Search

Background Lymphatic venous anastomosis (LVA) is a widely accepted surgical procedure for lymphedema. To obtain the best outcomes, surgeons should be well trained. A recent study introduced an LVA training model using pig trotters for their utility and structural similarity to human tissues. However, details regarding the utilization of anastomosis models, such as feasible points for training based on vessel anatomy, have not been clarified. Therefore, we assessed the anatomical details of lymphatic vessels and veins of trotters to establish a practical training model of LVA. Methods Ten frozen trotters were used. After thawing at room temperature, indocyanine green fluorescent lymphography was used to visualize the lymphatic course. To dissect the lymphatic vessels and veins from the distal to the proximal end, whole skins were detached thoroughly from the plantar side. Data from the lymphatic vessels and veins were collected based on their courses, diameters, and layouts to clarify adjacent points feasible for LVA training. Results Both lymphatic vessels and veins were classified into four major courses: dorsal, medial, lateral, and plantar. The majority were dorsal vessels, both lymphatic vessels and veins. The adjacent points were always found in the distal dorsum center and were especially concentrated between the metacarpophalangeal (MP) joint and central interphalangeal crease, followed by the medial and lateral sides. Conclusion The most relevant point for LVA surgical training in the trotter was the dorsal center distal to the MP joint, where parallel vessels of similar sizes were found in all cases. This practical LVA surgical model would improve surgeon skills in not only anastomosis but also preoperative fluorescent lymphography.

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin The research leading to these results has received funding from the following FEDER cofounded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD and ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, and Interreg AlertoxNet EAPA-317-2016. ABJ is recipient of a predoctoral fellowship from the Spanish Ministry of Education SI

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Aldehyde dehydrogenases (ALDHs) play a major role in detoxification of aldehydes. High expression of ALDHs is a marker for stem cells of many organs including the lungs. A common polymorphism in ALDH2 gene (ALDH2*2) results in inactivation of the enzyme and is associated with alcohol flushing syndrome and increased risk for cardiovascular and Alzheimer’s diseases and some cancers. The effect of this ALDH2 polymorphism on the lung and its stem cells has not been thoroughly examined. We examined the association between the ALDH2*2 allele and lung function parameters in a population of healthy individuals. We also examined its association with the incidence of asthma and COPD in patient cohorts. We used the in vitro colony forming assay to detect the effect of the polymorphism on lung epithelial stem cells from both primary human surgical samples and Aldh2*2 transgenic (Tg) and Aldh2 −/− mice. Response to acute and chronic lung injuries was compared between wild type (WT), Aldh2*2 Tg and Aldh2 −/− mice. In humans, the ALDH2*2 allele was associated with lower FEV1/FVC in the general population, but not with the development of asthma or COPD. Both the bronchial and lung epithelium carrying the ALDH2*2 allele showed a tendency for lower colony forming efficiency (CFE) compared to ALDH2 allele. In mice, the tracheal epithelial thickness, nuclear density, and number of basal stem cells were significantly lower in Aldh2 −/− and Aldh2*2 Tg adult mice than in WT. Electron microscopy showed significantly increased number of morphologically abnormal mitochondria in the trachea of Aldh2 −/− mice. Aldh2 −/− tracheal and lung cells showed higher ROS levels and fewer functional mitochondria than those from WT mice. No significant differences were detected when tracheal and lung epithelial stem cells were examined for their in vitro CFE. When exposed to chronic cigarette smoke, Aldh2*2 Tg mice were resistant to emphysema development, whereas influenza infection caused more epithelial damage in Aldh2 −/− mice than in WT mice. ALDH2 polymorphism has several subtle effects on the lungs, some of which are similar to changes observed during normal aging, suggesting a “premature lung aging” effect.

Ciguatoxins (CTXs) and maitotoxins (MTXs) are polyether ladder shaped toxins derived from the dinoflagellate Gambierdiscus toxicus. Despite the fact that MTXs are 3 times larger than CTXs, part of the structure of MTXs resembles that of CTXs. To date, the synthetic ciguatoxin, CTX 3C has been reported to activate voltage-gated sodium channels, whereas the main effect of MTX is inducing calcium influx into the cell leading to cell death. However, there is a lack of information regarding the effects of these toxins in a common cellular model. Here, in order to have an overview of the main effects of these toxins in mice cortical neurons, we examined the effects of MTX and the synthetic ciguatoxin CTX 3C on the main voltage dependent ion channels in neurons, sodium, potassium, and calcium channels as well as on membrane potential, cytosolic calcium concentration ([Ca2+] c ), intracellular pH (pH i ), and neuronal viability. Regarding voltage-gated ion channels, neither CTX 3C nor MTX affected voltage-gated calcium or potassium channels, but while CTX 3C had a large effect on voltage-gated sodium channels (VGSC) by shifting the activation and inactivation curves to more hyperpolarized potentials and decreasing peak sodium channel amplitude, MTX, at 5 nM, had no effect on VGSC activation and inactivation but decreased peak sodium current amplitude. Other major differences between both toxins were the massive calcium influx and intracellular acidification produced by MTX but not by CTX 3C. Indeed, the novel finding that MTX produces acidosis supports a pathway recently described in which MTX produces calcium influx via the sodium-hydrogen exchanger (NHX). For the first time, we found that VGSC blockers partially blocked the MTX-induced calcium influx, intracellular acidification, and protected against the short-term MTX-induced cytotoxicity. The results presented here provide the first report that shows the comparative effects of two prototypical ciguatera toxins, CTX 3C and MTX, in a neuronal model. We hypothesize that the analogies and differences in the bioactivity of these two toxins, produced by the same microorganism, may be strongly linked to their chemical structure. © 2014 American Chemical Society.

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

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 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 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.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL.

Our aim was to test the influence of cold ischaemia on replanted limbs, focusing on muscular atrophy and neurological recovery. Inbred wild-type and green fluorescent protein (GFP) transgenic (Tg) Lewis rats aged 8-10 weeks were used. The amputated limbs were transplanted at several cold ischaemic times (0, 1, 8, 12, 24, 48, and 72 hours). An arterial ischaemic model and a denervation model were used as controls. To study nerve regeneration, a GFP limb was transplanted on to the syngenic wild Lewis rat. These animals were evaluated histologically, electrophysiologically, and immunohistochemically. The longer the ischaemic time, the more evident was atrophy of the muscles. Electrophysiological investigation showed that the latency at 3 weeks was longer in the transplantation models than in the normal controls, particularly in the longer ischaemia group. Larger numbers of migrating Schwann cells were seen in the group with no delay than in the group that had been preserved for 12 hours. Ischaemia after amputation of a limb causes muscle cells to necrose and atrophy, and these changes worsen in proportion to the ischaemic preservation time. A delay in nerve regeneration and incomplete paralysis caused by malregeneration also affect muscular atrophy.

This paper describes the details of our synthetic studies on the marine steroidal alkaloids cortistatins A and J. The key features of our strategy include (i) an efficient Knoevenagel/electrocyclic strategy to couple the diketone and the CD-ring fragment, (ii) a chemoselective radical cyclization to construct the oxabicyclo[3.2.1]octene B-ring system, (iii) a highly stereocontrolled installation of the isoquinoline unit, and (iv) a late-stage functionalization of the A-ring.

The purpose of this study was to determine the usefulness of a new flap model, the superficial inferior epigastric artery (SIEA) flap for supermicrosurgical training. Experimental groups were randomly divided into three groups of 10 rats each. In each group SIEA flaps were elevated and then returned to their original locations with or without vascular anastomosis of the superficial inferior epigastric vessels. Group 1: free SIEA flap, group 2: free SIEA flap with 1 hour ischemia time, group 3: free SIEA flap with 4 hours ischemia time, group 4: SIEA flap without vascular anastomosis. The viability rate was 80% with group 1, 50% with group 2, and 40% with group 3. All nonvascularized flaps (group 4) underwent complete necrosis. These findings suggest that preservation of blood flow in a flap has a beneficial effect on the prevention of microthrombosis in the subcutaneous capillary network of the skin and increases the flap survival rate. The SIEA flap with preserved circulation is an ideal model for developing supermicrosurgical skills.

