Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA₃ gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b₅₅₉ that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ?Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b₅₅₉ into the low potential form. Structural reasons for such effects are discussed.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

Copper(II) complexes based on a new chelating 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine ligand: Synthesis and crystal structures. Lone pair-π, C-H···π, π-π and C-H···A (A = N, Cl) non-covalent interactions

A new bidentate chelating pyrazolylpyrimidine ligand bearing a strong electron-donating substituent, i.e. 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine (L) (Scheme 1), has been synthesized and used to obtain the copper(II) complexes by reaction with CuCl₂. The molar ratio Cu:L = 1:2 leads to isolation of a complex having CuL₂Cl₂ empirical formula, while the molar ratio Cu:L = 1:1 gives a complex with CuLCl₂ empirical formula. The crystal structure of L as well as the structures of both complexes were studied by single crystal X-ray diffraction. The crystal structure of CuL₂Cl₂ compound is formed by trans-[CuL₂Cl₂] mononuclear molecules. Surprisingly, in contrast to the previous compound having molecular structure, the crystal structure of CuLCl₂ consists of mononuclear [CuL₂Cl]⁺ complex cations and dinuclear [Cu₂Cl₆]²⁻ anions. Thus, formula of CuLCl₂ complex can be represented as [CuL₂Cl]₂[Cu₂Cl₆]. In both complexes molecules of L adopt bidentate chelating coordination mode through N² atom of pyrazole and N³ atom of pyrimidine rings forming five-membered CuN₃C metallocycles. Owing to C-H···N interactions and π-π-stacking L molecules form 2D network. In the structure of trans-[CuL₂Cl₂] there exist double lone pair(N(piperidine))-π(pyrimidine) interactions and C-H···Cl contacts resulting in the formation of 1D chains. Layered 2D structure of [CuL₂Cl]₂[Cu₂Cl₆] results from C-H···Cl, C-H···π and double lone pair(Cl([CuL₂Cl]⁺ complex cation)-π(pyrimidine) interactions.     

Effects of various iron supply on oxidative stress development and ferritin formation in the common ice plants

Mesembryanthemum crystallinum L. plants were grown from seeds in perlite. At the age of 4 weeks (juvenile plants) or 6 weeks (adult plants), they were transferred on nutrient media with different Fe³⁺ content brought in as Fe₂(SO₄)₃—EDTA complex (pH 6.0): control, iron deficit, and iron “excess”. Adult plants grown in media differing in iron content were subjected to salinity (300 mM NaCl) during the last 8 days of growth. Biochemical analyses were performed after plant fixation in liquid nitrogen; simultaneously, the samples for electron microscopy were taken. Different content of available Fe³⁺ in medium, especially under salinity conditions, changed sharply the content of chlorophyll and proline, the rate of lipid peroxidation, the level of H₂O₂, the activities of antioxidant enzymes in the leaves and roots, the number and sizes of plastoglobules, and ferritin formation in plastids. Joint action of salinity and iron deficit enhanced oxidative stress development, whereas iron excess hampered oxidative reaction development, reduced the rate of lipid peroxidation, and increased the chlorophyll content. At iron excess, plastoglobule lysis in plastids did not occur, their number and sizes increased, and ferritin deposits appeared, whereas the latter were absent at iron deficit.     

Response of photosynthetic apparatus to moderate high temperature in contrasting wheat cultivars at different oxygen concentrations

The photosynthetic responses to moderately high temperatures (38 degrees C, imposed at 21% or 2% O(2) in air and 1500 mumol m(-2) s(-1)) were compared in wheat (Triticum aestivum L.) cultivars grown in northern regions of Ukraine and expected to be relatively sensitive to high temperatures ('North' cultivars) and in cultivars grown in southern regions and expected to be relatively heat-tolerant ('South' cultivars). Heating intact leaves in 21% O(2) for 1 h decreased CO(2) assimilation by c. 63% in 'North' cultivars and only c. 32% in 'South' cultivars, with a decrease in PSII activity being observed in only one of the 'North' cultivars. Carboxylation efficiency was decreased by about 2.7-fold in 'North' cultivars with no significant effect in 'South' cultivars. The maximum rates of carboxylation by Rubisco in vivo, V(cmax), estimated from Farquhar's model, increased more than 2-fold in 'South' cultivars and remained unchanged in 'North' cultivars while the maximum rate of RuBP regeneration, J(max), decreased by 53% and 21% in 'North' and 'South' cultivars, respectively. Where the heat treatment was imposed in 2% O(2) this increased (as compared with treatment at 21% O(2)) the inhibitory effect on CO(2) assimilation in tolerant cultivars, but decreased it in sensitive ones. The results suggested that differences in tolerance of moderately high temperatures in wheat relate to the stability of the Rubisco function and to RuBP regeneration activity rather than to the effects on PSII activity or stomatal control.