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Ring‐shaped silica nanoparticles are synthesized with a high tetrakis(4‐carboxyphenyl)porphyrin (TCPP) content or silica/TCPP hybrid nanorings (HNRs) using a one‐pot sol‐gel reaction with a TCPP‐binding silica precursor for fluorescence imaging of tumor. The shape of the HNRs is a reflection of abundant ring‐shaped TCPP aggregates in the silica matrix. The HNRs are of a size that makes them susceptible to the enhanced permeability and retention effect. For comparison, the TCPP‐doped silica nanoparticles are synthesized using a conventional method. The nanoparticles are spherical in shape because little TCPP is contained in the silica matrix and are designated as TCPP‐containing silica nanospheres (NSs). The absorption bands of the HNRs shift by about 20 nm toward longer wavelengths compared with the TCPP bands. This redshift leads the excitation wavelength of the HNRs into the near‐infrared (NIR) region. Therefore, the HNRs are excited by NIR light to emit strong fluorescence, although the NSs emit no fluorescence. The PEGylated HNRs (PEG‐HNRs) are uncharged and possess a significantly longer blood circulation time than PEG‐NSs. The PEG‐HNRs accumulate in tumor through multiple factors including their size, uncharged surface, unique shape, and long circulation time in blood, resulting in the acquisition of clear images of tumor.

Novel organosilica nanoparticles made of 3-mercaptopropyltrimethoxysilane as the single silica source (MPS NPs) have been synthesized successfully by using a Stöber method. The MPS NPs were well dispersed in solution and have unique surface properties such as thiol residues on the surface and reduced zeta potential compared with the nanoparticles made of tetraethoxysilane. The MPS NPs with fluorescent dye were synthesized by a method that deposited fluorescent dye on the silica network. The resulting MPS NPs with fluorescence were bright, nonaggregated in solution, and photostable. The fluorescence intensity and photostability of fluorescent MPS NPs were sufficient for detection as a single fluorescent particle using flow cytometry and fluorescence microscopy. Protein-modified MPS NPs were prepared easily by absorption and by maleimide coupling. In this paper, we demonstrate the usefulness of MPS NPs and their surface properties and discuss their advantages for biological applications.

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL6J and 129SV mice, Tcore was continuously recorded under 1212 h lightdark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity andor cortisol regulation.

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin–biotin–peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin–avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11β-hydroxylase (P45011β), 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase (3βHSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17α-Hydroxylase/C17-20 lyase (P45017α, lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011β, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011β are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3βHSD, P450C21, and P45017α, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes. Microsc. Res. Tech. 36:445–453, 1997. © 1997 Wiley-Liss, Inc.

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.

The fine structural development of the intercellular junctions in the interrenal tissue of chick embryos and hens was studied using freezefracture and thin section images.

We immunohistochemically studied the localization of 5a-reductase type 1 in combination with androgen receptor (AR) expression in individual lobes of the prostates of intact and castrated rats. In the normal rat prostate, 5a-reductase was localized in the cytoplasm of most epithelial cells in the ventral, dorsal, and lateral type 1 (L1) lobes. Epithelial cells of lateral type 2 (L2) lobes were negative for 5a-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5a-reductase-immunoreactive cells rapidly decreased in the ventral and L1 lobes after castration, whereas many positive cells remained in the dorsal lobe even at 4 weeks after castration. AR immunostaining was lost in the ventral, dorsal, and L1 lobes at 1 week after castration, but remained in the L2 lobe of 4-week-castrated rats. Electron microscopic immunocytochemistry showed that 5a-reductase was exclusively localized in the rough endoplasmic reticulum membranes and that there were no distinct structural differences between the positively and negatively stained epithelial cells. These findings suggested that the expression of 5a-reductase type 1 in the epithelial cell is heterogeneous within and among the individual lobes of the rat prostate, and does not correspond to AR expression.

Thymic stromal cell lines, termed MRL104 and MRL28, have been isolated from long‐term liquid cultures of thymic stromal cells from MRL/1 mice. The capacities of these parental lines and derived clones to express la antigens and to produce cytokines involved in T‐cell proliferation and/or differentiation were investigated. Parental lines and their clones did not exhibit a typical fibroblastic, macrophage‐like, or epithelial appearance in electron as well as phase‐contrast micrographs. These thymic stromal cells seemed to differ from established fibroblast lines in that these thymic stromal cells expressed la antigens after exposure to interferon‐γ (IFN‐γ), whereas fibroblast lines did not They also appeared to differ from macrophage cell lines in that they lacked the expression of Mac‐1 antigens on their cell surface and produced no detectable level of interteukin‐1 (IL1) before or even after exposure to lipopolysaccharide. When these parental lines and its clones were tested for their ability to produce various types of cytokines, it was revealed that they were capable of producing colony‐stimulating factor (CSF), IL6, and thymic stroma‐derived T cell growth factor (TSTGF). which was recently described, but were unable to generate other lymphokines and IFNs. Thus these cell lines and clones represent unique features in that they have potentials to express la antigens and to produce CSF, IL6, and TSTGF. The biological significance for the expression of these features is discussed in the context of intrathymic T‐cell maturation and T‐cell repertoire selection.

