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Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are large ladder-like polycyclic ethers with the 13 ether rings ranging from five- to nine-membered. In this paper, we describe the total synthesis of the two most toxic members of the ciguatoxin family, ciguatoxin 1 and 51-hydroxyCTX3C 2, based on a unified synthetic strategy. The key features in our syntheses were (i) direct construction of the O,S-acetal from the corresponding left and right wing fragments (3, 4, 14); (ii) stereo- and chemoselective radical reaction of the alpha-oxyradical with pentafluorophenyl acrylate to achieve cyclization of the seven-membered G-ring; (iii) ring-closing metathesis reaction to build the nine-membered F-ring; and (iv) an efficient protective group strategy using the oxidatively removable 2-naphthylmethyl groups.

We prepared a monoclonal antibody (mAb), IgM class, to rabbit muscle aldolase (ALD) A4. The mAb reacted with the ALD A subunit but not with the B and C subunits. Distribution of the ALD A subunit was studied employing immunohistochemistry, with the ABC procedure in the rabbit tissues. The ALD A subunit was localized in the l-band of the skeletal muscle fibers. In the liver, endothelial cells showed strong immunoreactivity while parenchymal cells were negative or yielded only a faint reaction. The collecting tubules were positively stained in the kidney. Strong reactivity was seen in the proximal tubules of the outer medulla but not in those of the cortex. In the adrenal gland, cells in the cortex exhibited strong immunostaining, while medullary cells were negative. In the testis, most of the seminiferous epithelium was moderately immunoreactive. Intense staining was present in interstitial cells. The ALD A subunit was localized in the interstitial cells and follicular cells of primary follicles in the ovary. In the cerebellum, the ALD A subunit was localized exclusively in nerve fibers of the white matter; Purkinje cells and nerve cells in the granular layer were negative.

A concise radical route to the framework of azadiradione is developed, in which the four carbon—carbon bonds and seven stereocenters featuring the C13,14-cis configuration are constructed in one step.

Perinatal estrogen exposure elicits a wide range of abnormalities in the female genital tract. Since angiogenesis is essential for morphogenesis, we investigated the vascular density, integrity of vasculatures, and expression of angiogenic factors and their receptors in the uteri of mice treated with diethylstilbestrol (DES) neonatally (DES-mice); the uteri were collected from Day 4 to Day 20. DES treatment reduced the number and density of vasculatures immunostained with PECAM1 (platelet and endothelial cell adhesion molecule 1) in the stroma. Horseradish peroxidase injected into the left ventricle leaked into the endometrium and myometrium on Day 10 in the DES-mice but not in the controls. Electron microscopy confirmed the immaturity of the capillaries, which had an incomplete basal lamina and fewer pericytes. Immunohistochemical studies demonstrated that VEGFA (vascular endothelial growth factor A) expression and ANGPT1 (angiopoietin 1) expression were down-regulated in the stromal cells until Days 20 and 10, respectively. The number of vasculatures with ANGPT2 immunoreaction was reduced in the DES-mice. DES treatment suppressed the expression of VEGFR2 (VEGF receptor 2) and the co-receptor NRP1 (neuropilin 1) as well as TEI2 in the vasculatures. The results of RT-PCR and Western blotting supported the down-regulation of the expression of angiogenic factors and their receptors in DES-mice, whereas the VEGFR1 protein expression was up-regulated. These results suggested that the low concentration of angiogenic factors in the stroma was primarily responsible for the low vascular density in the stroma of the DES-mice, and that the low vascular density and immature vasculatures resulted in uterine malformations.

Perinatal estrogen exposure elicits a wide range of abnormalities in the female genital tract. Since angiogenesis is essential for morphogenesis, we investigated the vascular density, integrity of vasculatures, and expression of angiogenic factors and their receptors in the uteri of mice treated with diethylstilbestrol (DES) neonatally (DES-mice); the uteri were collected from Day 4 to Day 20. DES treatment reduced the number and density of vasculatures immunostained with PECAM1 (platelet and endothelial cell adhesion molecule 1) in the stroma. Horseradish peroxidase injected into the left ventricle leaked into the endometrium and myometrium on Day 10 in the DES-mice but not in the controls. Electron microscopy confirmed the immaturity of the capillaries, which had an incomplete basal lamina and fewer pericytes. Immunohistochemical studies demonstrated that VEGFA (vascular endothelial growth factor A) expression and ANGPT1 (angiopoietin 1) expression were down-regulated in the stromal cells until Days 20 and 10, respectively. The number of vasculatures with ANGPT2 immunoreaction was reduced in the DES-mice. DES treatment suppressed the expression of VEGFR2 (VEGF receptor 2) and the co-receptor NRP1 (neuropilin 1) as well as TEI2 in the vasculatures. The results of RT-PCR and Western blotting supported the down-regulation of the expression of angiogenic factors and their receptors in DES-mice, whereas the VEGFR1 protein expression was up-regulated. These results suggested that the low concentration of angiogenic factors in the stroma was primarily responsible for the low vascular density in the stroma of the DES-mice, and that the low vascular density and immature vasculatures resulted in uterine malformations.

Employing immunohistochemistry and in situ hybridization, we studied the temporal and cell type specific localization of c-Fos and c-Jun proteins and the corresponding messenger RNAs (mRNAs) elicited by a single 17beta-estradiol (E2) injection in the uteri of castrated adult mice. Cellular expression of mRNAs was in parallel with the synthesis of proteins within 1 h. E2 stimulated the c-fos expression rapidly and transiently in the epithelium and vascular endothelium. A second small peak of c-Fos protein and c-fos mRNA expression occurred around 11-13 h in the epithelium. No detectable amount of c-fos transcript and protein was present throughout the time course (0-24 h) in the stromal and myometrial cells. E2 treatment caused differential c-jun expression in all uterine cell types. In the epithelium, c-jun mRNA and protein expression was decreased during 1-6 h post injection, and thereafter returned showing small peak around 11-13 h. Induction of c-Jun protein and c-jun mRNA was evident in the stromal and myometrial cells at 2-3 h, and then the expression gradually decreased and returned to nearly control level by 24 h. E2 treatment induced rapid and transient activation of c-jun in the vascular endothelium. Present results suggest that transient increase of c-Fos and decrease of c-Jun protein at the early phase and coexpression of these proteins at the late phase contribute the proliferation of endometrial epithelium in mature mice. Furthermore, c-Fos and c-Jun expression in the vascular endothelium at the early phase may participate in the uterine imbibition.

In this study, the detailed horizontal structure, i.e. cliffs and plateaux of the heat island of the Metropolitan Tokyo area is investigated. According to Oke (1977), cliff is steep temperature gradient at the rural/urban boundary and plateau is a steady but weaker horizontal gradient of increasing temperature towards the city center. However, these features are not always evident, e.g. large city like Tokyo. To elucidate such aspects, moving observations of the horizontal distribution of air temperature from electric trains of the transportation network of Metropolitan Tokyo during late evening or early morning were thus conducted. In total, 16 railroad lines were used for the moving observations. The observations were done in two phases for sectional and horizontal distributions. Results show that three cliffs exist in the heat island of Metropolitan Tokyo, although the location of these cliffs should be taken into consideration for urban planning or urban redevelopment.

Lactoferrin (LF) is known as an estrogen-inducible protein in the murine uterus. This study, employing immunoelectron microscopy with the pre-embedding methods, was carried out to elucidate temporal LF induction, the process of the induction and intracellular localization after 17βestradiol (E2) stimulation in the enodometrial epithelium of ovariectomized adult mice. By single i. p. injection of E2 (20μg/kg b. w.), LF was rapidly induced, even after 1 hr, and was exclusively localized in the nucleoli of surface and glandular epithelium. At 7 hr after E2 injection, strong immunostaining was recognized in the amorphous cytoplasm and in nucleoli, especially in the dense fibrillar component of the epithelium. From 13 hr to 23 hr after E2 administration, strong reaction was observed in the secretory pathway, i. e., cisternae of endoplasmic reticulum and the Golgi apparatus, and in vesicles and vacuoles in the apical cytoplasm, in addition to the nucleolar staining. LF-immunoreaction was also detected in the nucleoli and cytoplasm of stromal and muscle cells; it was demonstrated in the epithelium at the earliest period, subsequently in the stromal cells (7 hr), and finally in the muscle cells (13 hr). After three days of consecutive E2 stimulation, many secretory granules in the apical cytoplasm and apical cell membrane showed intense LF immunoreaction. The present study suggests that LF in the nucleolus plays an important role in activation of ribosomal biogenesis preceding the cell differentiation and proliferation in the mouse uterus.