Since the introduction of the fluorescence-labeled antibody method by Coons et al. [Immunological properties of antibody containing a fluorescent group. Proc Soc Exp Biol Med 47, 200-2002], many immunohistochemical methods have been refined to obtain high sensitivity with low background staining at both light and electron microscopic levels. Heat-induced antigen retrieval (HIAR) reported by Shi et al. in the early 1990s has greatly contributed to immunohistochemical analysis for formalin-fixed and paraffin-embedded (FFPE) materials, particularly in the field of pathology. Although antigen retrieval techniques including enzyme digestion, treatment with protein denaturants and heating have been considered tricky and mysterious techniques, the mechanisms of HIAR have been rapidly elucidated. Heating cleaves crosslinks (methylene bridges) and add methylol groups in formaldehyde-fixed proteins and nucleic acids and extends polypeptides to unmask epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In buffers having an appropriate pH and ion concentration, epitopes are exposed without entangling the extended polypeptides during cooling process, since polypeptides may strike a balance between hydrophobic attraction force and electrostatic repulsion force. Recent studies have demonstrated that HIAR is applicable for immunohistochemistry with various kinds of specimens, i.e., FFPE materials, frozen sections, plastic-embedded specimens, and physically fixed tissues at both the light- and electron-microscopic levels, and have suggested that the mechanism of HIAR is common to aldehyde-fixed and aldehyde-unfixed materials. Furthermore, heating has been shown to be effective for flow cytometry, nucleic acid histochemistry (fluorescein in situ hybridization (FISH), in situ hybridization (ISH), and terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL)), and extraction and analysis of macromolecules in both FFPE archive materials and specimens processed by other procedures. In this article, we review mechanism of HIAR and application of heating in both immunohistochemistry and other histochemical reactions.

Ciguatera is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs), sodium channel activator toxins. Symptoms of ciguatera include neurological alterations such as paraesthesiae, dysaesthesiae, depression, and heightened nociperception, among others. An important issue to understand these long-term neurological alterations is to establish the role that changes in activity produced by CTX 3C represent to neurons. Here, the effects of synthetic ciguatoxin CTX 3C on membrane potential, spontaneous spiking, and properties of synaptic transmission in cultured cortical neurons of 11-18 days in vitro (DIV) were evaluated using electrophysiological approaches. CTX 3C induced a large depolarization that decreased neuronal firing and caused a rapid inward tonic current that was primarily GABAergic. Moreover, the toxin enhanced the amplitude of miniature postsynaptic inhibitory currents (mIPSCs), whereas it decreased the amplitude of miniature postsynaptic excitatory currents (mEPSCs). The frequency of mIPSCs increased, whereas the frequency of mEPSCs remained unaltered. We describe, for the first time, that a rapid membrane depolarization caused by CTX 3C in cortical neurons activates mechanisms that tend to suppress electrical activity by shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition. Indeed, these results suggest that the acute effects of CTX on synaptic transmission could underlie some of the neurological symptoms caused by ciguatera in humans.

Neonatal diethylstilbestrol (DES) exposure elicits a wide range of abnormalities in the female mouse genital tract. This animal model system is suitable for investigating the mechanism of DES syndrome in humans. Accumulated evidence has shown that critical periods in development are present for distinct and permanent alterations in the female genital tract of mice exposed to DES neonatally (DES-mice). These effects of DES and other estrogens are mainly mediated by estrogen receptor alpha (ER alpha) through multiple pathways. Induction of ER alpha by DES exposure in neonatal stromal and epithelial cells, and successive premature activation of estrogen-regulated genes are thought to be essential to induce the abnormalities. Induction of malformation, permanent changes in estrogen-regulated genes, such as protooncogenes and growth factors, and carcinogenesis are assumed to be interdependent. This review focuses the following topics to discuss the molecular basis of DES-induced abnormalities mainly based on the results by histochemical techniques in the uterus: spatiotemporal expression of ER alpha and coactivators, proteins relating morphogenesis, and estrogen-regulated protooncogenes and growth factors.

