The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Plant action: Multiple levels of regulation

This lecture is devoted to the relative contribution of various levels of regulation of the actin cytoskeleton functioning in the cell. Regulation at the levels of gene expression, mRNA and protein synthesis and stability, processes of actin polymerization/depolymerization and actin structures reorganization is briefly considered. Novel information about the pathways of signal transduction to the actin cytoskeleton with the involvement of Arp2/3 complex and RIC proteins is highlighted.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

Aminoadamantanes containing monoterpene-derived fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors

The ability of a number of nitrogen-containing compounds that simultaneously carry the adamantane and monoterpene moieties to inhibit Tdp1, an important enzyme of the DNA repair system, is studied. Inhibition of this enzyme has the potential to overcome chemotherapeutic resistance of some tumor types. Compound (+)-3c synthesized from 1-aminoadamantane and (+)-myrtenal, and compound 4a produced from 2-aminoadamantane and citronellal were found to be most potent as they inhibited Tdp1 with IC₅₀ values of 6 and 3.5 µM, respectively. These compounds proved to have low cytotoxicity in colon HCT-116 and lung A-549 human tumor cell lines (CC₅₀ > 50 µM). It was demonstrated that compound 4a at 10 µM enhanced cytotoxicity of topotecan, a topoisomerase 1 poison in clinical use, against HCT-116 more than fivefold and to a lesser extent of 1.5 increase in potency for A-549.     

NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice

Parkin mutations produce Parkinson’s disease (PD) in humans and nigrostriatal dopamine lesions related to increased free radicals in mice. We examined the effects of NP7, a synthetic, marine derived, free radical scavenger which enters the brain, on H₂O₂ toxicity in cultured neurons and glia from wild-type (WT) and parkin null mice (PK-KO).NP7, 5-10 μM, prevented the H₂O₂ induced apoptosis and necrosis of midbrain neuronal and glial cultures from WT and PK-KO mice. NP7 suppressed microglial activation and the H₂O₂ induced drop-out of dopamine neurons_. Furthermore, NP7 prevented the increased phosphorylation of ERK and AKT induced by H₂O₂. NP7 may be a promising neuroprotector against oxidative stress in PD.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Intrinsically Unstructured Sequences in the mRNA 3ʹ UTR Reduce the Ability of Poly(A) Tail to Enhance Translation

The 5? cap and 3? poly(A) tail of mRNA are known to synergistically stimulate translation initiation via the formation of the cap?eIF4E?eIF4G?PABP?poly(A) complex. Most mRNA sequences have an intrinsic propensity to fold into extensive intramolecular secondary structures that result in short end-to-end distances. The inherent compactness of mRNAs might stabilize the cap?eIF4E?eIF4G?PABP?poly(A) complex and enhance cap-poly(A) translational synergy. Here, we test this hypothesis by introducing intrinsically unstructured sequences into the 5? or 3? UTRs of model mRNAs. We found that the introduction of unstructured sequences into the 3? UTR, but not the 5? UTR, decreases mRNA translation in cell-free wheat germ and yeast extracts without affecting mRNA stability. The observed reduction in protein synthesis results from the diminished ability of the poly(A) tail to stimulate translation. These results suggest that base pair formation by the 3? UTR enhances the cap-poly(A) synergy in translation initiation.     

Focal contacts of spreading platelets with the substratum

Contacts with glass substratum formed by the spreading rabbit platelets were examined by an antibody-exclusion method; monoclonal antibodies against 80 kD bovine serum protein were used. It was found that platelets form focal contacts in the course of spreading. The size of the largest focal contacts formed by platelets is smaller than that of the contacts formed by fibroblasts. The antibody-exclusion method revealed focal contacts of platelets much more clearly than interference reflection microscopy (IRM). The similarity of reactions involved in spreading platelets and of large nucleus-containing tissue cells is discussed.     

Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development

The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly.