Development of a novel method to determine very low density lipoprotein kinetics

Isotopic tracer methods of determining triglyceride-rich lipoprotein (TRL) kinetics are costly, time-consuming, and labor-intensive. This study aimed to develop a simpler and cost-effective method of obtaining TRL kinetic data, based on the fact that chylomicrons compete with large VLDL (VLDL(1); S(f) = 60-400) for the same catalytic pathway. Ten healthy subjects [seven men; fasting triglyceride (TG), 44.3-407.6 mg/dl; body mass index, 21-35 kg/m(2)] were given an intravenous infusion of a chylomicron-like TG emulsion (Intralipid; 0.1 g/kg bolus followed by 0.1 g/kg/h infusion) for 75-120 min to prevent the clearance of VLDL(1) by lipoprotein lipase. Multiple blood samples were taken during and after infusion for separation of Intralipid, VLDL(1), and VLDL(2) by ultracentrifugation. VLDL(1)-apolipoprotein B (apoB) and TG production rates were calculated from their linear increases in the VLDL(1) fraction during the infusion. Intralipid-TG clearance rate was determined from its exponential decay after infusion. The production rates of VLDL(1)-apoB and VLDL(1)-TG were (mean +/- SEM) 25.4 +/- 3.9 and 1,076.7 +/- 224.7 mg/h, respectively, and the Intralipid-TG clearance rate was 66.9 +/- 11.7 pools/day. Kinetic data obtained from this method agree with values obtained from stable isotope methods and show the expected relationships with indices of body fatness and insulin resistance (all P < 0.05). The protocol is relatively quick, inexpensive, and transferable to nonspecialist laboratories.     

Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress

Hepatic steatosis is a common histological feature of chronic hepatitis C. Hepatitis C virus (HCV) gene expression has been shown to alter host cell cholesterol/lipid metabolism and thus induce hepatic steatosis. Since sterol regulatory element binding proteins (SREBPs) are major regulators of lipid metabolism, we sought to determine whether genotype 2a-based HCV infection induces the expression and posttranslational activation of SREBPs. HCV infection stimulates the expression of genes related to lipogenesis. HCV induces the proteolytic cleavage of SREBPs. HCV core and NS4b derived from genotype 3a are also individually capable of inducing the proteolytic processing of SREBPs. Further, we demonstrate that HCV stimulates the phosphorylation of SREBPs. Our studies show that HCV-induced oxidative stress and subsequent activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt pathway and inactivation (phosphorylation) of PTEN (phosphatase and tensin homologue) mediate the transactivation of SREBPs. HCV-induced SREBP-1 and -2 activities were sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca(2+) chelator 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl) ester (BAPTA-AM), and PI3-K inhibitor (LY294002). Collectively, these studies provide insight into the mechanisms of hepatic steatosis associated with HCV infection.     

Mycobacterium leprae inhibits dendritic cell activation and maturation

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.     

Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress

The human hepatitis B virus (HBV) X protein (HBx) plays a crucial role(s) in the viral life cycle and contributes to the onset of hepatocellular carcinoma (HCC). HBx caused the mitochondrial translocation of Raf-1 kinase either alone or in the context of whole-viral-genome transfections. Mitochondrial translocation of Raf-1 is mediated by HBx-induced oxidative stress and was dependent upon the phosphorylation of Raf-1 at the serine338/339 and Y340/341 residues by p21-activated protein kinase 1 and Src kinase, respectively. These studies provide an insight into the mechanisms by which HBV induces intracellular events relevant to liver disease pathogenesis, including HCC.     

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge. Molecular modeling of three thioether containing type-3 copper proteins using the dead-end elimination method showed that the concerned histidine would be very flexible if not linked to the cysteine. Also, the side chain orientation of the histidine is rather exceptional, as evidenced by statistical data from the protein databank. It is suggested that the role of the bridge is to fix the histidine in an orientation that is optimal for coordination of the Cu(A) atom.     

Interactive Effects of Abiotic Stress and Elevated CO2 on Physio-Chemical and Photosynthetic Responses in Suaeda Species

Suaeda fruticosa and S. monoica are important halophytes for ecological rehabilitation of saline lands. We report differential physio-chemical, photosynthetic, and chlorophyll fluorescence responses in these halophytes under 100 mM sodium chloride (NaCl), 50% strength (16.25 ppt) of seawater (SW)-imposed salinity, and 10% polyethylene glycol 6000 imposed osmotic stress at 380 (ambient) and 1200 (elevated) µmol mol^–1 CO₂ concentrations. SW salinity enhanced the growth in both species; however, compared with S. fruticosa, the S. monoica exhibited comparatively better growth and biomass accumulation under saline conditions at elevated CO₂. Results demonstrated better photosynthetic performances of S. monoica under stress conditions at both levels of CO₂, and this resulted in higher accumulation of carbon, nitrogen, sugar, and starch contents. S. monoica exhibited improved antenna size, electron transfer at PSII donor side, and efficient working of photosynthetic machinery at elevated CO₂, which might be due to efficient upstream utilization of reducing power to fix the CO₂. The δ¹³C results supported the operation of C₄ CO₂ fixation in S. monoica and C₃ or intermediate pathway of CO₂ fixation in S. fruticosa. Lower accumulation of reactive oxygen species, reduced membrane damage, lowered solute potential, and higher accumulation of proline and polyphenol contents indicated elevated CO₂-induced abiotic stress tolerance in Suaeda. Higher activity of antioxidant enzymes in both species at both levels of CO₂ help plants to combat the oxidative stress. Upregulation of NADP-dependent malic enzyme and NADP-dependent malate dehydrogenase genes indicated their role in abiotic stress tolerance as well as photosynthetic carbon (C) sequestration. Operation of C₄ type CO₂ fixation in S. monoica and an intermediate CO₂ fixation in S. fruticosa could be the possible reason for the superior photosynthetic efficiency of S. monoica under stress conditions at elevated CO₂.

     

Systematic analysis of HD-ZIP transcription factors in sesame genome and gene expression profiling of SiHD-ZIP class I entailing drought stress responses at early seedling stage

Molecular Biology Reports - Sesame is an ancient oilseed crop, known for its high oil content and quality. Its sensitivity to drought at early seedling stage is one of the limiting factors...     

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.