The authors intend to develop the environmental education through satellite images, especially in using by LANDSAT images, as one of the best ways for visualized educational means. In recent years, many kinds of remote sensing systems are improved remarkably and are indispensable for understanding physical system of the earth. Especially, the LANDSAT images prepare the information for large area simultaneously and repeatedly.The understanding of physical system means the understanding of earth's radiative equilibrium because solar energy is the only one energy that the earth receives within the solar systems, the earth-atmosphere system, or solar-earth system as more global physical system. The information obtained from images by TM (Thematic Mapper), on-board LANDSAT 4 and 5, are very valuable for understanding the system because each band of the TM are reflected solar energy (Bands 1 through 5 and 7) and long wave radiation (Band 6) from the earth-atmosphere system.The purpose of the environmental education is to bring up environmental ethics according to the understanding of physical system. For this purpose, learning the urban heat island phenomena, the most serious problem for the disruption of the physical system as thermal pollution, is the one of the best ways to experience the disruption of the system. Therefore, to recognize the urban heat island phenomena visually, from the LANDSAT images is the very excellent way for understanding the thermal pollution of the system from personal experiences.

A simple security system based on acceleration sensors for orchards is proposed to detect malicious picking activities by humans. The system reliably detects and accurately recognizes abnormal movements of tree branches. Experiments are conducted to verify its feasibility, especially its ability to discriminate normal movements, e.g. by wind, from abnormal ones. The results show that abnormal movements can be detected reliably if an adequate threshold is set. System considerations for realizing an actual system are also discussed.

In our previous study, we introduced a combination methodology of Fluorescence Correlation Spectroscopy (FCS) and Transmission Electron Microscopy (TEM), which is powerful to investigate the effect of intracellular environment to biochemical reaction processes. Now, we developed a reconstruction method of realistic simulation spaces based on our TEM images. Interactive raytracing visualization of this space allows the perception of the overall 3D structure, which is not directly accessible from 2D TEM images. Simulation results show that the diffusion in such generated structures strongly depends on image post-processing. Frayed structures corresponding to noisy images hinder the diffusion much stronger than smooth surfaces from denoised images. This means that the correct identification of noise or structure is significant to reconstruct appropriate reaction environment in silico in order to estimate realistic behaviors of reactants in vivo. Static structures lead to anomalous diffusion due to the partial confinement. In contrast, mobile crowding agents do not lead to anomalous diffusion at moderate crowding levels. By varying the mobility of these non-reactive obstacles (NRO), we estimated the relationship between NRO diffusion coefficient (Dnro) and the anomaly in the tracer diffusion (α). For Dnro=21.96 to 44.49 μm2/s, the simulation results match the anomaly obtained from FCS measurements. This range of the diffusion coefficient from simulations is compatible with the range of the diffusion coefficient of structural proteins in the cytoplasm. In addition, we investigated the relationship between the radius of NRO and anomalous diffusion coefficient of tracers by the comparison between different simulations. The radius of NRO has to be 58 nm when the polymer moves with the same diffusion speed as a reactant, which is close to the radius of functional protein complexes in a cell.

The intracellular environment is known to be a crowded and inhomogeneous space. Such an in vivo environment differs from a well-diluted, homogeneous environment for biochemical reactions. However, the effects of both crowdedness and the inhomogeneity of environment on the behavior of a mobile particle have not yet been investigated sufficiently. As described in this paper, we constructed artificial reaction spaces with fractal models, which are assumed to be non-reactive solid obstacles in a reaction space with crevices that function as operating ranges for mobile particles threading the space. Because of the homogeneity of the structures of artificial reaction spaces, the models succeeded in reproducing the physiological fractal dimension of solid structures with a smaller number of non-reactive obstacles than in the physiological condition. This incomplete compatibility was mitigated when we chose a suitable condition of a perimeter-to-area ratio of the operating range to our model. Our results also show that a simulation space is partitioned into convenient reaction compartments as an in vivo environment with the exact amount of solid structures estimated from TEM images. The characteristics of these compartments engender larger mean square displacement of a mobile particle than that of particles in smaller compartments. Subsequently, the particles start to show confined particle-like behavior. These results are compatible with our previously presented results, which predicted that a physiological environment would produce quick response and slow exhaustion reactions.

In vivo environments are highly crowded and inhomogeneous, which may affect reaction processes in cells. In this study we examined the effects of intracellular crowding and an inhomogeneity on the behavior of in vivo reactions by calculating the spectral dimension (ds), which can be translated into the reaction rate function. We compared estimates of anomaly parameters obtained from Fluorescence Correlation Spectroscopy (FCS) data with fractal dimensions derived from Transmission Electron Microscopy (TEM) image analysis. FCS analysis indicated that the anomalous property was linked to physiological structure. Subsequent TEM analysis provided an in vivo illustration; soluble molecules likely percolate between intracellular clusters, which are constructed in a self-organizing manner. We estimated a cytoplasmic spectral dimension ds to be 1.39 ± 0.084. This result suggests that in vivo reactions initially run faster than the same reactions in a homogeneous space; this conclusion is consistent with the anomalous character indicated by FCS analysis. We further showed that these results were compatible with our Monte-Carlo simulation in which the anomalous behavior of mobile molecules correlates with the intracellular environment, leading to description as a percolation cluster, as demonstrated using TEM analysis. We confirmed by the simulation that the above-mentioned in vivo like properties are different from those of homogeneously concentrated environments. Additionally, simulation results indicated that crowding level of an environment might affect diffusion rate of reactant. Such knowledge of the spatial information enables us to construct realistic models for in vivo diffusion and reaction systems.

A secure and stereocontrolled route to the seven-membered M-ring moiety of Caribbean ciguatoxin C-CTX-1 is developed featuring i) the photoinduced [2 + 2]cyclization to construct the cyclobutene structure (III), ii) the enzymatic desymmetrization of bis-acetate (III), and iii) the stereoselective reduction of diketone (VII).

Gold nanorods have strong absorption bands in the near-infrared region, in which light penetrates deeply into tissues. The absorbed light energy is converted into heat by gold nanorods, the so-called 'photothermal effect'. Hence, gold nanorods are expected to act not only as on-demand thermal converters for photothermal therapy but also as controllers of a drug-release system responding to irradiation by near-infrared light. To achieve a controlled-release system that can be triggered by light irradiation, double-stranded DNA (dsDNA) was modified on gold nanorods. When the dsDNA-modified gold nanorods were irradiated by near-infrared light, the single-stranded DNA (ssDNA) was released from gold nanorods due to the photothermal effect. The amount of released ssDNA was dependent upon the power and exposure time of light irradiation. Release of ssDNA was also observed in tumors grown on mice after light irradiation. Such a controlled-release system of oligonucleotide triggered by the photothermal effect could expand the applications of gold nanorods that have unique optical characteristics in medicinal fields.

Parkinson's disease (PD), which is characterized by degeneration of mesencephalic dopaminergic neurons of unclear etiology, is primarily an age-related neurodegenerative disorder, while the normal process of aging is also known to decrease the number of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA). However, no consensus exists regarding how advancing age may predispose the dopaminergic system to PD. The Klotho-insufficient (klotho) mouse exhibits a syndrome that resembles human aging. Recent studies have revealed that abnormal activation of vitamin D is the major cause of this phenotype. In this study, we examined mesencephalic dopaminergic neurons of klotho mice and identified tyrosine hydroxylase-positive neurons in the SNc and VTA, and found that levels of striatal dopamine were significantly decreased with aging in klotho mice. Notably, these phenotypes were rescued by vitamin D restriction, suggesting that abnormal activation of vitamin D due to Klotho insufficiency leads to degeneration of the dopaminergic system. The present study provides new insights into the pathology of age-related degeneration of dopaminergic neurons possibly related to Klotho-mediated regulation of vitamin D.

The total synthesis of a 10-membered F-ring analogue of 51-hydroxyCTX3C is described which constitutes the first example of an F-ring modified ciguatoxin that exhibits potent cytotoxicity as well as mouse acute toxicity.

We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 20 mM Tris-HCl buffer (pH 9.0) for 1 h at 95 degrees C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO(4) solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures.

Ciguatoxins, principal causative toxins of ciguatera seafood poisoning, are large ladder-like polycyclic ethers that exert their toxicities by binding to the voltage-sensitive sodium channels. We r...

We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 20 mM Tris-HCl buffer (pH 9.0) for 1 h at 95 degrees C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO(4) solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.

