Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.     

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level ($～$100–1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.     

Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed to shear stress

Morphology and mechanical properties of cultured endothelial cells were measured, using a novel atomic force microscope (AFM) system, developed in our laboratory, in conjunction with an inverted confocal laser scanning microscope. We used this system to examine endothelial cell both in static cultures and exposed to a shear stress of 2 Pa. Initially, the three-dimensional topography of a cell was measured by the AFM and a location was selected for the subsequent measurement of the mechanical response of the cell. The surface of statically cultured cell was smooth. The cell height was not altered by the exposed duration of shear stress. A relationship between external force, F, and the indentation depth, delta, was obtained for several different locations on a cell. This force-indentation response was modelled using a quadratic equation, F = adelta2 + bdelta, indicating that two parameters, a and b, will be constants which are representative of the mechanical response. Endothelial cells cultured at static conditions demonstrated a polygonal shape and less stiff mechanical characteristics around the nucleus compared to those at peripheral regions. The stiffness of the endothelial cells exposed to shear stress increased with the duration time of exposure. At 6-h exposures, the stiffness was higher at upstream side of the cell than the downstream side. However, after 24-h exposure, the stiffness was similar on both sides of the cell. These changes in the stiffness of endothelial cells when exposed to shear stress were suggested to correspond with the distribution of stress fibers in the cell.     

Modulation of synaptic plasticity by brain estrogen in the hippocampus

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention.     

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of α-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that α-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that α-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.