Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements

As the first step toward understanding the involvement of endogenous brassinosteroids (BRs) in cytodifferentiation, we analyzed biosynthetic activities of BRs in zinnia (Zinnia elegans L. cv Canary Bird) cells differentiating into tracheary elements. The results of feeding experiments suggested that both the early and late C6-oxidation pathways occur during tracheary element differentiation. Gas chromatography-mass spectrometry analysis revealed that five BRs, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol, and 6-deoxoteasterone, actually existed in cultured zinnia cells and culture medium. Quantification of endogenous BRs in each stage of tracheary element differentiation by gas chromatography-mass spectrometry exhibited that they increased dramatically prior to the morphogenesis, which was consistent with the idea that BRs are necessary for the initiation of the final stage of tracheary element differentiation. Moreover, the proportion of each BR in culture medium was quite different from that in cells, suggesting that specific BRs are selectively secreted into medium and may function outside the cells.     

Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts

Both prostaglandin E2 (PGE2) and low intensity pulsed ultrasound (U/S) exposure have been reported to accelerate fracture repair. We hypothesized that these two pathways are interactive. To verify this hypothesis, we examined the regulation of PGE2 production by U/S exposure (sine wave of 1.5MHz repeating at 1kHz, 30mW/cm2, 20 minutes) in mouse osteoblastic cell line, MC3T3-E1. The production of PGE2 in osteoblasts was augmented by U/S, which was threefold at 60 min. in comparison with unexposed samples. Then we evaluated the expression of cyclooxygenase-2 (COX-2) mRNA, which is a critical enzyme for PGE2 production. U/S rapidly up-regulated the expression of COX-2 mRNA in a time dependent manner. In addition, PGE2 production by U/S was drastically suppressed by a selective inhibitor of COX-2. These results provide strong evidence that PGE2 production in osteoblasts is dependent upon the induction of COX-2 mRNA expression by U/S and offer a mechanistic insight into how U/S accelerates fracture repair.     

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.     

Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.)

A rice nuclear gene, Rf-1, restores the pollen fertility disturbed by the BT-type male sterile cytoplasm, and is widely used for commercial seed production of japonica hybrid varieties. Genomic fragments carrying Rf-1 were identified by conducting chromosome walking and a series of complementation tests. Isolation and analysis of cDNA clones corresponding to the fragments demonstrated that Rf-1 encodes a mitochondrially targeted protein containing 16 repeats of the 35-aa pentatricopeptide repeat (PPR) motif. Sequence analysis revealed that the recessive allele, rf-1, lacks one nucleotide in the putative coding region, presumably resulting in encoding a truncated protein because of a frame shift. Rice Rf-1 is the first restorer gene isolated from cereal crops that has the property of reducing the expression of the cytoplasmic male sterility (CMS)-associated mitochondrial gene like many other restorer genes. The present findings may facilitate not only elucidating the mechanisms of male sterility by the BT cytoplasm and its restoration by Rf-1 but also isolating other restorer genes from cereal crops, especially rice.     

Localized compliance of small airways in excised rat lungs using microfocal X-ray computed tomography.

Airway compliance is a key factor in understanding lung mechanics and is used as a clinical diagnostic index. Understanding such mechanics in small airways physiologically and clinically is critical. We have determined the "morphometric change" and "localized compliance" of small airways under "near"-physiological conditions; namely, the airways were embedded in parenchyma without dehydration and fixation. Previously, we developed a two-step method to visualize small airways in detail by staining the lung tissue with a radiopaque solution and then visualizing the tissue with a cone-beam microfocal X-ray computed tomography system (Sera et al. J Biomech 36: 1587-1594, 2003). In this study, we used this technique to analyze changes in diameter and length of the same small airways ( approximately 150 microm ID) and then evaluated the localized compliance as a function of airway generation (Z). For smaller (<300-microm-diameter) airways, diameter was 36% larger at end-tidal inspiration and 89% larger at total lung capacity; length was 18% larger at end-tidal inspiration and 43% larger at total lung capacity than at functional residual capacity. Diameter, especially at smaller airways, did not behave linearly with V(1/3) (where V is volume). With increasing lung pressure, diameter changed dramatically at a particular pressure and length changed approximately linearly during inflation and deflation. Percentage of airway volume for smaller airways did not behave linearly with that of lung volume. Smaller airways were generally more compliant than larger airways with increasing Z and exhibited hysteresis in their diameter behavior. Airways at higher Z deformed at a lower pressure than those at lower Z. These results indicated that smaller airways did not behave homogeneously.     

Characterization of a new double-filament model of focal cerebral ischemia in heme oxygenase-2-deficient mice

Variations in vascular anatomy in knockout mouse strains can influence infarct volume after middle cerebral artery (MCA) occlusion (MCAO). In wild-type (WT) and heme oxygenase-2 gene-deleted (HO2-/-) mice, infarcts were not reproducibly achieved with the standard intraluminal filament technique. The present study characterizes a double-filament model of MCAO, which was developed to produce consistent infarcts in both WT and HO2-/- mice. Diameters of most cerebral arteries were similar in WT and HO2-/- mice, although the posterior communicating artery size was variable. In halothane-anesthetized mice, two 6-0 monofilaments with blunted tips were inserted into the left internal carotid artery 6.0 and 4.5 mm past the pterygopalatine artery junction to reside distal and proximal to the origin of the MCA. The tissue "volume at risk" determined by brief dye perfusion in WT (59 +/- 2% of hemisphere; +/-SE) was similar to HO2-/- (62 +/- 4%). The volume of tissue with cerebral blood flow <50 ml.min(-1).100 g(-1) was similar in WT (35 +/- 9%) and HO2-/- (36 +/- 11%) during MCAO and at 3 h of reperfusion (<2%). After 1 h MCAO, infarct volume was greater in HO2-/- (44 +/- 6%) than WT (25 +/- 3%). After increasing MCAO duration to 2 h, the difference between HO2-/- (47 +/- 4%) and WT (36 +/- 3%) diminished, but infarct volume remained substantially less than the volume at risk. Infusion of tin protoporphyrin IX, an HO inhibitor, during reperfusion after 1 h MCAO increased infarct volume in WT but not significantly in HO2-/- mice, although infarct volume remained less than the volume at risk. Thus greater infarct volume in HO2-/- mice is not attributable to a greater volume at risk, lower intraischemic blood flow, or poor reflow, but rather to a neuroprotective effect of HO2 activity. The double-filament model may be of use as an alternative in other murine knockout strains in which the standard filament model does not yield consistent infarcts.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

The Sgp3 locus on mouse chromosome 13 regulates nephritogenic gp70 autoantigen expression and predisposes to autoimmunity

By interval mapping of a backcross progeny between New Zealand White (NZW) and C57BL/6 (B6) mice bearing the Y chromosome-linked autoimmune acceleration gene Yaa, we previously identified a genetic locus on mid-chromosome 13, here designated as Sgp3, showing a major effect on the expression of a nephritogenic autoantigen, gp70. In this study, the NZW-derived Sgp3 region was transferred by backcross procedure and marker-assisted selection on the B6 background to produce three independent congenic strains B6.NZW-Sgp3/1, -Sgp3/2, and -Sgp3/3. We show that NZW homozygosity at a single 3 centiMorgans ( approximately 12 megabases (Mb)) interval between markers D13Mit142 and D13Mit254 mediates increased basal serum levels of gp70 in B6.NZW-Sgp3/1 and B6.NZW-Sgp3/2 mice and with a higher degree in males ( approximately 15 micro g/ml) than in females ( approximately 9 micro g/ml) as compared with B6 ( approximately 2 micro g/ml), revealing a gender effect. However, their gp70 levels are still lower than that of NZW mice ( approximately 60 micro g/ml). In addition, B6.NZW-Sgp3/1 and B6.NZW-Sgp3/2 mice showed a moderate 2- to 3-fold increase in serum gp70 in response to LPS, which contrasted with over a 10-fold increase in NZW mice. Although both B6.NZW-Sgp3/1 and B6.NZW-Sgp3/2 mice failed to produce significant amounts of gp70 anti-gp70 immune complexes, unexpectedly, aged B6.NZW-Sgp3/2 congenic males bearing the Yaa gene developed increased titers of IgG autoantibodies to DNA and chromatin. Our data indicate that Sgp3 is involved in a complex process of gp70 production under polygenic control and may provide a significant contribution to lupus susceptibility not only through up-regulation of gp70 autoantigen production but also predisposition to autoimmunity.