Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.     

The role of parasites and pathogens in influencing generalised anxiety and predation-related fear in the mammalian central nervous system

This article is part of a Special Issue "Neuroendocrine-Immune Axis in Health and Disease." Behavioural and neurophysiological traits and responses associated with anxiety and predation-related fear have been well documented in rodent models. Certain parasites and pathogens which rely on predation for transmission appear able to manipulate these, often innate, traits to increase the likelihood of their life-cycle being completed. This can occur through a range of mechanisms, such as alteration of hormonal and neurotransmitter communication and/or direct interference with the neurons and brain regions that mediate behavioural expression. Whilst some post-infection behavioural changes may reflect 'general sickness' or a pathological by-product of infection, others may have a specific adaptive advantage to the parasite and be indicative of active manipulation of host behaviour. Here we review the key mechanisms by which anxiety and predation-related fears are controlled in mammals, before exploring evidence for how some infectious agents may manipulate these mechanisms. The protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, is focused on as a prime example. Selective pressures appear to have allowed this parasite to evolve strategies to alter the behaviour in its natural intermediate rodent host. Latent infection has also been associated with a range of altered behavioural profiles, from subtle to severe, in other secondary host species including humans. In addition to enhancing our knowledge of the evolution of parasite manipulation in general, to further our understanding of how and when these potential changes to human host behaviour occur, and how we may prevent or manage them, it is imperative to elucidate the associated mechanisms involved.     

Gender discrimination in undernutrition with mediating factors among Bengalee school children from Eastern India

This study was undertaken to determine age and sex variations in the prevalence of underweight and stunting, and to assess the impact of some socio-economic variables on undernutrition among 6–16 year old school children of Bengalee ethnicity in Chapra, West Bengal, India. The subjects were selected randomly from various schools and madrassas of the Chapra Block. A total of 725 children (342 boys and 383 girls) aged 6–16 years were measured and data on their socio-economic status were collected. Age and sex combined rates of underweight and stunting were 44.40% and 37.20%, respectively. Weight-for-age Z-score (WAZ) showed significant association with per-capita income (PCI) among boys (F = 5.45) and girls (F = 8.14). Height-for-age Z-score (HAZ) has also shown the association with per-capita income among boys (F = 4.43) and girls (F = 9.69). The WAZ was significantly associated with fathers’ educational status (FOS) (t = ?2.95) and the number of living rooms (NLR) (t = ?2.91) among girls. The HAZ showed significant association with number of siblings (NS) among girls (F = 4.25). Linear regression analyses revealed that NLR (t = 2.04) and NS (t = 1.95) had a significant impact on HAZ among boys. Among girls, PCI (t = 3.38), FOS (t = 2.87) and NLR (t = 2.81) had a significant impact on WAZ and also PCI (t = 3.28) and FOS (t = 2.90) had a significant impact on HAZ. NLR had significant associations with underweight (χ² = 3.59) and stunting (χ² = 4.20) among boys. Among girls, PCI had significant associations with underweight (χ² = 11.15) and stunting (χ² = 11.64). FOS also showed significant associations with underweight (χ² = 8.10) as well as stunting (χ² = 8.28) among girls. NLR showed a significant association with underweight (χ² = 7.75). Logistics regression analyses revealed that FOS (Wald = 8.00) and NLR (Wald = 4.09) were significant predictors of stunting among boys. Among girls, PCI was a significant predictor of underweight (Wald = 10.95) as well as stunting (Wald = 10.45). FOS, NLR and NS were also significant predictors of stunting (Wald = 8.16), underweight (Wald = 7.68) and stunting (Wald = 6.97) respectively. The present study revealed that the nutritional status of the children was unsatisfactory and it is of paramount importance not only to increase the amount of food supplementation given but also to promote gender equality.     

Synthesis and structure-activity relationships of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones as novel series of potent β isoform selective phosphatidylinositol 3-kinase inhibitors

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (～ 0.100 μM IC(50)) growth inhibition in a PTEN deficient cancer cell line.     