We examined the suitability of the freeze-substitution and paraffin embedding technique for the im-munolocalization of membrane-bound and soluble antigens in the rat at the light microscopic level. Frozen tissues were substituted at -80°C in methanol or acetone containg chemical fixatives, i. e., formaldehyde, glutaraldehyde, acrolein, hexamethylene diisocyanate (HMD), diethyl malonimide (DEM) and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (water soluble carbodiimide, WSC). The following antigens were localized using monoclonal antibodies and antisera: A) membrane-bound antigens; γ-glutamyl transpeptidase (γ-GTP), common antigen of secretory granule membrane (SG 170 antigen) and Golgi associated antigen (GAA 108); and B) soluble antigens; branched-chain amino acid transferase type I isozyme (BAT), glutamate dehydrogenase (GDH), pancreatic amylase, proliferating cell nuclear antigen (PCNA), rat serum albumin and IgG.1) The freeze-substitution technique maintained an excellent tissue structure and conservation of antigenicity. By using this method, BAT was localized in mitochondria in iver cells, and γ-GTP was demonstrated in the secretory granule membrane of pancreatic acinar cells, although conventional fixation methods provided negative reaction. 2) In general, the membrane-bound antigens were localized in detail with a strong immunoreaction in the tissues substituted in solvent alone; however, for the localization of soluble antigens, tissues substituted in solvents containing chemical fixatives revealed a strong and precise antigen localization. 3) Formaldehyde and glutaraldehyde proved to be better fixatives concerning the conservation of structure and antigenicity than the other chemical reagents. 4) The choice of substitution solvent was important for the immunohistochemistry of some antigens. For example, methanol was suitable for PCNA, and substitution in acetone was essential for γ-GTP using one of the monoclonal antibodies to γ-GTP.

To assess the effect of carteolol, a beta-blocker, on ischemia and reperfusion, changes in the ultrastructure of myocytes and energy metabolism were studied by 31P-NMR in 41 pig hearts without collateral circulation. The left anterior descending coronary artery was occluded for 20 min and reperfused for 120 min in three groups: seven pigs (group 1, no treatment with carteolol; group 2, pre-ischemia treatment with carteolol (10 micrograms/kg); group 3, post-ischemia treatment with carteolol before reperfusion). Other groups of five pigs were killed after 120 min of ischemia (group 4, no treatment; group 5, pre-ischemia treatment) or 20 min of ischemia (group 6, no treatment; group 7, pre-ischemia treatment). After 20 min of ischemia, ATP was higher in groups 2 (76 +/- 9% of the baseline value) than in group 1 (59 +/- 5%) and group 3 (60 +/- 10%). However, the difference disappeared after 30 min of ischemia. After 120 min of reperfusion, ATP showed much better recovery in group 2 (92 +/- 9%) than in groups 1 (66 +/- 7%) and 3 (68 +/- 10%). Ischemic injury, as viewed by light and electron microscopy, was milder in group 7 than in group 6 after 20 min occlusion, but the myocytes were almost normal after 120 min reperfusion in groups 1 to 3. The heart rate, blood pressure and rate pressure product showed no significant difference among the groups. These results indicate that pre-ischemia treatment with carteolol provided protection against ischemic cellular injury and accelerated the repletion of ATP during reperfusion, but the post-ischemia treatment did not lead to recovery of ATP. Therefore, the favorable effect during reperfusion of pre-ischemia treatment with carteolol depends on its protective effect during ischemia.

Microwave (MW) has the advantage of accelerating infiltration of fixatives into tissue blocks, but the disadvantage of causing the temperature of the fixatives and tissue blocks to rise. We tried to minimize the temperature elevation during MW irradiation by applying ultrasound (US) in combination with MW oven furnishing US generator. US was uneffective in reducing the rise of temperature, however, it was able to enhance the rapid tissue fixation. Electron microscopic examination showed that the fine structure of organs, such as liver, pancreas, submandibular gland, small intestine, and kidney, was well preserved. Characteristically, the matrix of mitochondria appeared electron-dense, and the marginal zone of zymogen granules in the pancreas was more electron-opaque than the core of the granule. The immunoreactivity of zymogen granules to anti-amylase antibody was strong. The deformation or disorder of cell structure due to the thermal effect and cavitation that can be caused by US was not seen. US can be useful for histologic and immunohistochemical study.