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

Ontogenic expression of p300, steroid receptor coactivator-1 (SRC-1), GR-interacting protein-1 (GRIP1) and p300/CBP cointegrator associate protein (p/CIP) was examined using an immunohistochemical method in the genital tract of female mice neonatally exposed to diethylstilbestrol (DES). Mice were injected with 4 μg of DES for 5 days and killed on days 10, 15 and 21; some mice were killed on day 4 after three injections. The p300 immunoreactive protein in the epithelial cells of the oviducts, uteri, and vaginae was almost constant during development independent of neonatal DES exposure, while the stromal cells exhibited a weaker reaction than that of the vehicle-treated controls on days 4 and 10. Neonatal DES exposure caused no significant changes in the SRC-1 and p/CIP expression patterns but decreased the GRIP1 expression with development in the reproductive tract. The constitutive expression of coactivators in both epithelial and stromal cells may play a role in the estrogen imprints through ERα systems during the period of DES treatment.

Using immunohistochemical methods, we studied the cell-type- and species-specific expressions of estrogen receptor (ER) isoforms (ER alpha and ER beta) and androgen receptors (ARs) in the male reproductive tract and accessory sex glands of mature mice and rats. ER alpha and ER beta showed cell-type- and species-specific distributions, respectively. In contrast, AR was localized in the epithelial and stroma cells of all tissues examined in this study, in both species. In mice, the epithelial cells of the ductuli efferentes showed a strong ER alpha-immunoreaction, and those of the caput epididymis, coagulating glands, and prostate also exhibited a positive reaction. Stroma cells, except in the ductuli efferentes, showed a positive ER alpha-immunostaining. In rats, ER alpha was detected in very few cell types: the epithelial cells of the ductuli efferentes showed a strong reaction, and the stroma cells of the ampullary and urethral glands exhibited a weak reaction. ER beta was localized in the epithelial cells of the prostate in mice, while the reaction was faint or negative in both the epithelial and stroma cells of other tissues. In rats, the ER beta-immunoreaction was strongest in the epithelial cells of the ventral prostate. The epithelial cells of the corpus and cauda epididymis, ductus deferens, and urethral glands, and the stroma cells of the urethral glands were also positively ER beta-immunostained. Almost the same AR distribution pattern was observed in both species. In particular, strong AR-immunostaining was present in the epithelial cells of the caput and corpus epididymis, seminal vesicle, and ventral prostate. These results indicate that species and tissues differences should be taken into careful consideration in assessing the physiological and pharmacological effects of sex steroids (particularly estrogens) on the reproductive tissues of male rodents.

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.

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.

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.

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.

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 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.

Expression of a previously cloned secretory protein named adrenocortical zonation factor 1 (AZ-1, also called Tin-ag-RP or lipocalin 7) is tightly linked with the zonal differentiation of adrenocortical cells. It is also present in vascular smooth muscle (VSM), although its function has remained unknown. In this study, the location of AZ-1 was specified to the basal laminae along adrenocortical sinusoidal capillaries and surrounding VSM cells in the arterial system, consistent with the fact that AZ-1 was extractable under denaturing conditions as a 52 kDa polypeptide. Purified recombinant AZ-1 exhibited abilities to bind to fibronectins via the first type III repeat (anastellin) and to collagens with affinities in submicromolar ranges. AZ-1 immobilized on substratum or bound to collagens or anastellin promoted adhesion and spreading of adrenocortical cells. Although VSM cells spread on AZ-1 slowly, AZ-1 bound to anastellin facilitated the spreading. The adhesion activity of AZ-1 was mediated by a subset of integrins, including alpha(1)beta(1), alpha(2)beta(1), and alpha(5)beta(1), in a cell type-specific manner. Collectively with the putative role of AZ-1 in the adrenocortical zonation, we propose that AZ-1 potentially regulates functions of adrenocortical and VSM cells by modulating cell-matrix interactions.

