Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

Thyroid hormones and cognitive functioning in healthy, euthyroid women: a correlational study

Thyroid hormones (THs) play a critical role in differentiation, growth, and metabolism of animal and human organ systems, including the brain. Although associations between normal levels of THs and cognitive functions in healthy elderly individuals have been reported, the findings are inconsistent, possibly due to differences in study designs. Because thyroid disease occurs more frequently in women, the goal of the present study was to examine the relationship between levels of THs and performance on neuropsychological tests in 122 healthy, euthyroid women whose mean age was 51 years. Higher levels of free T3 were positively associated with longer completion times (slower performance) on Trail Making Test - Part A (p = 0.006) and Part B (p = 0.032) and on the Tower of London test (p = 0.002). Higher levels of thyroglobulin antibodies (TgAb) were positively correlated with more errors on the Trail Making Test Part B (p = 0.000), on the Word Fluency test (p = 0.023), and on the Design Fluency test (p = 0.045). No significant correlations between TH levels and scores on mood, verbal memory, or working memory measures were observed. The findings point to a possible link between THs and cognitive processes that are mediated primarily by frontal cortex, areas associated with executive function tasks, and suggest that elevations in levels of free T3 and TgAB within the normal range may negatively influence executive functions.     

Genetic polymorphism of NK receptors and their ligands in melanoma patients: prevalence of inhibitory over activating signals

Antitumor cytotoxicity of NK cells and T cells expressing NK-associated receptors is regulated by interaction between their cell surface killer immunoglobulin-like receptors (KIRs) and CD94/NKG2 heterodimers with MHC class I ligands on target cells. To test the hypothesis that KIR and/or HLA polymorphisms, and KIR/HLA combinations could contribute to the tumorigenesis, association studies were performed in 50 patients with malignant melanoma (MM) in different stages of disease and 54 controls. Our data showed that the frequency of inhibitory and activating KIR genes and KIR genotypes did not differ significantly between healthy individuals and melanoma patients. HLA haplotype distribution showed statistically significant increased frequencies of A*01-B*35-Cw*04 (0.069 vs 0.000; pc<0.05; OR=19.9), A*01-B*08-DRB1*03 (0.079 vs 0.019; pc<0.05; OR=4.5), and A*24-B*40-DRB1*11 (0.026 vs 0.000; pc<0.05; OR=7.1) in melanoma patients compared with healthy controls. Individuals homozygous for group 2 HLA-C ligands were less frequent in the patient group compared with the control cohort (12% vs 31.5%; p<0.017). In addition, we observed an increased frequency (88.0% vs 68.5%; p=0.017; OR=2.80) of KIR2DL2/2DL3 in combination with their group 1 HLA-C ligands, while the presence of these KIRs in the absence of the putative ligands was decreased (12.0% vs 31.5%; p=0.017) in the patient group. Furthermore, an increased frequency of activating KIR2DS1 in the absence of the putative HLA-C^Lys80 ligands was found in melanoma patients (16.0% vs 9.2%). In contrast, KIR2DS2 was absent in patients more often (38.0% vs 25.9%) when the presumptive HLA-C^Asn80 ligands were present. A slightly higher incidence of KIR3DL1 in combination with the less effective Bw4^Thr80 ligands was seen in patients with primary (20.8%) compared with metastatic (4.2%) disease. The data obtained in this study imply that there may not be a direct association between KIR gene content in the genome and the presence of malignant melanoma, or melanoma progression. However, some HLA haplotypes could be predisposing to MM in the Bulgarian population. Furthermore, distinct KIR/HLA ligand combinations may be relevant to the development of malignancy whereby inhibition overrides activation of NK cells and T cells expressing NK-associated receptors, which in turn might facilitate tumor escape and progression.     

Crystal structure of Escherichia coli penicillin-binding protein 5 bound to a tripeptide boronic acid inhibitor: a role for Ser-110 in deacylation

Penicillin-binding protein 5 (PBP 5) from Escherichia coli is a well-characterized d-alanine carboxypeptidase that serves as a prototypical enzyme to elucidate the structure, function, and catalytic mechanism of PBPs. A comprehensive understanding of the catalytic mechanism underlying d-alanine carboxypeptidation and antibiotic binding has proven elusive. In this study, we report the crystal structure at 1.6 A resolution of PBP 5 in complex with a substrate-like peptide boronic acid, which was designed to resemble the transition-state intermediate during the deacylation step of the enzyme-catalyzed reaction with peptide substrates. In the structure of the complex, the boron atom is covalently attached to Ser-44, which in turn is within hydrogen-bonding distance to Lys-47. This arrangement further supports the assignment of Lys-47 as the general base that activates Ser-44 during acylation. One of the two hydroxyls in the boronyl center (O2) is held by the oxyanion hole comprising the amides of Ser-44 and His-216, while the other hydroxyl (O3), which is analogous to the nucleophilic water for hydrolysis of the acyl-enzyme intermediate, is solvated by a water molecule that bridges to Ser-110. Lys-47 is not well-positioned to act as the catalytic base in the deacylation reaction. Instead, these data suggest a mechanism of catalysis for deacylation that uses a hydrogen-bonding network, involving Lys-213, Ser-110, and a bridging water molecule, to polarize the hydrolytic water molecule.     

pH,inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure-function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five alpha- and epsilon-substituted L-Lys-D-Ala-D-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6-12.3, and the k(cat)/K(m) vs. pH profile for activity against Ac(2)-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed DD-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A beta-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.     

Membrane deformation under local pH gradient: mimicking mitochondrial cristae dynamics

Mitochondria are cell substructures (organelles) critical for cell life, because biological fuel production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to showing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis.