Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus.

Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

Identification of Magnaporthe oryzae Avirulence Genes to Three Rice Blast Resistance Genes.

The segregation of avirulence/virulence was studied in 115 F1 progeny isolates of Magnaporthe oryzae from a cross of two field isolates on three Japanese race-differential rice cultivars Kanto 51, Fukunishiki, and Toride 1. The χ2 tests of goodness-of-fit for a 1:1 ratio indicated that avirulence on cvs. Kanto 51, Fukunishiki, and Toride 1 was under monogenic control. The relationship between the avirulence (Avr) gene in the parental isolate and the Avr gene in the standard isolate was investigated by using 100 lines each of three F3 families from the crosses of the rice cultivars Norin 3/Kanto 51, AK61/Fukunishiki, and Norin 3/Toride 1, respectively. Based on the resistant reactions of the F3 rice lines to the parental isolates and the standard isolates harboring three known Avr genes, three genetically independent Avr genes, AvrPik, AvrPiz, and AvrPiz-t, were identified. The three identified Avr genes were mapped using random amplified polymorphic DNA (RAPD) analysis, and a partial linkage map was constructed with 17 RAPD markers closely linked to the Avr genes. Twelve markers and AvrPik, three markers and AvrPiz, and two markers and AvrPiz-t, as well as mating locus MAT1, constructed linkage groups A, B, and C, respectively.

Uterine angiomyolipoma: case report and review of the literature.

Extrarenal angiomyolipomas (AML) have been reported at various anatomical sites, but infrequently in the gynecological region. In the uterus, only a few cases have been described. We describe a uterine angiomyolipoma occurring in a 40-year-old woman without evidence of tuberous sclerosis. The tumor arose on the right wall of the uterine body and was partially cystic, and it was associated with marked degeneration. It was composed of mature adipose tissue, anomalous blood vessels and non-vascular smooth muscle cells. Immunohistochemistry revealed that non- vascular smooth muscle cells were positive for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, antihuman muscle actin (HHF35) and progesterone receptor (PR), and negative for cytokeratin, antihuman melanoma (HMB45), CD34, S-100 and estrogen receptor (ER). It is of particular interest that non-vascular smooth muscle cells were negative for HMB45, in contrast to renal and other extrarenal AML in which HMB45 immunoreactivity has been demonstrated in these cells.

Cross-reactive and major virus-specific epitopes are located at the N-terminal halves of the cylindrical inclusion proteins of turnip mosaic and zucchini yellow mosaic potyviruses.

To investigate the antigenic nature of cylindrical inclusion proteins (CIPs) of the potyviruses Turnip mosaic virus (TuMV) and Zucchini yellow mosaic virus (ZYMV), monoclonal antibodies (MAbs) against the two CIPs were produced and epitopes on the CIPs were localized using Escherichia coli-expressed CIP fragments in Western blot analysis. All 23 MAbs against ZYMV CIP reacted only with ZYMV CIP. In contrast out of the 18 MAbs produced against TuMV CIP, 14 MAbs were TuMV CIP-specific while the remaining four MAbs cross-reacted with both CIPs. The four cross-reactive MAbs recognized two distinct epitopes in the N-terminal half of TuMV CIP corresponding to amino acid residues 103-119 and 224-237. Thirteen out of 14 TuMV CIP-specific MAbs recognized two distinct epitopes within residues 1-102 and 120-214, while the other one recognized an epitope within residues 301-644. On the other hand, 21 out of 23 ZYMV CIP-specific MAbs recognized epitopes within residues 1-118, while the remaining two recognized epitopes within residues 301-522. These results suggest that cross-reactive and major virus-specific epitopes are located at the N-terminal half of the respective CIPs.

Computer-assisted 3D mapping and morphometry of dysplastic zones in endoscopically resected colonic adenomas.

Three-dimensional (3D) reconstruction and morphometry of resected colonic adenomas were undertaken to extend current knowledge of clinically significant features such as the frequency of occurrence of cancer, and the size and spatial distribution of dysplastic zones in these tumours. Fifty endoscopically resected colonic adenomas were serially sectioned at intervals of 0.2 mm and the sectional images were loaded into a computer system in order to visualize the spatial distribution of dysplastic zones. These were graded into five groups according to the criteria of Morson and Dawson: normal mucosa, mild dysplasia, moderate dysplasia, severe dysplasia, and cancer. The way in which zones of different grades are distributed in a polyp was visualized in a computer display and the volume of each dysplastic zone was estimated by the Cavalieri principle. In five polyps, adenocarcinoma was found growing in an adenoma. In all of these, the cancer was surrounded by less dysplastic zones, in the form of 'cancer in adenoma'. In pedunculated polyps, submucosal invasion could occur even if the volume percent of severe dysplasia was less than 10%. In such a case, multiple biopsy specimens are advisable. Semipedunculated polyps smaller than 200 mm(3) can also harbour submucosal invasion. In this study it was found that if the adenomas had been examined by only a single section, as many as one in five of the cases in which submucosal invasion had already developed would have escaped microscopic confirmation. To prevent such diagnostic failure, it is advisable to add a few deeper sections. Thus, 3D reconstruction and morphometry have been helpful in establishing a better standard for the diagnostic histopathology of colonic tumours.

Application of tyramide signal amplification system to immunohistochemistry: a potent method to localize antigens that are not detectable by ordinary method.

Tyramide signal amplification-avidin-biotin complex (TSA-ABC) method is a powerful technique used to detect antigens that are not detectable by ordinary immunohistochemistry. It is worth trying in cases where localization of antigens by the conventional method has failed and antibodies are precious.

Changing distribution of islets in the developing human pancreas: a computer-assisted three-dimensional reconstruction study.

Tissue specimens of nine normal human pancreata from fetuses, neonates, and adults were subjected to serial sectioning and computer-assisted three-dimensional (3-D) reconstruction in an effort to study the growth of Langerhans' islets based on their distribution in the lobule and their relationship with the ducts and arterioles. Also, islet volumes were obtained by 3-D morphometry. In 24-week-old fetuses, the islets were shown to be in direct contact with the ducts, whereas in the neonates, they gradually became separated from the ducts, finally dispersing throughout the lobule in the adults. This transition seemed to allow islet hormones to have physiological effects on the exocrine function of the acinar tissue, making the pancreas achieve the endocrine-exocrine correlation. However, half of the islets remained next to the ducts even in the adult pancreata. With regard to the relationship between the islets and arterioles, "arteriolar" islets having an afferent arteriole accounted for approximately 20% in number, but their volume cumulated to approximately 70% of the total, comprising a greater part of the pancreatic endocrine gland.

Nucleotide sequences of the cylindrical inclusion protein genes of two Japanese zucchini yellow mosaic virus isolates.

The nucleotide sequences of the cylindrical inclusion protein (CIP) genes of two Japanese zucchini yellow mosaic virus (ZYMV) isolates (ZYMV-169 and ZYMV-M) were determined. The CIP genes of both isolates comprised 1902 nucleotides and encoded 634 amino acids containing consensus nucleotide binding motif. The sequence similarities between the two isolates at the nucleotide and amino acid levels were 91% and 98%, respectively. When the CIP gene sequences of the Japanese ZYMV isolates were compared with those of previously reported ZYMV isolates, the nucleotide and amino acid sequence similarities ranged between 81% and 97%, and between 95% and 97%, respectively. Phylogenetic analysis of the deduced amino acid sequences of the CIP genes indicated that the Japanese ZYMV isolates were closely related to those of other ZYMV isolates.

Effects of Nd:YAG laser irradiation on morphometry and lung function in elastase-induced emphysema in rats.

BACKGROUND & OBJECTIVE: Although thoracoscopic laser ablation therapy has been hailed as an effective surgical treatment for diffuse emphysema, no one has as yet made an in-depth study of the efficacy of this treatment. This investigation was undertaken to research the effects of laser pneumoplasty on an animal model of emphysema. STUDY DESIGN/MATERIALS AND METHODS: Eight weeks after elastase treatment, the rats' left lungs were irradiated using contact Nd:YAG laser. Pulmonary function tests were performed 4 weeks after irradiation and the lungs were prepared for histologic examination. RESULTS: Dense fibrous scars beneath the pleura were observed at 4 weeks after irradiation. Although mean linear intercept values of irradiated lungs were not much lower than those in the non-irradiated elastase-treated group, laser irradiation caused a significant decrease in lung volume. While there was no significant difference in quasistatic compliance, elastic recoil pressure of the lung increased to control levels at total lung capacity volume. CONCLUSION: We conclude that laser therapy does not cause normalization of compliance, or improvement in the deeper part of the emphysematous lung, but rather a peripheral volume reduction and "encasement effect" on the lungs as a result of fibrotic scars.

Experimental ablation of emphysematous rat lung with Nd: YAG laser: lung changes studied by histopathology and SEM.

Laser ablation has been employed as a therapeutic measure for chronic pulmonary emphysema. As yet, however, its effect is not understood on firm pathological basis. We aimed to study, both histopathologically and using Scanning Electric Microscopy (SEM), the changes produced by irradiation with contact Neodymium-yttrium aluminum garnet laser (Nd: YAG laser) in rat lungs with experimentally induced emphysema. Emphysema was produced in 34 rats by instilling elastase via airways. Eight weeks after the instillation, the emphysematous left lung was irradiated under thoracotomy with contact Nd: YAG laser at a power of 5 watts. The animals were sacrificed in acute as well as chronic phase for histopathological observation of lung and scanning electron microscopy. Laser caused necrotic and inflammatory changes in the subpleural zone of lung. Immediately after irradiation, the alveolar septa were destroyed as visualized by SEM, only leaving the elastic skeleton. In a chronic phase, the necrotic zone was collapsed and replaced with a thick fibrous scar which seemed to serve more or less to keep the organ from being excessively inflated. In this model, irradiation induces subpleural dense scarring, which, by "encasing" an emphysematous lung, is expected to more or less normalize the excessive compliance.

Systemic-to-pulmonary vascular malformation of lung visualized by computer-assisted 3-D reconstruction.

AIMS: While a growing number of cases with pulmonary arteriovenous malformation (PAVM) have been reported, detailed analysis has yet to be found on the relation of abnormal vessels with the whole lung vasculature and airways. To gain more insight into the structure-function interrelation of this disease, we attempted to visualize the vessels in and around the arteriovenous malformation, resorting to computer-aided 3-D reconstruction. METHODS AND RESULTS: The material was the upper lobe of the right lung from a 44-year-old man resected for recurrent haemoptysis. On pre-surgical selective angiography, an arteriovenous communication was suggested to exist between a tributary of the right 3rd intercostal artery and pulmonary vein. Semi-serial sections were prepared from the material and submitted to 3-D reconstruction of blood vessels and airways. In 3-D images, branches of the 3rd intercostal artery proved to be forming a plexus of abnormally dilated, thin-walled vessels in the subepithelial layer of a membranous bronchiole, a situation clearly explaining the mechanism of haemoptysis. There was no capillary bed interventing between the afferent arteries and draining vessels leading to the pulmonary vein. CONCLUSIONS: This presents the first overall visualization of PAVM, allowing comparison of 2-D microscopy with the corresponding 3-D morphology.

The lobular architecture of the normal human pancreas: a computer-assisted three-dimensional reconstruction study.

Tissue specimens of the normal pancreata from three adult patients were subjected to serial sectioning and computer-assisted three-dimensional reconstruction of the lobules, blood vessels, pancreatic ducts, and islets, to establish the anatomical relation among these structures. Most lobules, although more or less imperfectly demarcated by septa, were found to receive a single duct (lobular duct), justifying their definition as "glandular lobules." However, there were also lobules receiving not only a lobular duct of their own but a small accessory duct from a neighboring lobule. On the other hand, these lobules were shown not to correspond to the territories of arterial supply, leaving no ground for defining them as "vascular lobules."

Assessment of cancer recurrence in residual tumors after fractionated radiotherapy: a comparison of fluorodeoxyglucose, L-methionine and thymidine.

UNLABELLED: This study evaluates the midterm follow-up of tumor and normal tissue uptake of deoxyglucose, thymidine and methionine after fractionated radiotherapy to assess cancer recurrence in residual tumors. METHODS: AH109A tumor-burdened rats were treated with one to eight doses of 5Gy 60Co radiation. Tissue distribution study with 18F-FDG, 3H-thymidine and 14C-methionine, double-tracer autoradiography with 18F-FDG and 14C-methionine, and single-tracer autoradiography with 14C-labeled deoxyglucose, thymidine and methionine were performed 6 days after the end of therapy. RESULTS: Dose response study shows a significant decrease of tumor uptake of all tracers after two and more doses, even in the case of later recurrence. Whereas 3H-Thd and 14C-Met tumor uptake was similar to that of normal muscle, 18F-FDG tumor uptake remains higher than that of muscle, even in the case of complete tumor cure. The irradiated muscle shows a higher 18F-FDG uptake than the nonirradiated muscle. Autoradiography after eight doses (100% tumor cure) reveals elevated 14C-DG tumor uptake to be ascribable to nonmalignant cellular elements, in particular to a macrophage layer at the rim of necrotic areas. Autoradiography after four and six doses (33% and 57% tumor cure) shows the highest methionine and thymidine uptake in viable cancer cells, whereas deoxyglucose uptake did not differ between viable cancer cells and macrophages. CONCLUSION: To detect and differentiate viable cancer cells in a residual tumor mass after radiotherapy, PET using 11C-methionine or 11C-thymidine may have some advantages over 18F-FDG, especially if the residual tumor includes larger areas of necrosis.

[A clinicopathological study of 3 dimensional structure in adenoma of large intestine].

The 3 dimensional reconstruction electrocomputor was used to study the distribution of atypia epithelia in 50 cases of large intestinal adenoma which were taken by fibercoloscopic resection. The 0.2 mm interval serial sections were stained with HE. Carcinomatous changes in adenomas were found in 17 cases, the rate being 34%. The average size of intraadenoma carcinoma being 3.4 times that of adenoma. The larger the adenoma, the higher the cancer rate. But the adenoma to cancer rate is high (25%) in Ips type adenoma of small size with pedicle. The size of adenoma is not associated with allotype incidence. 14 of the 17 cases were detected by study of conventional paraffin block slides, missed diagnosis rate being 18%. The serial sections must be made with intervals less than 0.6 mm in order to prevent missed diagnosis. This method is also of significance to determine if the excision site is cancer free.