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.

In this study, we examined the mechanism of heat-induced antigen retrieval using analytical procedures involving SDS-PAGE, Western blotting, and immunohistochemistry. Five proteins were treated with 4% formaldehyde in the presence or absence of 25 mM CaCl2, then heated under various conditions after removal of formaldehyde and analyzed on SDS-PAGE. Formaldehyde produced inter- and intramolecular cross-links in the proteins. Heating at high temperatures cleaved these cross-links at all pH ranges examined (pH 3.0, 6.0, 7.5, 9.0) and produced almost the same electrophoregrams as the native proteins. Proteins treated with formaldehyde containing CaCl2 showed similar electrophoretic patterns, observed without heating or after heating at pH 6.0 and pH 9.0 in the presence or absence of 10 mM EDTA. Western blot analyses demonstrated that the soluble forms of β-actin (monomer and oligomers) and fibronectin were present in extracts from deparaf-finized mouse uterine sections autoclaved for 15 min but not in extracts from unheated specimens. Nine of ten antigens, independent of their isoelectric points, exhibited much stronger immunoreaction in the sections heated at pH 9.0 than in those heated at pH 6.0. The second heating at pH 6.0 significantly decreased the immunostaining of the antigens that had been boiled at pH 9.0, but the immunostaining was recovered after a third heating at pH 9.0. These results suggest that the main mechanism of heat-induced antigen retrieval is disruption of the cross-links and that pH is an essential factor for a proper refolding of epitopes.

Receptors of steroid hormones, thyroid hormones and several kinds of vitamins have been shown to act as nuclear transcription factors and to form a nuclear receptor (NR) family. Histochemical techniques including autoradiography using radio-labeled ligands, immunohistochemistry and in situ hybridization histochemistry, have displayed that target cells of these receptors are distributed not only in the classical target organs but also widely in a variety of tissues; these techniques can demonstrate the presence of receptor proteins and mRNAs, even though they are expressed in a small cell population of tissues. On the other hand, many studies have been performed to demonstrate the interaction between NRs and nuclear and cytoplasmic proteins, and to clarify the mechanism of transcriptional regulation through NRs in artificial conditions which are created in gene transfer experiments or under cell-free conditions. Some data coincide with those obtained from histochemical techniques, however, some histochemical data do not support the results of studies in vitro. This review focuses on the following topics: histochemical methodologies to detect NRs, the distribution and function of NRs in the tissues, the intracellular and intranuclear localization of NRs, roles of gonadal steroid receptors and their ligands on developing tissues including cell communications such as mesenchymal-stromal interaction, and the interaction between other cellular components and NRs. In addition, the agreement and disagreement between the results of histochemical studies and those from the experiments in the model systems or in vitro are discussed.

Preventive effects of dehydroepiandrosteone acetate (DHEA-A) and clofibrate (positive control substance) on the fatty liver induced by orotic acid (OA) were examined on the male Sprague-Dawley rats fed a high sucrose based diet containing 1% OA and this diet further mixed with 0.5% DHEA-A or 0.5% clofibrate for 2 weeks. Numerous lipid droplets were observed in the hepatocytes of the rats treated with OA alone, but not in those treated with DHEA-A or clofibrate. In comparison to the group with OA alone, the DHEA-A or clofibrate treated rats showed a larger relative liver weight (to body weight) which was accompanied by increased peroxisomes in the hepatocytes. These results indicate that DHEA-A, as well as clofibrate, may prevent OA-induced fatty liver.

