Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100-115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Ac as a tool for the functional genomics of rice

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.     

Interaction of granulocyte colony-stimulating factor (G-CSF) with its receptor. Evidence that Glu19 of G-CSF interacts with Arg288 of the receptor.

Granulocyte colony-stimulating factor (G-CSF) forms a tetrameric complex with its receptor, comprising two G-CSF and two receptor molecules. The structure of the complex is unknown, and it is unclear whether there are one or two binding sites on G-CSF and the receptor. The immunoglobulin-like domain and the cytokine receptor homologous module of the receptor are involved in G-CSF binding, and Arg288 in the cytokine receptor homologous module is particularly important. To identify residues in G-CSF that interact with Arg288, selected charged residues in G-CSF were mutated to Ala. To clarify whether there are two binding sites, a chimeric receptor was created in which the Ig domain was replaced with that of the related receptor gp130. This chimera bound G-CSF but could not transduce a signal, consistent with failure of dimerization and loss of one binding site. The G-CSF mutants had reduced mitogenic activity on cells expressing wild-type receptor. When tested with the chimeric receptor, all G-CSF mutants except one (E46A) showed reduced binding, suggesting that Glu46 is important for interaction with the Ig domain. On cells expressing R288A receptor, all the G-CSF mutants except E19A showed reduced mitogenic activity, indicating that Glu19 of G-CSF interacts with Arg288 of the receptor.     

Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitals

Sixty Gram-negative bacterial isolates were collected from Palestinian hospitals in 2006. Thirty-two (53.3%) isolates showed multidrug resistance phenotypes. PCR and DNA sequencing were used to characterize integrons and antimicrobial resistance genes. PCR screening showed that 19 (31.7%) and five (8.3%) isolates were positive for class 1 and class 2 integrons, respectively. DNA-sequencing results for the captured antimicrobial resistance gene cassettes within class 1 integrons identified the following genes: dihydrofolate reductases, dfrA1 , dfrA5 , dfrA7 , dfrA12, dfrA17 and dfrA25 ; aminoglycoside adenyltransferases, aadA1, aadA2 , aadA5, aadA12 and aadB ; aminoglycoside acetyltransferase, aac(6')-Ib ; and chloramphenicol resistance gene, cmlA1 . ESBL were identified in 25 (41.7%) isolates. The identified ESBL were bla _CTX-M-15, bla _CTX-M-56, bla _OXA-1, bla _SHV-1, bla _SHV-12, bla _SHV-32 and bla _TEM-1 genes. Moreover, we characterized the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr and qnrB2 , which were detected in seven (11.7%) and two (3.3%) isolates, respectively. In this study various types of antibiotic resistance genes have been identified in Gram-negative bacteria from Palestinian hospitals, many of which are reported in the Middle East area for the first time.     

Molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Japan

Aims:  To investigate the prevalence of integrons and antimicrobial resistance genes in Salmonella recovered from animals in Japan.
Methods and Results:  Forty-eight out of ninety-four (51·1%) Salmonella isolates showed multidrug resistance phenotypes and harboured at least one antimicrobial resistance gene. Twenty-two out of forty-seven (46·8%) Salmonella enterica serovar Typhimurium that were multidrug-resistant were of definitive phage type DT104. Class 1 integrons were identified in 34/94 isolates (36·2%): 21 isolates containing two gene cassettes, aadA2 and bla _PSE–1, and 13 containing one gene cassette, aadA1 , aadA2 or bla _PSE–1. Class 2 integrons containing estX - sat2 - aadA1 gene cassettes were only identified in Salmonella Enteritidis. The β-lactamase-encoding gene, bla _TEM, was only detected in S. Typhimurium. The plasmid-mediated quinolone resistance gene, qnrS1 , was identified in S. Typhimurium and Salmonella Thompson.
Conclusions:  Our results characterized integrons and antimicrobial resistance genes in Salmonella of animal origin. To the best of our knowledge, this is the first report of qnrS in Salmonella from Japan and also the first report of qnrS in S . Thompson.
Significance and Impact of the Study:  Little is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals. This study provides useful data on the incidence of integrons and resistance genes in Salmonella of animal origin.     

Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla _TEM-1 and bla _CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

Design, total synthesis, and biological evaluation of neodysiherbaine A derivative as potential probes

To enable studies to elucidate the detailed biological function of dysiherbaine and neodysiherbaine A, potent and subunit-selective agonists for ionotropic glutamate receptors, the derivative with a hydroxymethyl substituent at the C10 position has been developed. Preliminary biological evaluation of the analogue showed that a C10 hydroxymethyl substituent produced significant alterations in binding affinities for the ionotropic glutamate receptor subtypes.     

Three-dimensional reconstruction of the human capillary network and the intramyocardial micronecrosis

Three-dimensional reconstruction of the human heart was performed to define the structure of the intramyocardial microvasculature. A total of 200 consecutive serial sections of 6 μm each were prepared from the left ventricular tissue of an autopsied human heart with normal coronary arteries. The corresponding arteriole, venule, and all capillaries were reconstructed using three-dimensional software. The capillary network extended right and left along the cardiomyocyte with major and minor axes of about 130 and 120 μm, respectively. The capillary length from an arteriole to an adjacent venule was about 350 μm. Two types of sack-like structures, the precapillary sinus and the capillary sinus, were present in the capillary network, and many capillaries diverged from these sinuses. The cardiomyocytes were covered with reticular capillaries. In contrast, the precapillary and capillary sinuses were surrounded by many cardiomyocytes. The arterial and venous capillaries were positioned alternately, forming a lattice pattern. Intramyocardial microcirculatory units forming a capillary network from an arteriole to adjacent venules on both sides were present. The sizes of myocardial micronecroses corresponded to that of the intramyocardial microcirculatory unit. These results show that the capillary network is an ordered and anatomically regulated structure and that the microcirculatory unit and the precapillary and capillary sinuses may play an important role in maintaining the intramyocardial microcirculation during contraction and relaxation.