The airway dimension in ordinary human lungs. A standardized morphometry of lung sections.

In chronic obstructive pulmonary disease, obscurities remain in the degree of obstruction and its distribution in the airway tree. To overcome these obscurities, a technique was introduced to estimate the original diameter of an airway in lung sections; this technique involved measurement of the accompanying artery. Ordinary lungs had been obtained at autopsy from 17 deceased patients of various ages; from these lungs, 695 pairs of airways and pulmonary arteries were subjected to computer-assisted measurement of their diameters: the whole range of airways from the segmental bronchi to the respiratory bronchioles was sampled. We proved that a log-log regression existed between the diameter of an airway and that of the pulmonary artery; this regression was sufficiently close to allow one to estimate the diameter of an airway from the diameter of the pulmonary artery, even in chronic obstructive pulmonary disease, in which airways are usually strongly stenosed or dilated. In an additional study of three lungs with various types of chronic obstructive pulmonary disease, the diameters of the pulmonary arteries were shown to remain essentially unaffected.

The site, severity, and distribution of bronchiolar obstruction in lungs with chronic obstructive pulmonary disease. Morphometry and computer-assisted three-dimensional reconstruction of airways.

Autopsy lungs from 10 patients with various types of chronic obstructive pulmonary disease were submitted to morphometry and computer-aided three-dimensional reconstruction of airways to study, by the former technique, the severity and the site of obstruction in the bronchioles and to visualize, by the latter, how far the whole airway bed was involved. The site of obstruction in the bronchioles was shown to be different between diffuse panbronchiolitis and bronchobronchiolitis obliterans. There was also a case where the entire bronchiolar tree was mildly but uniformly stenosed with mucous hypersecretion, chronic inflammation, and fibrosis, a pattern considered to correspond to small airways disease. Thus, the combination of the above techniques proved to be useful in studying the state of the airway bed in chronic obstructive pulmonary disease, a task not accessible by the usual microscopic examination of a single lung section.

Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens.

Ustiloxins A (1a), B (1b), C (1c), D (1d) and E (1e), antimitotic peptides, have been isolated from the water extract of false smut balls caused on the panicles of rice plant by a fungus Ustilaginoidea virens. The structure of 1b was assigned from its spectral property and its amino acid analysis in relation to 1a whose structure was determined previously by a combination of X-ray crystallographic and amino acid analyses. Structures of 1c and 1d were elucidated by their spectroscopic data, specially based on their 1H and 13C NMR spectra. Bioactivities of these compounds against microtubule assembly as well as mammal, plant and fungal cells have been studied.

"Lupinosis"-like lesions in mice caused by ustiloxin, produced by Ustilaginoieda virens: a morphological study.

A crude toxin, obtained by methanol-extraction of a water extract of false smut balls (INA-KOUJI in Japanese) caused by Ustilaginoidea virens on rice panicles, was injected into mice intraperitoneally. Single injection of 100 or 200 mg/kg body weight of the extract was not lethal but caused acute, occasional necrosis of hepatocytes and renal tubular cells, followed by increased number of mitotic figures with occasional multinuclear giant cells. Erosions and ulceration of the forestomach and atrophy of the thymus were observed a week later. Repeated intraperitoneal injection of the crude toxin at the levels of 12 and 25 mg/kg body weight induced more severe necrosis of the liver and kidneys, with delayed occurrence of mitosis. Forestomach erosion also occurred. Serial injection for 10-12 days of either 3 or 6 mg/kg of the crude extract or 400 micrograms/kg of ustiloxin A, using the purified crystals, caused relatively mild but definite liver and kidney lesions similar to those described above. The lesions in the liver and kidney were quite similar to those observed in lupinosis caused by phomopsin A, a mycotoxin produced by Phomopsis leptostromiformis. Isolation of the toxic substance indicates that the contaminated rice panicles may cause toxicosis of cattle, although no field outbreaks have occurred yet.

Estimating neuron dendritic length in 3D from total vertical projections and from vertical slices.

The recently developed method of total vertical projections is illustrated to estimate the total dendritic length of a human Substantia Nigra neuron. Next, the length of the different orders of dendritic branches, and the mean segment length for each order--commonly regarded as important parameters in neuron physiology--are also estimated. Finally, it is shown how to estimate the mean dendritic length in a population of neurons from vertical slices of arbitrary and unknown thickness. Being unbiased and highly efficient, the proposed methods offer interesting alternatives to current procedures used for the metric analysis of neuron arborizations.