Structural and functional studies on the tetraheme cytochrome subunit and its electron donor proteins: the possible docking mechanisms during the electron transfer reaction

The photosynthetic reaction centers (RCs) classified as the group II possess a peripheral cytochrome (Cyt) subunit, which serves as the electron mediator to the special-pair. In the cycle of the photosynthetic electron transfer reactions, the Cyt subunit accepts electrons from soluble electron carrier proteins, and re-reduces the photo-oxidized special-pair of the bacteriochlorophyll. Physiologically, high-potential cytochromes such as the cytochrome c₂ and the high-potential iron–sulfur protein (HiPIP) function as the electron donors to the Cyt subunit. Most of the Cyt subunits possess four heme c groups, and it was unclear which heme group first accepts the electron from the electron donor. The most distal heme to the special-pair, the heme-1, has a lower redox potential than the electron donors, which makes it difficult to understand the electron transfer mechanism mediated by the Cyt subunit. Extensive mutagenesis combined with kinetic studies has made a great contribution to our understanding of the molecular interaction mechanisms, and has demonstrated the importance of the region close to the heme-1 in the electron transfer. Moreover, crystallographic studies have elucidated two high-resolution three-dimensional structures for the RCs containing the Cyt subunit, the Blastochloris viridis and Thermochromatium tepidum RCs, as well as the structures of their electron donors. An examination of the structural data also suggested that the binding sites for both the cytochrome c₂ and the HiPIP are located adjacent to the solvent-accessible edge of the heme-1. In addition, it is also indicated by the structural and biochemical data that the cytochrome c₂ and the HiPIP dock with the Cyt subunit by different mechanisms although the two electron donors utilize the same region for the interactions; cytochrome c₂ is recognized through electrostatic interactions while hydrophobic interactions are important in the HiPIP docking.     

Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf

The growth and survival of pathogenic and nonpathogenic Pseudomonas syringae strains and of the nonpathogenic species Pantoea agglomerans, Stenotrophomonas maltophilia, and Methylobacterium organophilum were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic P. syringae strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic P. syringae strains than for nonpathogenic P. syringae strains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic P. syringae strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of P. syringae, P. agglomerans, and S. maltophilia decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.     

Nuclear matrix contains novel WD-repeat and disordered-region-rich proteins

To find novel proteins predicted to participate in the formation of nuclear bodies, nuclear speckles, and nuclear macro-protein complexes, we applied proteome analysis to a HeLa cell nuclear matrix fraction. Proteins in the fraction were separated by SDS-PAGE, digested with trypsin, and analyzed by nanoflow liquid chromatography-iontrap-tandem mass spectrometry. Three hundred and thirty three proteins including 39 novel ones were identified. Seven WD-repeat proteins and 16 disordered region-rich proteins, which act frequently as scaffolding proteins for macro-protein complexes, were found amongst the novel proteins.     

Structural studies on extracellular acidic polysaccharides secreted by three non-nodulating rhizobia

The structures of extracellular, acidic polysaccharides from three non-nodulating rhizobia, Rhizobium trifolii AHU 1134, Rhizobium phaseoli AHU 1133, and Rhizobium lupini KLU were studied by a method involving successive fragmentation with specific two β-d-glycanases of Flavobacterium M64. These three polysaccharides are composed of repeating units of the octassacharide shown. Half of the terminal d-galactose residues are substituted by pyruvic acid acetal groups.

     

Functional cDNA library for efficient expression of measles virus-specific gene products in primate cells.

A cDNA library designed for high-level expression of measles virus-specific gene products in mammalian cells was generated. From this library, functional clones which contained the entire protein-coding sequences of the nucleocapsid (N) and the phosphoprotein (P) genes were isolated. By DNA-mediated gene transfer into a line of simian virus 40-transformed monkey kidney cells, the N-specific cDNA was expressed into a single polypeptide of about 60,000 Mr, which was immunoprecipitated by monoclonal antibodies against the measles virus N protein. In contrast, the P-specific cDNA could be expressed into either one or two species of polypeptides of 75,000 or 70,000 Mr, both of which were immunoprecipitated by monoclonal antibodies against the measles virus P protein.     