Monoclonal antibodies were prepared to branched-chain amino acid transferase (BAT) type I isozyme purified from rat kidney. Two stable clones producing specific antibodies to the enzyme were obtained. Specificity was confirmed by immunoblots of purified BAT after polyacrylamide gel electrophoresis and testing enzyme inhibition by the antibodies. In the immunohistochemical study, type I BAT was localized in the mitochondria of the proximal tubular cells of the rat kidney. In the liver, reaction was observed in the mitochondria of parenchymal cells. The cells surrounding the central veins exhibited strong immunoreaction. Immunoelectron microscopy using a rapid freezing and substitution technique revealed type I BAT localization in the mitochondria matrices of rat liver parenchymal cell. A faint reaction was present in the cytoplasm. Immunoblot analysis of mitochondrial and post-microsomal fractions from rat liver and kidney also indicated that the enzyme is chiefly present in the mitochondria.The monoclonal antibodies we prepared may provide a useful tool for studying changes in both isozyme pattern and localization of type I isozyme in the prenatal and neonatal rat and in tumor cells.

We have developed a new standardized method for the post-embedding immunoelectron microscopy using the same fixation, antigen retrieval and image contrasting procedures. Tissues were fixed with 4% formaldehyde containing 2.5 mM CaCl(2), 1.25 mM MgCl(2) in a 0.1 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) for 2 h and then with the same fixative composition in 0.1 M HEPES buffer (pH 8.5) overnight at room temperature. Vehicle osmolarity of fixatives was adjusted to 300-330 mOsm by adding glucose. The specimens were dehydrated with dimethylformamide on ice and embedded in LR-White resin. Ultrathin sections were heated in a 20 mM Tris-HCl buffer (pH 9.0) for 1-2 h at 95 degrees C. After immuno-gold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in a 0.1 M phosphate buffer (pH 5.5) for 5 min and with a 1% OsO(4)/0.1 M phosphate buffer (pH 7.4) for 5 min, and then they were double stained with uranyl acetate and lead citrate. The standardized method yielded strong and reproducible immunoreactions for soluble, membrane-bound and filamentous proteins showing an excellent image contrast without destruction of the fine structures.

We treated 2 different types of intersexual patients who underwent a vaginoplasty with the pudendal-thigh flap. One was a female with testicular feminization syndrome for whom we reconstructed the total vagina with a pudendal-thigh flap, and the other was a female with an adrenogenital syndrome for whom we enlarged the introitus of the vagina with the same approach. There were no complications such as a flap necrosis. In addition, there was no stricture of the neo-vagina and no urinary problem.

An efficient synthesis of the pentacyclic framework of cortistatins has been developed. The key strategy comprises assembly of the A- and the CD-ring fragments by Knoevenagel reaction, facile formation of the pyran ring via electrocyclization, and construction of the seven-membered B-ring by radical addition to an alpha,beta-unsaturated ketone.

We have examined the relationship between localization of estrogen receptor (ER) and selected cell responses induced by estrogen in the neonatal CD-1 mouse uterus. The following simultaneous staining techniques were used to determine whether only uterine epithelial cells with ER are capable of showing proliferative and secretory activities after estrogen treatment: 1) ER localization and [3H]thymidine incorporation using immunohistochemistry and autoradiography; and 2) immunohistochemical double staining of ER and an estrogen-induced secretory protein lactoferrin (LF). Uterine tissues from two strains (CD-1 and BALB/c) mice on day 4 of age showed detectable ER by immunostaining in both epithelial and stromal cells. Day 4 CD-1 mice received a single injection of diethylstilbestrol (DES; 20 micrograms/kg BW). The percentage of ER-immunostained uterine epithelial cells and the intensity of the staining rapidly increased from 6-36 h, indicating that estrogen stimulates the expression and detectability of ER during the course of hormone treatment. Twenty-eight and 81% of epithelial cells showed positive ER immunostaining 6 and 24 h, respectively, after the DES treatment. The intensity of ER-immunostained stromal cells, however, was slightly decreased by the treatment. DES administration elicited epithelial cell proliferation. Labeling indices of epithelial cells reached a maximum (approximately 44%) 12 h after an injection of DES, and a second small peak was recognized 24 h after the treatment. Labeling indices of positively ER-immunostained epithelial cells and those of negatively stained epithelial cells showed almost the same pattern for 6-18 h after treatment. The respective maximum values were 45% and 43%. When day 4 mice were given three daily injections of DES in saline and killed 12 and 24 h after the last injection, significant amounts of LF were recognized in the apical cytoplasm of the epithelial cells. The epithelial cells that showed LF immunostaining always exhibited ER immunostaining in the nuclei. These results suggest that exogenous estrogen elicits increased expression of ER, DNA synthesis, and cell proliferation in neonatal uterine epithelial cells associated with low levels of ER, while there was a direct relationship with the presence of ER in epithelial cells and the induction of LF.

