SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.     

Distinct role of calmodulin and calmodulin-dependent protein kinase-II in lipopolysaccharide and tumor necrosis factor-alpha-mediated suppression of apoptosis and antiapoptotic c-IAP2 gene expression in human monocytic cells

Exposure of phagocytic cells to bacterial endotoxin (lipopolysaccharide; LPS) or inflammatory cytokines confers antiapoptotic survival signals; however, in the absence of the appropriate stimulus, monocytes are programmed to undergo apoptosis. Macrophage survival may thus influence inflammatory and immune responses and susceptibility to microbial pathogens. Herein, we demonstrate that LPS and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), enhance monocytic cell survival through the induction of the antiapoptotic c-IAP2 gene in a human promonocytic THP-1 cell line. We also investigated the role of upstream signaling molecules including the mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and the calcium signaling pathways in the regulation of c-IAP2 expression and eventual survival of monocytic cells. Our results suggest that LPS and TNF-alpha-induced c-IAP2 expression was regulated by calmodulin (CaM) through the activation of calmodulin-dependent protein kinase-II (CaMKII). In addition, CaM and CaMKII regulated c-IAP2 expression in LPSand TNF-alpha-stimulated cells through NF-kappaB activation. Moreover, the CaM/CaMKII pathway also regulated LPS- and TNF-alpha-mediated inhibition of apoptosis in these cells. Taken together, these results suggest that LPS- and TNF-alpha-induced c-IAP2 expression and its associated antiapoptotic survival signals in THP-1 cells are regulated selectively by CaM/CaMKII through NF-kappaB activation.     

STAT-1 mediates the stimulatory effect of IL-10 on CD14 expression in human monocytic cells

IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human monocytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK.     

Elevated CO2 affects plant responses to variation in boron availability

Aim

Effects of elevated CO₂ on N relations are well studied, but effects on other nutrients, especially micronutrients, are not. We investigated effects of elevated CO₂ on response to variation in boron (B) availability in three unrelated species: seed geranium (Pelargonium x hortorum), barley (Hordeum vulgare), and water fern (Azolla caroliniana).

Methods

Plants were grown at two levels of CO₂ (370, 700?ppm) and low, medium, and high B. Treatment effects were measured on biomass, net photosynthesis (P_n) and related variables, tissue nutrient concentrations, and B transporter protein BOR1.

Results

In geranium, there were interactive effects (P?<?0.05) of B and CO₂ on leaf, stem, and total plant mass, root:shoot ratio, leaf [B], B uptake rate, root [Zn], and P_n. Elevated CO₂ stimulated growth at 45?μM B, but decreased it at 450?μM B and did not affect it at 4.5?μM B. P_n was stimulated by elevated CO₂ only at 45?μM B and chlorophyll was enhanced only at 450?μM B. Soluble sugars increased with high CO₂ only at 4.5 and 45?μM B. High CO₂ decreased leaf [B] and B uptake rate, especially at 450?μM B. Though CO₂ and B individually affected the concentration of several other nutrients, B x CO₂ interactions were evident only for Zn in roots, wherein [Zn] decreased under elevated CO₂. Interactive effects of B and CO₂ on growth were confirmed in (1) barley grown at 0, 30, or 1,000?μM B, wherein growth at high CO₂ was stimulated more at 30?μM B, and (2) Azolla grown at 0, 10, and 1,000?μM B, wherein growth at high CO₂ was stimulated at 0 and 10?μM B.

Conclusion

Thus, low and high B both may limit growth stimulation under elevated vs. current [CO₂], and B deficiency and toxicity, already common, may increase in the future.     

Overexpression of plastidic maize NADP-malate dehydrogenase (<Emphasis Type="Italic">ZmNADP-MDH</Emphasis>) in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> confers tolerance to salt stress

The plastidic C4 Zea mays NADP-malate dehydrogenase (ZmNADP-MDH), responsible for catalysis of oxaloacetate to malate, was overexpressed in Arabidopsis thaliana to assess its impact on photosynthesis and tolerance to salinity stress. Different transgenic lines were produced having ~3–6-fold higher MDH protein abundance and NADP-MDH enzyme activity than vector control. The overexpressors had similar chlorophyll, carotenoid, and protein content as that of vector control. Their photosynthetic electron transport rates, carbon assimilation rate, and consequently fresh weight and dry weight were almost similar. However, these overexpressors were tolerant to salt stress (150 mM NaCl). In saline environment, the Fv/Fm ratio, yield of photosystem II, chlorophyll, and protein content were higher in ZmNADP-MDH overexpressor than vector control. Under identical conditions, the generation of reactive oxygen species (H₂O₂) and production of malondialdehyde, a membrane lipid peroxidation product, were lower in overexpressors. In stress environment, the structural distortion of granal organization and swelling of thylakoids were less pronounced in ZmNADP-MDH overexpressing plants as compared to the vector control. Chloroplastic NADP-MDH in consort with cytosolic and mitochondrial NAD-MDH plays an important role in exporting reducing power (NADPH) and exchange of metabolites between different cellular compartments that maintain the redox homeostasis of the cell via malate valve present in chloroplast envelope membrane. The tolerance of NADP-MDH overexpressors to salt stress could be due to operation of an efficient malate valve that plays a major role in maintaining the cellular redox environment.     

Dendritic Cell-Mediated,DNA-Based Vaccination against Hepatitis C Induces the Multi-Epitope-Specific Response of Humanized,HLA Transgenic Mice

Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.     

Omega-3 fatty acid supplementation and cardiovascular disease: Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases

Epidemiological studies on Greenland Inuits in the 1970s and subsequent human studies have established an inverse relationship between the ingestion of omega-3 fatty acids [C_20–22 ω 3 polyunsaturated fatty acids (PUFA)], blood levels of C_20–22 ω 3 PUFA, and mortality associated with cardiovascular disease (CVD). C_20–22 ω 3 PUFA have pleiotropic effects on cell function and regulate multiple pathways controlling blood lipids, inflammatory factors, and cellular events in cardiomyocytes and vascular endothelial cells. The hypolipemic, anti-inflammatory, anti-arrhythmic properties of these fatty acids confer cardioprotection. Accordingly, national heart associations and government agencies have recommended increased consumption of fatty fish or ω 3 PUFA supplements to prevent CVD. In addition to fatty fish, sources of ω 3 PUFA are available from plants, algae, and yeast. A key question examined in this review is whether nonfish sources of ω 3 PUFA are as effective as fatty fish-derived C_20–22 ω 3 PUFA at managing risk factors linked to CVD. We focused on ω 3 PUFA metabolism and the capacity of ω 3 PUFA supplements to regulate key cellular events linked to CVD. The outcome of our analysis reveals that nonfish sources of ω 3 PUFA vary in their capacity to regulate blood levels of C_20–22 ω 3 PUFA and CVD risk factors.