Regioselective synthesis and antimicrobial studies of ester linked 1,4-disubstituted 1,2,3-bistriazoles

A series of 1,4-disubstituted 1,2,3-bistriazoles was synthesized via click chemistry by cycloaddition of various bisalkynes with benzyl/2-phenylethyl azide. Synthesized triazoles were characterized by IR, (1)H NMR, (13)C NMR and mass spectral techniques. All the compounds were evaluated for antibacterial/antifungal activities and found to possess moderate to good antimicrobial activities. Further the docking study for the most active compound against DNA Gyrase was also carried out.     

Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships

Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.     

Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability

Acid aspiration, a common cause of acute lung injury, leads to alveolar edema. Increase in lung vascular permeability underlies this pathology. To define mechanisms, isolated rat lungs were perfused with autologous blood. Hydrochloric acid and rhodamine-dextran 70 kDa (RDx70) were coinstilled into an alveolus by micropuncture. RDx70 fluorescence was used to establish the spatial distribution of acid. Subsequently, FITC-dextran 20 kDa (FDx20) was infused into microvessels for 60 min followed by a 10-min HEPES-buffered saline wash. During the infusion, FITC fluorescence changes were recorded to quantify the ratio of peak to postwash fluorescence. The ratio, termed normalized fluorescence, was low for acid compared with buffer instillation both in microvessels abutting acid-treated alveoli and those located more than 700 μm away. In contrast, the normalized fluorescence was similar to buffer controls when a higher molecular weight tracer (FITC-dextran 70 kDa) was infused instead of FDx20, suggesting that normalized FDx20 fluorescence faithfully represented microvascular permeability. Inhibiting endothelial connexin43 (Cx43) gap junction communication with Gap27 blunted the acid-induced reduction in normalized fluorescence, although scrambled Gap27 did not have any effect. The blunting was evident not only in microvessels away from the site of injury, but also in those abutting directly injured alveoli. Thus the new fluorescence-based method reveals that acid increases microvascular permeability both at acid-instilled and away sites. Inhibiting endothelial Cx43 blocked the permeability increase even at the direct injury sites. These data indicate for the first time that Cx43-dependent mechanisms mediate acid-induced increases in microvascular permeability. Cx43 may be a therapeutic target in acid injury.     

Modulation of the IgM gene expression and IgM immunoreactive cell distribution by the nutritional background in gilthead sea bream (Sparus aurata) challenged with Enteromyxum leei (Myxozoa)

The aim of the present work was to determine if a plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the distribution of IgM immunoreactive cells (IRCs) and the IgM expression pattern in the intestine and haematopoietic tissues of gilthead sea bream (GSB) (Sparus aurata) challenged with the myxosporean Enteromyxum leei. In a first trial (T1), GSB fed for 9 months either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) was challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to know their infection status. After 102 days of exposure, samples of intestine and head kidney were obtained for IgM expression and immunohistochemical detection (IHC). Additional samples of spleen were taken for IHC. Fish were categorized as control (C, not exposed), and early (E), or late (L) infected. The 66VO diet had no effect on the number of IgM-IRCs in any of the tissues or on IgM expression in C fish, whereas the infection with E.?leei had a strong effect on the intestine. A combined time-diet effect was also observed, since the highest expression and IRCs values were registered in the posterior intestine (Pi) of E-66VO fish. A positive correlation was found between IgM expression and the presence of IgM-IRCs in the Pi. The effect of the time of infection was studied more in detail in a second trial (T2) in which samples of Pi were taken at 0, 24, 51, 91 and 133 days after exposure to the parasite. A significant increase of the IgM expression was detected only in parasitized fish, and very late after exposure. These results show that the duration of the exposure to the parasite is the most determinant factor for the observed intestinal IgM increased phenotype which gets magnified by the feeding of a high VO-based diet.     

Antineoplastic and Apoptotic Potential of Traditional Medicines Thymoquinone and Diosgenin in Squamous Cell Carcinoma

Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180–induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G₁ population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.