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.

The temperature during microwave irradiation in the core of tissue blocks, either 1cm or 2cm cubes of rat kidney and liver, was measured by plunging a sensor into the tissue blocks. The temperature of the tissue blocks was much higher than the outer media, i.e., chemical fixatives. Thus, the temperature of the outer media did not reflect that in the tissue block to be fixed. The temperature of tissue reached 40°C within 5-10sec of irradiation. The highest temperature of tissuee block at 15-second irradiation, for example, was 92.5°C (1cm cube of kidney) and 98°C (2cm cube of liver), using 4% PFA with CaCl2 as a fixative. Microwave fixation is a practical tool for the preparation of tissue spections for either histology or cytology. However, it must be required that the irradiation is controlled so as not to produce high temperature within specimens.

The recent studies of the earth's surface conditions relating to heat islands in Japan are presented from geographical viewpoints as an activity of the Association of Japanese Geographers. Urban climate is defined as the result of urbanization, which is expressed as concentration of population, modification of earth's surface constituent materials, and expansion of living space onto and under the ground. These bring about changes in morphological and physical features of the urban surface, and its energetic conditions, which consequently and unintentionally modify radiation, heat and water balance in urban areas. Some estimations of urban surface conditions are at first explained. Surface conditions treated in this study are morphology of urban surface (mainly building height), roughness, sky view and energy consumption. Next, the relationships were considered between air temperature and surface morphology, heat island intensity and population, population and sky view factor, respectively. Finally the regional differences in heat islands were discussed, and the necessity of urban climate studies to ameliorate human and global conditions.

Gamma-glutamyl transpeptidase (γ-GTP) was localized in prenatal and neonatal rat kidneys using an immunohistochemical method with monoclonal antibodies to rat kidney γ-GTP. γ-GTP was demonstrated on the brush border and basolateral membrane of proximal convoluted tubules from the 18th fetal day. The basolateral membrane of proximal tubules revealed strong reaction at 1 and 3 days after birth. The reaction of basolateral membrane decreased with the maturation of nephrons, however, moderate reaction was still observed at 30 days of age in the S3 segment. Throughout the postnatal development of the kidney, the S3 exhibited the strongest immunostainingand S1 showed the weakest reaction, and proximal tubules in the deep layer of the cortex always provided a stronger reaction than those in the superficial layer of the cortex. Maturating cuboidal and squamous epithelial cells of descending Henle's loop showed a positive reaction on the luminal and lateral cell membranes. On immunoelectron microscopy, γ-GTP was found on the brush border, basolateral membrane, membranes of pinocytotic vesicles and apical invaginations in proximal tubular cells at 1-7 days after birth. However, tight junctions were devoid of the reaction. The development of basal infolding and localization of γ-GTP there seemed to relate closely to the development of surrounding capillaries. γ-GTP was localized on the cell membrane and occasionally in the cytoplasm of interstitial cells of renal interstitium.

Ciguatoxins, the causative toxins of ciguatera seafood poisoning, are large ladder-like polycyclic ethers, and known to have more than 20 structurally distinct congeners. In this paper, we describe a new concise synthesis of the two E-ring structures, of different ring sizes, found in ciguatoxins. From the common oxepane intermediate, the seven-membered E-ring was prepared in three steps, and the eight-membered E-ring was synthesized in seven steps using ring expansion and olefin isomerization as key reactions.

A new convergent strategy for assembling 6/6- and 6/7-fused ether ring systems was developed. The key features in our method include Ag+-promoted facile formation of chemically labile enol ether from O,S-acetal and addition of an acyl radical to unactivated enol ether to cyclize a six- or seven-membered ether ring. [reaction: see text]

Ciguatoxins, principal causative toxins of ciguatera seafood poisoning, are large ladderlike polycyclic ethers. Here, we report two convergent routes to synthesis of the multiolefinic left half of ciguatoxins based on a newly developed acyl radical strategy. Remarkably, only 13 steps from the monocyclic E-ring were required to construct the left wing. [reaction: see text]

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.