the SCN^– ion are also identical to that observed in the structure of LPO·SCN.Lactoperoxidase (LPO⁴; EC 1.11.1.7) is a Fe³⁺ heme enzyme that belongs to the mammalian peroxidase family (1). The family of mammalian peroxidases comprises lactoperoxidase (2), eosinophil peroxidase (3), thyroid peroxidase (4), and myeloperoxidase (MPO) (5). LPO, eosinophil peroxidase, and MPO are responsible for antimicrobial function and innate immune responses (6–8), whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis (9). These peroxidases are different from plant and fungal peroxidases because unlike plant and fungal enzymes, the prosthetic heme group in mammalian peroxidases is covalently linked to the protein (10). There are also several striking structural and functional differences among the mammalian peroxidases (11). The heme group in MPO is attached to the protein via three covalent linkages (12), whereas LPO (12, 13), eosinophil peroxidase (12), and thyroid peroxidase (12) contain only two ester linkages. These covalent and various non-covalent linkages contribute differentially to the high stability of the heme core as well as for the peculiar values of their redox potentials (2, 14). Furthermore, MPO consists of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase, and thyroid peroxidase are single chain proteins, although their chain lengths differ greatly. In addition, their sequences contain several critical amino acid differences that may also contribute to the variations in the stereochemical environments of the substrate-binding sites. As a consequence of these differences, the mammalian enzymes oxidize various inorganic ions such as SCN^–, Br^–, Cl^–, and I^– with differing specificities and potencies. Biochemical studies have shown that LPO catalyzes preferentially the conversion of SCN^– to OSCN^– (15, 16), whereas MPO uses halides (17, 18) with a preference for chloride ion as the substrate. The preferences of eosinophil peroxidase and thyroid peroxidase are bromide and iodide, respectively. However, the stereochemical basis of the reported preferences for the substrates by mammalian heme peroxidases is still unclear. So far, the structures of only two mammalian enzymes, MPO and LPO, have been determined (12, 13). It is of considerable importance to identify the structural parameters that are responsible for the subtle specificities. In the present work, we have attempted to address this question through the new crystal structures of LPO complexes with SCN^– ions using goat, bovine, and buffalo lactoperoxidases. Because the overall structures of complexes of SCN^– with LPO from all three species were found to be identical, the structure of the complex of buffalo LPO with SCN^– and the ternary complex with SCN^– and CN^– will be discussed here, and buffalo LPO will be termed hereafter as LPO. To highlight the factors pertaining to binding specificity of SCN^–, a comparison of the structures of LPO·SCN^– and MPO·SCN^– has also been made, revealing many valuable differences pertaining to the observed orientations of the common substrate, SCN^– ion, when bound at the substrate-binding site in the distal heme cavity of the two structures. The structures of LPO·SCN^– and MPO·SCN^– clearly show that the bound SCN^– ions are present in the distal heme cavity of two enzymes with opposite orientations. In the structure of LPO·SCN^–, the sulfur atom is closer to the heme iron than the nitrogen atom, whereas in that of MPO·SCN^–, the nitrogen atom is closer to the heme iron than the sulfur atom. As a result of this, the interactions of the SCN^– ion in the distal site of two proteins differ drastically. Gln⁴²³, a conserved water (Wat) molecule at position 6′, and a well aligned carbonyl oxygen of Phe⁴²² in the proximity of the substrate-binding site in LPO against a protruding Phe⁴⁰⁷ in MPO seem to play the key roles in inducing the observed orientations of SCN^– ions in LPO and MPO. The structure of LPO·SCN^– has also been compared with the structure of its ternary complex with SCN^– and CN^– ions.     

Determinants of exposure to mass media family planning messages among indigenous people in Bangladesh: a study on the Garo

This paper evaluates exposure to mass media family planning (FP) messages among the Garo, an indigenous community in Bangladesh. A sample of 223 currently married Garo women were selected purposively from two districts where most of the Garo population live. The analysis demonstrated that television was the most significant form of mass media to disseminate FP messages among the recipients - more so than radio and newspapers. About 80.6% of the respondents had heard of FP messages through television, while for the radio and newspapers the percentages were 55.3% and 22.7% respectively. The contraceptive prevalence rate is much higher (79.5%) in the study area than the national level (55.8%). A linear logistic regression model was employed to identify the confluence of different demographic and socioeconomic characteristics on mass media FP messages. Regarding exposure to FP messages, four independent variables out of six had significant effects on the exposure to FP messages through any one of the types of media, i.e. radio, television and newspapers. These independent variables were age, level of education, occupation and number of children.     

Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors

Abnormal immune responses have been reported to be associated with autism. A number of studies showed that cytokines were increased in the blood, brain, and cerebrospinal fluid of autistic subjects. Elevated IL-6 in autistic brain has been a consistent finding. However, the mechanisms by which IL-6 may be involved in the pathogenesis of autism are not well understood. Here we show that mice with elevated IL-6 in the brain display many autistic features, including impaired cognitive abilities, deficits in learning, abnormal anxiety traits and habituations, as well as decreased social interactions. IL-6 elevation caused alterations in excitatory and inhibitory synaptic formations and disrupted the balance of excitatory/inhibitory synaptic transmissions. IL-6 elevation also resulted in an abnormal change in the shape, length and distributing pattern of dendritic spines. These findings suggest that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity.     

Chemical analysis of an immunostimulating (1→4)-, (1→6)-branched glucan from an edible mushroom, Calocybe indica

An immunostimulating water-soluble glucan was isolated from hot aqueous extract of fruit bodies of an edible mushroom Calocybe indica. Structural investigation of the glucan was carried out using acid hydrolysis, methylation analysis, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments, the structure of the repeating unit of the polysaccharide was established as [see figure in text]. This glucan stimulated the splenocytes and thymocytes.     

A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.