Proteomic analysis of growth phase-dependent proteins of Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen that causes a variety of diseases, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis, in both adults and children. The global pattern of growth phase-dependent protein expression of S. pneumoniae during in vitro culture was analyzed using 2-DE combined with MALDI-TOF MS and LC/ESI-MS/MS. Several protein production patterns were observed at four time points throughout the growth stage, although some protein levels did not change significantly. We focused on the switch in protein expression at the transition from log growth phase to stationary phase. Proteins that were significantly induced or repressed at this point are likely to be involved in central intermediary metabolism, amino acid synthesis, nucleotide, and fatty acid metabolism, cell wall synthesis, protein degradation, and stress responses. This global expression profiling approach has revealed previously unrecognized relationships between proteins in the life of this pathogen.     

Binding model for eriodictyol to Jun-N terminal kinase and its anti-inflammatory signaling pathway

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-α, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signalregulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, 8.79 × 10⁵ M^-1. Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK. [BMB Reports 2013; 46(12): 594-599]     

Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes

Chronic inflammation is closely associated with metabolic disorders such as obesity and type 2 diabetes, however, the underlying mechanism is unclear. Toll-like receptors (TLRs) play a key role in innate immune response as well as inflammatory signals. Here, we observed that mRNA level of TLR4 was induced during adipocyte differentiation and remarkably enhanced in fat tissues of obese db/db mice. In addition, activation of TLR4 with either LPS or free fatty acids stimulated NFkappaB signaling and expression of inflammatory cytokine genes, such as TNFalpha and IL-6 in 3T3-L1 adipocytes. Furthermore, we discovered that TLR4 activation in 3T3-L1 adipocytes provoked insulin resistance. Taken together, these results suggest that activation of TLR4 in adipocyte might be implicated in the onset of insulin resistance in obesity and type 2 diabetes.     

Structure-activity relationship of 5'-substituted fluoro-neplanocin a analogues as potent inhibitors of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues la-d were designed and synthesized, and the inhibitory activity against SAH was in the following order: NH2 > SH > F, N3, indicating a hydrogen bonding donor is essential for inhibitory activity.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Synthesis and evaluation of nanoglobular macrocyclic Mn(II) chelate conjugates as non-gadolinium(III) MRI contrast agents

Because of the recent observation of the toxic side effects of Gd(III) based MRI contrast agents in patients with impaired renal function, there is strong interest on developing alternative contrast agents for MRI. In this study, macrocyclic Mn(II) chelates were conjugated to nanoglobular carriers, lysine dendrimers with a silsesquioxane core, to synthesize non-Gd(III) based MRI contrast agents. A generation 3 nanoglobular conjugate of Mn(II)-1,4,7-triaazacyclononane-1,4,7-triacetate-GA amide (G3-NOTA-Mn) was also synthesized and evaluated. The per ion T(1) and T(2) relaxivities of G2, G3, G4 nanoglobular Mn(II)-DOTA monoamide conjugates decreased with increasing generation of the carriers. The T(1) relaxivities of G2, G3, and G4 nanoglobular Mn(II)-DOTA conjugates were 3.3, 2.8, and 2.4 mM(-1) s(-1) per Mn(II) chelate at 3 T, respectively. The T(1) relaxivity of G3-NOTA-Mn was 3.80 mM(-1) s(-1) per Mn(II) chelate at 3 T. The nanoglobular macrocyclic Mn(II) chelate conjugates showed good in vivo stability and were readily excreted via renal filtration. The conjugates resulted in much less nonspecific liver enhancement than MnCl(2) and were effective for contrast-enhanced tumor imaging in nude mice bearing MDA-MB-231 breast tumor xenografts at a dose of 0.03 mmol Mn/kg. The nanoglobular macrocyclic Mn(II) chelate conjugates are promising nongadolinium based MRI contrast agents.     

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.