High frequency of common DNA copy number abnormalities detected by bacterial artificial chromosome array comparative genomic hybridization in 24 breast cancer cell lines

Breast cancer is a widespread disease in Japan and across the world. Breast cancer cells, as well as most other types of cancer cells, have diverse chromosomal aberrations. Clarifying the character of these chromosomal aberrations should contribute to the development of more suitable therapies, along with the predictions of metastasis and prognosis. Twenty-four breast cancer cell lines were analyzed by bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH). The array slide contained duplicate spots of 4030 BAC clone DNAs covering the entire human genome with 1 Mbp resolution. In all 24 breast cancer cell lines, frequent and significant amplifications as well as deletions were detected by BAC array CGH. Common DNA copy number gains, detected in 60% (above 15 cell lines) of the 24 breast cancer cell lines were found in 76 BAC clones, located at 1q, 5p, 8q, 9p, 16p, 17q, and 20q. Moreover, common DNA copy number loss was detected in 136 BAC clones, located at 1q, 2q, 3p, 4p, 6q, 8p, 9p, 11p, 13q, 17p, 18q, 19p, Xp, and Xq. The DNA copy number abnormalities found included abnormality of the well-known oncogene cMYC (8q24.21); however, most of them were not reported to relate to breast cancer. BAC array CGH has great potential to detect DNA copy number abnormalities, and has revealed that breast cancer cell lines have substantial heterogeneity.     

Low-volume muscular endurance and strength training during 3-week forearm immobilization was effective in preventing functional deterioration

Purpose

The purpose of this study was to determine whether endurance and strength hand grip exercises during 3-week upper limb immobilization preserve muscle oxidative capacity, endurance performance and strength.

Methods

Ten healthy adult men underwent non-dominant forearm immobilization by plaster cast for 21 days. Five healthy adult subjects were designated as the immobilization (IMM) group and five were designated as the immobilization + training (IMM+TRN) group. Grip strength, forearm circumference, dynamic handgrip endurance and muscle oxygenation response were measured before and after the 21 day immobilization period. Using near-infrared spectroscopy (NIRS), muscle oxygen consumption recovery (VO₂mus) was recorded after a submaximal exercise and the recovery time constant (TcVO₂mus) was calculated. Reactive hyperemic oxygenation recovery was evaluated after 5 minutes ischemia. Two training programs were performed by the IMM+TRN group twice a week. One exercise involved a handgrip exercise at 30% maximum voluntary contraction (MVC) at a rate of 1 repetition per 1 second until exhaustion (about 60 seconds). The other involved a handgrip exercise at 70% MVC for 2 seconds with a 2 second rest interval, repeated 10 times (40 seconds).

Results

There was a significant group-by-time interaction between the IMM and IMM+TRN groups in the TcVO₂mus (p = 0.032, F = 6.711). A significant group-by-time interaction was observed between the IMM and IMM+TRN groups in the MVC (p = 0.001, F = 30.415) and in grip endurance (p = 0.014, F = 9.791). No significant group-by-time interaction was seen in forearm circumference and reactive hyperemic oxygenation response either in IMM or IMM+TRN group.

Conclusion

The training programs during immobilization period used in this experiment were effective in preventing a decline in muscle oxidative function, endurance and strength.

     

The role of alternative translation start sites in the generation of human protein diversity

According to the scanning model, 40S ribosomal subunits initiate translation at the first (5 proximal) AUG codon they encounter. However, if the first AUG is in a suboptimal context, it may not be recognized, and translation can then initiate at downstream AUG(s). In this way, a single RNA can produce several variant products. Earlier experiments suggested that some of these additional protein variants might be functionally important. We have analysed human mRNAs that have AUG triplets in 5 untranslated regions and mRNAs in which the annotated translational start codon is located in a suboptimal context. It was found that 3% of human mRNAs have the potential to encode N-terminally extended variants of the annotated proteins and 12% could code for N-truncated variants. The predicted subcellular localizations of these protein variants were compared: 31% of the N-extended proteins and 30% of the N-truncated proteins were predicted to localize to subcellular compartments that differed from those targeted by the annotated protein forms. These results suggest that additional AUGs may frequently be exploited for the synthesis of proteins that possess novel functional properties.Electronic Supplementary Material Supplementary material is available for this article at