Dehydroepiandrosterone (DHEA) is a steroid hormone which induces the peroxisome proliferation in rodents. The fatty liver induced by orotic acid and a high sucrose diet in male rats was prevented by the administration of DHEA and/or phenobarbital (PB). A significant increase in the liver weight was induced in the DHEA group (relative weight) and the DHEA + PB group (absolute and relative weight). The liver weight increased more conspicuously in the DHEA + PB group than in the DHEA group. The increase in the liver weight was caused by an increase in the cell size and peroxisome number. In addition, the administration of DHEA alone and the combination of DHEA and PB prevented the lipid droplet accumulation in hepatocytes. The administration of PB alone also prevented the accumulation of lipid droplets without any increase in the liver weight.

Employing immunoelectron microscopy with ultrathin frozen sections, intranuclear localization of hormone-occupied and -unoccupied estrogen receptors (ER) was examined in the uterine epithelium; after 1 h of 17 beta-estradiol (E2, 20 micrograms/kg b.w.) or saline intraperitoneal injection, adult ovariectomized mice were sacrificed. ER were localized with both anti-ER monoclonal antibody and polyclonal antibody (AS 409) and 1 nm immunogold followed by silver enhancement. No significant differences in ultrastructure were recognized in either the nuclei or the cytoplasm of the uterine epithelium in E2-treated and control animals. E2-occupied and -unoccupied ER showed the same distribution pattern in the nuclei of both luminal and glandular epithelium. ER were exclusively localized in the dispersed and slightly condensed chromatin; the margins of the condensed chromatin also showed strong immunoreaction. Little immunolabeling was observed in the highly condensed chromatin associated with nuclear membranes. The reaction was negative in the nucleoli. Both antibodies provided the same ER intranuclear localization. It is suggested that marked change in the distribution of steroid receptors, i.e. rapid translocation from condensed chromatin to dispersed chromatin or the margins of condensed chromatin as described in some previous reports, probably takes place as a consequence of conformational changes in the chromatin-binding receptors.

The effects of zinc hydroxide on the respiratory burst and phagocytosis by rat neutrophils were examined. Zinc hydroxide induced an increase in oxygen consumption and O2- production. Electronmicroscopy showed that neutrophils engulfed zinc hydroxide particles by phagocytosis. Pertussis toxin (0.25, 0.5, 1.0 micrograms/ml) and EGTA (1, 2, 5 mM) inhibited zinc hydroxide-induced O2- production in a dose-dependent manner. The inhibitors of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide inhibited zinc hydroxide-induced O2- production with IC50 values ranging between 10 microM and 25 microM. The inhibitory study using an inhibitor of myosin light chain kinase, 1-(5-iodo-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine, showed IC50 values ranging from 5 microM to 10 microM. These findings indicate that zinc hydroxide induces respiratory burst and phagocytosis by rat neutrophils.

We demonstrated that a common antigen (Golgi-associated antigen, GAA 108) is present in the Golgi apparatus of serous exocrine cells, using an immunohistochemical method with monoclonal antibodies (MAb) 108 (IgG1) and 18 (IgM), raised to the microsomal fractions of rat parotid gland. The MAb reacted with polypeptides of molecular weights in the 58-170 KD range in parotid gland on Western blot analysis. The Golgi apparatus of the following cells was immunostained with these MAb: acinar cells of parotid gland, pancreas, and exorbital lacrimal gland, serous cells of sublingual gland, chief cells of stomach, and epithelial cells of rat prostate. However, positive reaction occurred throughout the entire cytoplasm of submandibular gland acinar cells. Immunoelectron microscopy (IM) revealed antigen (GAA 108) localization in the medial and trans-Golgi cisternae and trans-Golgi network (TGN), including condensing vacuoles, in parotid, exorbital lacrimal, and pancreatic acinar cells, and serous acinar cells of sublingual gland. Lysosomes and apical cell membranes also stained positively in some cells. In the submandibular gland reactions were observed in the medial and trans-Golgi cisternae, condensing vacuoles, secretory granule contents, cell membrane, and in some duct lumens. These results suggest that although GAA 108 is found in the Golgi apparatus of most serous exocrine cells, it is secreted by a regulated pathway in the acinar cells of submandibular gland.

Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are potent neurotoxic polycyclic ethers. We report herein the total synthesis of a 10-membered F-ring analogue of 51-hydroxyCTX3C, which constitutes the first example of an F-ring modified ciguatoxin that exhibits potent cytotoxicity as well as mouse acute toxicity.

The distribution of alcohol dehydrogenase in the hepatic acinus was examined by both histochemical and immunohistochemical approaches. The immunohistochemical method using anti–alcohol dehydrogenase antibody indicated zone 3 predominance of this enzyme in the hepatic acinus, whereas a conventional histochemical method showed slight zone 1 predominance. However, when the histochemical technique was improved by using 2% glutaraldehyde instead of formalin for fixation and by adding phenazine methosulfate (0.33 mmol/L) to the staining incubation mixture, this method also supported zone 3 predominance of alcohol dehydrogenase. Evidence for zone 3 distribution of alcohol dehydrogenase may be of value in elucidating the mechanism of zone 3 liver damage by alcohol. (HEPATOLOGY 1990;12:66–69).

Summary The antihypertensive action of OPC-13340, a new dihydropyridine, was studied in rats and compared with the action of nicardipine and other dihydropyridines. OPC-13340 showed more potent and longer hypotensive action than nicardipine when administered either intraveneously (i.v.) or orally in normotensive and hypertensive rats. Among 6 compounds tested, (OPC-13340, nifedipine, nitrendipine, nisoldipine, nicardipine and diltiazem), OPC-13340 was the most potent and long-acting when administered orally to spontaneously hypertensive rats (SHR). Tachycardia after administration of OPC-13340 was less or diminished earlier than that of nicardipine. Oral administration of OPC-13340 (3 mg/kg) once daily for 13 days did not cause any rebound phenomena in SHR. The compound inhibited Ca- or K-induced contractions in isolated rat aorta and shortened action potential duration in guinea pig papillary muscle, suggesting Ca channel blocking action. OPC-13340 might be useful as a drug for once-daily therapy of essential hypertension.

A series of 1-heteroaroylpiperazines (V-VII, XI) was synthesized and examined for positive inotropic activities on the canine heart. The key intermediates, heteroarenecarboxylic acids (III, X), were prepared by two different methods, and were condensed with substituted piperazines to give 1-heteroaroylpiperazines V-VII and XI. Among them, quinazoline derivatives (XI) were found to have potent positive inotropic activities.

In the isolated rabbit papillary muscle, adenosine (1-300 microM) alone scarcely affected the basal tension developed. The positive inotropic action of isoprenaline, mediated via beta-adrenoceptors, was inhibited by adenosine in a concentration-dependent manner. Atropine (0.3 microM) abolish the inhibitory action of carbachol on the isoprenaline-induced positive inotropic action but not affect the inhibitory asction of adenosine. Adenosine failed to inhibit the positive inotropic action exerted by phenylephrine via stimulation of alpha-adrenoceptors in the presence of pindolol (30 nM). The present results indicate that the positive inotropic action was mediated via alpha-adrenoceptors whose subecllular mechanism was not susceptible to the inhibitory action of adenosine as are beta-adrenoceptors.

L'amylase, la DNase et l'albumine sont localises a la fois dans la glande parotide et le foie du rat par utilisation d'anticorps monospecifiques purifies

We studied Japanese monkeys, an anubis baboon, and dogs to determine possible differences in the responsiveness of the simian and the canine coronary vasculature to autonomic drugs. Under ketamine and pentobarbital anesthesia the left anterior descending coronary artery was perfused with autologous blood at constant pressure. All drugs were injected intraarterially. In monkeys, acetylcholine and methacholine produced vasoconstriction followed by vasodilatation, and vasoconstriction was marked after large doses. Both responses were antagonized by atropine. In dogs, the two muscarinic receptor agonists produced only vasodilatation. In the baboon, acetylcholine injected into the femoral artery produced only vasodilatation. These results indicate that vasoconstriction mediated by muscarinic receptors is characteristic of the simian coronary vasculature. In monkeys, norepinephrine produced vasoconstriction followed by vasodilatation. The former response was abolished and the latter enhanced by phentolamine. In dogs, norepinephrine produced only vasodilatation. This response was reduced and preceded by vasoconstriction after atenolol. Methoxamine produced only vasoconstriction, which was weaker in dogs than in monkeys. These results indicate that alpha-adrenoceptors in the coronary vasculature are more important in subhuman primates than in dogs.

