PDK1 protein phosphorylation at Thr354 by murine protein serine-threonine kinase 38 contributes to negative regulation of PDK1 protein activity

Murine protein serine-threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family, which acts as cellular energy sensors. In this study, MPK38-induced PDK1 phosphorylation was examined to elucidate the biochemical mechanisms underlying phosphorylation-dependent regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) activity. The results showed that MPK38 interacted with and inhibited PDK1 activity via Thr(354) phosphorylation. MPK38-PDK1 complex formation was mediated by the amino-terminal catalytic kinase domain of MPK38 and the pleckstrin homology domain of PDK1. This activity was dependent on insulin, a PI3K/PDK1 stimulator, as well as various apoptotic stimuli, including TNF-α, H(2)O(2), thapsigargin, and ionomycin. MPK38 inhibited PDK1 activity in a kinase-dependent manner and alleviated PDK1-mediated suppression of TGF-β (or ASK1) signaling, probably via the phosphorylation of PDK1 at Thr(354). In addition, MPK38-mediated inhibition of PDK1 activity was accompanied by the modulation of PDK1 binding to its positive and negative regulators, serine/threonine kinase receptor-associated protein and 14-3-3, respectively. Together, these findings suggest an important role for MPK38-mediated phosphorylation of PDK1 in the negative regulation of PDK1 activity.     

A novel protecting strategy for internucleosidic phosphate and phosphorothioate groups

The utility of 2-(N-isopropyl-N-anisoylamino)ethyl group for protection of internucleosidic phosphate linkages in oligonucleotide synthesis was studied. The group demonstrated high coupling yields, favorable deprotection kinetics and a high hydrolytic stability of phosphoramidite building blocks. The mechanism of deprotection was established using a model phosphate triester.     

Lipid peroxidation, vitamins C, E and reduced glutathione levels in patients with pulmonary tuberculosis

The present study examined the relationship between lipid peroxidation and vitamin C, vitamin E and reduced glutathione levels in plasma, erythrocytes and erythrocyte membranes of pulmonary tuberculosis patients and an equal number of age-and sex-matched healthy subjects. Enhanced plasma, erythrocytes and erythrocyte membrane lipid peroxidation with concomitant decline in vitamin C, vitamin E and reduced glutathione levels were found in pulmonary tuberculosis patients. The elevated lipid peroxidation and decreased vitamin C, vitamin E and reduced glutathione levels indicate the potential of oxidative damage to erythrocytes and erythrocyte membranes of pulmonary tuberculosis patients.     

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).     

Lipid peroxidation and antioxidant status in patients with periodontitis

Our aim was to assess the degree of oxidative stress in patients with periodontitis by measuring their levels of thiobarbituric acid reactive substances (TBARS), enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GSHPx)), and non-enzymatic antioxidants (vitamins E and C, reduced glutathione (GSH)). This study was conducted on 25 adult chronic periodontitis sufferers who were patients in Rajah Muthiah Dental College and Hospital, Annamalai University. The levels of TBARS and non-enzymatic antioxidants, and the activities of enzymatic antioxidants in the patients' plasma, erythrocytes and gingival tissues were assayed using specific colorimetric methods. The periodontitis sufferers had a significantly higher TBARS level than the healthy subjects. In the plasma, erythrocytes, erythrocyte membranes and gingival tissues of the periodontitis sufferers, enzymatic antioxidant activities were found to be significantly higher, whereas the levels of non-enzymatic antioxidants were significantly lower (except for reduced glutathione in the gingival tissues) relative to the parameters found for healthy subjects. The disturbance in the endogenous antioxidant defense system due to over-production of lipid peroxidation products at inflammatory sites can be related to a higher level of oxidative stress in patients with periodontitis.     

Studies on heme release from normal and metal ion reconstituted hemoglobin mediated through ionic surfactant

The interaction of metal-substituted hemoglobin (MHb), where M = Ni and Cu (T-state with no O2 and CO binding capability) and Fe (R-state when CO is bound), with cationic cityl trimethyl ammonium bromide (CTAB) and anionic (sodium dodecyl sulfate-SDS) surfactants has been studied using spectroscopic techniques-UV-visible, electron paramagnetic resonance (EPR), and Fourier transform-Raman-with additional supportive evidence coming from conductivity measurements. We observed the loss of 5-coordination in all three hemoglobins below the critical micelle concentration (CMC) of surfactant, with noticeable differences, suggesting differing mechanisms involved in this process. In addition, above the CMC, Ni- and Cu-hemes were found to leave their proteins more easily than Fe-heme, presumably due to weaker or no bond with the proximal histidine in the former. The released heme is stabilized by micellar media through a hydrophobic interaction process. Of the two surfactants, CTAB seems to be capable of releasing the heme better than SDS and it is attributed to the greater hydrophobicity of CTAB though the charge of the surfactant plays an important role.     

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4′-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4′-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s²U) and 5-methyl-2-thiouridine (m⁵s²U) are natural nucleotide analogs. s²U in tRNA confers greater specificity of codon–anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2′-deoxyribose sugars for a C3′-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2′-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2′-O-[2-(methoxy)ethyl]-2-thiothymidine (m⁵s²Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m⁵s²Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m⁵Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter ‘opening’, an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m⁵s²Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2′-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.     

Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species.

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.