Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.     

FGF is an essential mitogen and chemoattractant for the air sacs of the drosophila tracheal system

The Drosophila adult has a complex tracheal system that forms during the pupal period. We have studied the derivation of part of this system, the air sacs of the dorsal thorax. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila, correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role.     

Quantitative determination of glufosinate in biological samples by liquid chromatography with ultraviolet detection after p-nitrobenzoyl derivatization

We have established a new HPLC method for derivatizing and quantifying glufosinate (GLUF) in human serum and urine using p-nitrobenzoyl chloride (PNBC). The p-nitrobenzoyl derivative of GLUF (PNB-GLUF) was produced quantitatively over 10 min at room temperature. PNB-GLUF possesses the property of ultraviolet (UV) light absorption with a lambda(max) of 272.8 nm, and was isolated from biological specimens by reversed-phase chromatography using Inertsil Ph-3. In experiments at a UV wavelength of 273 nm, GLUF has a quantitative detection limit of 0.005 microg/ml, and when it was added to both serum and urine to yield concentrations of 0.1-1000 microg/ml, its recovery rate was quite satisfactory: at least 93.8% in all cases. Further, the measured amounts of GLUF in 23 serum samples from patients intoxicated by ingestion of GLUF compared favorably with those obtained by fluorescence derivatization-HPLC using 9-fluorenylmethyl chloroformate (R=0.998). This technique of analysis is, in addition, applicable for Glyphosat, which possesses a chemical structure resembling that of GLUF, and it will be of great use in the determination of these two compounds.     

Enantioselective analysis of glufosinate using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography

We have developed a new analytical method to quantify the DL-homoalanine-4-yl(methyl)phosphinate (DL-GLUF) enantiomers in biological specimens using a reversed-phase high-performance liquid chromatography system with a fluorescence detection system. The derivatization of DL-GLUF enantiomers with (+)-1-(9-fluorenyl)ethyl chloroformate was carried out under mild conditions (40 degrees C for 30 min) without inducing racemization. The lower limit of quantitation was 0.01 microg/ml for both D-GLUF and L-GLUF, and the detection limit was 5 ng/ml. When DL-GLUF enantiomers were added to serum to produce concentrations between 0.1 and 100 microg/ml, the mean recovery rate was at least 93.8%. The recovery rate from urine was also satisfactory.     

Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases

The hydrolysis reaction of chitosan using immobilized chitosanases with regard to the composition of its products and the yield of the intermediate target products, pentamer and hexamer of chitosan oligosaccharides, was investigated. Chitosanase was immobilized onto agar or agarose gel particles by the multipoint attachment method. In batch experiments, surface enzyme density, support particle size, temperature, agitator speed, and initial substrate concentration significantly affected the composition of the oligosaccharides produced. It was believed that these factors all related to the reaction rate and mass transfer rate at the surface of the support materials immobilizing the enzymes. These effects were summarized as a correlation with Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The result showed that the reaction conditions that give a low value of Da provide a high yield of pentamer and hexamer oligosaccharides.     

Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase

Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori‐induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase. Materials and Methods. To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM. Results. We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours. Conclusions. Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori‐induced gastric disorders by SEM.     

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.     

The relationship and its change with aging between ADL and daily life satisfaction characteristics in independent Japanese elderly living at home

The purposes of this study were to examine the characteristics of the relationship between ADL ability and daily life satisfaction and the pattern change with aging in independent Japanese elderly, and to compare these tendencies between males and females. The characteristics of ADL ability and daily life satisfaction of 482 subjects (213 males, 269 females) were investigated in a self-response survey. Seventy-four ADL items, considered from previous studies, were selected from nine ADL domains of 1) movement, 2) going up and down stairs, 3) changing and holding posture, 4) bathing, 5) toileting, 6) dressing, 7) grooming, 8) eating, and 9) manual activities, and nine items of daily life satisfaction were selected from physical, psychological and sociological factors. Both ADL ability and life satisfaction of independent elderly tended to decline with aging. From correlation analysis, since life satisfaction of the elderly was higher with high ADL ability level, it was considered that ADL ability level is one of the important factors in providing for life satisfaction of independent elderly. The subjective symptoms of inconvenience in the lower extremity and lumbar region increased from the 70s in both genders, and the use of assisting devices for movement remarkably increased in the 80s in both genders. The use of assisting devices closely related to the activity area in daily life and influenced the characteristics of life satisfaction and its age-related change in the relationship between ADL ability and life satisfaction. The relationship between ADL ability and satisfaction with physical function was similar in both genders, while the relationship between ADL ability and satisfaction with sociological and psychological factors was different between males and females. Since the relationship between ADL ability and life satisfaction of independent elderly is influenced by a combination of personal, cultural, and environmental factors, additional study must investigate in detail the influence of these factors.     

Longitudinal assessment of ADL ability of partially dependent elderly people: examining the utility of the index and characteristics of longitudinal change in ADL ability

The present study aimed to evaluate the utility of the activities of daily living (ADL) index for partially dependent elderly people (ADL-PDI) when applied longitudinally to an institutionalized partially dependent (PD) group, and to determine the characteristics of the longitudinal change in ADL ability of the PD group. The subjects were ten Japanese PD living at welfare institutions for the aged such as accredited nursing homes and health facilities (mean age was 82.2 +/- 2.32 years in total; 84.3 +/- 4.18 years for five males; 80.3 +/- 2.33 years for five females). The questionnaire consisted of the ADL-PDI, the Barthel index (BI), physical independence, dementia independence, anamnesis, body impairments, use of assisting devices, the institutionalized period, and type of medical rehabilitation and medical treatment, and was administered to the subjects twice during their institutionalized period. All testers were staff working at the subjects' institution, such as occupational therapists, physiotherapists and nurses. The result of the longitudinal ADL assessment was that ADL-PDI may evaluate the longitudinal change in ADL ability on a unidimensional scale. The utility of the standard for discriminating the functional level of the elderly using the ADL-PDI score, which was indicated in our previous study (Sato et al., 2001), was supported by longitudinal data. Furthermore, the BI was superior to the ADL-PDI in evaluating the disabled elderly with lower functional levels. However, the ADL-PDI was better than the BI in evaluating the disabled elderly with a higher functional level and was considered to have wider applications in evaluating the ADL ability of the elderly.