On the long-wavelength component of the light-harvesting complex of some photosynthetic bacteria

The effect of the presence of a minor antenna component in light-harvesting complexes of photosynthetic bacteria is investigated with numerical simulation employing the transition probability matrix method. A model antenna system of hexagonal symmetry is adopted, using as a working hypothesis that the minor component forms a ring around the trap. Three cases have been considered: (a) the minor component is isoenergetic with the trap, which is at lower energy than the antennas (the “supertrap”), (b) the minor component is at lower energy than the trap, which is at lower energy than the antennas (the “asymmetric gutter”), (c) the minor component is at lower energy than the trap, which is isoenergetic with the antennas (the “gutter”). It is found that the supertrap speeds up the fluorescence decay and enhances the trapping efficiency, whereas the gutter slows down the fluorescence decay and decreases the trapping efficiency. It is concluded that, in contrast to a recent suggestion (Bergström, H., R. van Grondelle, and V. Sundström. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 250:503-508), concentrating excitations in the vicinity of the trap by the so-called long-wavelength minor antenna component purportedly present in Rhodobacter sphaeroides and Rhodospirillum rubrum instead of improving trapping actually impedes trapping.     

Development and characterization of microsatellite markers for endangered species Stipa pennata (Poaceae) and their usefulness in intraspecific delimitation

Molecular Biology Reports - Stipa pennata (Poaceae), has become a rare and endangered species in Central Europe due habitat loss and fragmentation. This species is characterized by high...     

Context-Dependency of Agricultural Legacies in Temperate Forest Soils

Ecosystems - Anthropogenic activities have affected forests for centuries, leading to persistent legacies. Observations of agricultural legacies on forest soil properties have been site specific...     

Pyrimidine pathways in health and disease

Genetic defects involving enzymes essential for pyrimidine nucleotide metabolism have provided new insights into the vital physiological functions of these molecules in addition to nucleic acid synthesis. Such aberrations disrupt the haematological, nervous or mitochondrial systems and can cause adverse reactions to analogue therapy. Regulation of pyrimidine pathways is also known to be disrupted in malignancies. Nine genetic defects have now been identified but only one is currently treatable. Diagnosis is aided by the accumulation of specific metabolites. Recently, progress has been made in understanding the molecular mechanisms underlying inborn errors of pyrimidine metabolism, together with the key clinical issues and the implications for the future development of novel drugs and therapeutic strategies.     

Mechanical coupling in the nitrogenase complex

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188^Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188^Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188^Ser) are important for communication between the two halves.     

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member.     

Mammalian Target of Rapamycin (mTOR) Activity Dependent Phospho-Protein Expression in Childhood Acute Lymphoblastic Leukemia (ALL)

Modern treatment strategies have improved the prognosis of childhood ALL; however, treatment still fails in 25–30% of patients. Further improvement of treatment may depend on the development of targeted therapies. mTOR kinase, a central mediator of several signaling pathways, has recently attracted remarkable attention as a potential target in pediatric ALL. However, limited data exists about the activity of mTOR. In the present study, the amount of mTOR activity dependent phospho-proteins was characterized by ELISA in human leukemia cell lines and in lymphoblasts from childhood ALL patients (n = 49). Expression was measured before and during chemotherapy and at relapses. Leukemia cell lines exhibited increased mTOR activity, indicated by phospho-S6 ribosomal protein (p-S6) and phosphorylated eukaryotic initiation factor 4E binding protein (p-4EBP1). Elevated p-4EBP1 protein levels were detected in ALL samples at diagnosis; efficacy of chemotherapy was followed by the decrease of mTOR activity dependent protein phosphorylation. Optical density (OD) for p-4EBP1 (ELISA) was significantly higher in patients with poor prognosis at diagnosis, and in the samples of relapsed patients. Our results suggest that measuring mTOR activity related phospho-proteins such as p-4EBP1 by ELISA may help to identify patients with poor prognosis before treatment, and to detect early relapses. Determining mTOR activity in leukemic cells may also be a useful tool for selecting patients who may benefit from future mTOR inhibitor treatments.     

Influence of manganese on morphology and cell wall composition of Aspergillus niger during citric acid fermentation

Morphology and cell wall composition of Aspergillus niger were studied under conditions of manganese sufficient or deficient cultivation in an otherwise citric acid producing medium. Omission of Mn²⁺ (less than 10^-7 M) from the nutrient medium of Aspergillus niger results in abnormal morphological development which is characterized by increased spore swelling, and squat, bulbeous hyphae. Fractionation and analysis of manganese deficient cell walls revealed increased chitin and reduced -glucan contents as well as reduction of galactose containing polymers, as compared to cell walls from manganese sufficient grown hyphae. Addition of copper induced the same effect as manganese deficiency, both on morphology and cell wall composition. Addition of cycloheximide also produced a very similar type of morphology with increased chitin and reduced -glucan contents of the cell wall but its effect on galactose was less pronounced.Dedicated to emer. Prof. Dr. J. Kisser on the occasion of his 80th birthday     

Polymerase chain reaction analysis of transgenic plants contaminated byAgrobacterium

Agrobacterium-mediated genetic transformation is a method of choice for the development of transgenic plants. The presence of latentAgrobacterium that multiplies in the plant tissue in spite of antibiotic application confounds the results obtained by polymerase chain reaction (PCR) analysis of putative transgenic plants. The presence ofAgrobacterium can be confirmed by amplification of eitherAgrobacterium chromosomal genes or genes present out of transfer DNA (T-DNA) in the binary vector. However, the transgenic nature ofAgrobacterium-contaminated transgenic plants cannot be confirmed by PCR. Here we report a simple protocol for PCR analysis ofAgrobacterium-contaminated transgenic plants. This protocol is based on denaturation and renaturation of DNA. The contaminating plasmid vector becomes double-stranded after renaturation and is cut by a restriction enzyme having site(s) within the PCR amplicon. As a result, amplification by PCR is not possible. The genomic DNA with a few copies of the transgene remains single-stranded and unaffected by the restriction enzyme, leading to amplification by PCR. This protocol has been successfully tested with 4 different binary vectors and 3Agrobacterium tumefaciens strains: EHA105, LBA4404, and GV3101.