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.

The tissue distribution and levels of estrogen receptor in neonatal mouse uterine tissue were determined in epithelial and stromal fractions separated by mild enzymatic treatment. Proteins of the isolated fractions were separated by gel electrophoresis and receptor was detected on immunoblots with monoclonal antibody H-222. Estrogen receptor protein was detectable in samples of reproductive tract tissue from 5- and 10-day-old mice. The level of receptor in 5-day-old animals was lower per unit DNA in epithelial cells than in stroma. Receptor levels were increased in both tissue types after treatment with diethylstilbestrol, but not with progesterone. Receptor protein present in these neonatal tissues was able to bind steroid as evidenced by affinity labeling with tamoxifen aziridine. Immunohistochemistry on sections of uteri from 4- and 10-day-old mice confirmed the biochemical results and indicated lower nuclear straining in epithelial cells than in stromal cells of uteri of 4-day-old mice. These results demonstrated that estrogen receptor protein is present in both epithelium and stroma of the neonatal mouse uterus, but at a higher level in stromal cells.

The distribution of the estrogen receptor (ER) was investigated in neonatal female genital tracts (uterus, oviduct, cervix, and vagina) from days 1-22 after birth, using immunohistochemistry employing an anti-ER monoclonal antibody. In uteri, the ER in epithelial cells began to be observed by day 4. The number of positive epithelial cells and the staining intensity gradually increased until day 22 of age. On the other hand, uterine stroma cells gave a strong ER immunostaining even on day 1. The staining intensity reached a maximum by days 4-7 and then slightly decreased with age. In the oviduct, cervix, and vagina, epithelial cells showed positive ER immunostaining on day 1, and the intensity increased gradually until day 22. ER immunostaining in stroma cells was almost constant during the development period. The ER in both epithelial and stroma cells from these younger animals showed similar biochemical properties, i.e. an increased affinity for nuclei and resistance to extraction with PBS. Thus, during neonatal development of the female reproductive tract, ER is present not only in stroma cells but also in epithelial cells. This ER protein exhibits properties and characteristics similar to those of adult mice. The presence of ER suggests that some of the estrogen actions of cell proliferation, differentiation, and tissue abnormalities resulting from prenatal and postnatal estrogen administration may be mediated by receptor interactions.

Many 5- (3-amino-2-hydroxypropoxy) -3, 4-dihydro-8-hydroxy-2 (1H) -quinolinone derivatives were synthesized and examined for positive inotropic activity on the canine heart. These compounds were prepared by the reaction of 8-alkoxy-5- (2, 3-epoxypropoxy) -2 (1H) -quinolinone derivatives and various amines. Among them, 3, 4-dihydro-8-hydroxy-5-[3- (1, 1, 3, 3-tetramethylbutylamino) propoxy] -2 (1H) -quinolinone hydrochloride (IIIf) was found to have potent positive inotropic activity with a relatively minor increase in heart rate.

Many (1-piperazinylcarbonyl)-2(1H)-quinolinone derivatives were synthesized and examined for positive inotropic activity on the canine heart. Among them, 3, 4-dihydro-6-[4-(3-oxo-3-phenylpropyl)-1-piperazinylcarbonyl]-2(1H)-quinolinone (XXIj) and 3, 4-dihydro-6-[4-(oxo-4-phenylbutyl)-1-piperazinylcarbonyl]-2(1H)-quinolinone (XXIl) were found to have potent activity.