The present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P?450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E?13), and additionally in the bed nucleus of the stria terminalis on E?15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E?16, and some were detected in the arcuate nucleus on E?19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and thereafter declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P?14) and 30th day (P?30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.

Arachidonate 12-lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-lipoxygenase cDNA or a vector without 12-lipoxygenase cDNA, these structural changes were not observed.

Freeze-fracture images of exocytosis and endocytosis were studied in various kinds of secretory cells of the anterior pituitary of mice and rabbits. Exocytotic figures are frequently observed in thin section of the anterior pituitary cells. In freeze-fracture images, small elevated membrane areas without membrane particles are often seen on the PF of the plasma membrane of the secretory cells. There is a secretory granule in the cytoplasm just beneath the particle-free membrane area, and limiting membrane of the granule is also devoid of the membrane particles at the part facing the plasma membrane. The fusion of membranes for exocytosis may occur at this particle-free area.

Freeze-etching images of the capillary endothelium in the thyroid of normal, TSH-treated and Thyradin (powdered thyroid)-treated mice were examined. Numerous pores represent vesicular stomata or fenestrations. The number of the pores and their population density are increased in TSH-treated mice, and decreased in Thyradin-treated animals. In addition, the width of the parajunctional zone and of the flat ray free from endothelial pores is smaller in TSH-treated mice and larger in Thyradin-treated animals. These facts indicate that the number of endothelial pores changes according to the functional activity of the gland.

We showed immunohistochemically the localization of 5 α-reductase-containing cells in the rat brain, using a rabbit antibody generated against 5α-reductase rat type 1. The antibody was produced by injecting the synthetic peptide corresponding to the amino acids 38–53 of 5α-reductase rat type 1, conjugated to keyhole limpet hemocyanin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Western blot analysis revealed that this antiserum recognized the protein with a molecular weight of 29 000 Da. The inununoreactive cells were distributed throughout the brain and they were preferentially located in the white matter rather than in the grey matter. These cells were mostly small and round and had a few fine processes. The immunoreaction was confined to the cytoplasm and processes. These findings indicate that 5α-reductase rat type 1-containing cells are widely distributed in the rat brain and are located preferentially in the white matter rather than in the grey matter.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side‐chain cleavage enzyme (P450scc) and of 11β‐hydroxylase (P45011β), 3β‐hydroxysteroid dehydrogenase/Δ5‐4isomerase (3βHSD), and 21‐hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata‐reticularis. 17α‐Hydroxylase/C17‐20 lyase (P45017α, lyase) is present in the zona fasciculata‐reticularis cells of human, bovine, pig, and guinea‐pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011β, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata‐reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011β are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3βHSD, P450C21, and P45017α, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata‐reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes. Microsc. Res. Tech. 36:445–453, 1997.© 1997 Wiley‐Liss, Inc.

Thyroid glands of normal, TSH-treated and Thyradin (powdered thyroid)-treated mice were examined by means of the freeze-etching method. Intramembranous particles on the PF (= A face) face of the apical plasma membrane often form aggregates especially in TSH-treated mice. Each aggregate, about 200 nm in diameter, and consisting of 15–25 large particles, corresponds to a depression of the apical cytoplasm, and the particles sometimes form rosettes. Particle-aggregates are very rare in the apical plasma membrane of the thyroid follicular cell of the Thyradin-treated animal. In the cytoplasm just beneath the particle-aggregate no secretory granules, reabsorbed colloid droplets or other special structures are found.

Recently, nitric oxide (NO) has been believed to act as a neuronal messenger to mediate penile erection. In the present study using human penile tissue, we investigated the coexistence of neuronal NO synthase (NOS), tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) by a triple staining method using NADPH diaphorase (ND) staining, a specific histochemical marker of neuronal NOS, and immunohistochemical staining for TH and VIP. Numerous ND-positive nerve fibers and TH-containing fibers were seen in axon bundles, but their distributions were different. Only a few axons in the bundles showed VIP immunoreactivity. Abundant fine varicose nerve terminals innervating cavernous smooth muscles and deep and helicine arteries were observed. The proportion of fibers showing TH-immunoreactivity in ND-positive terminals in the cavernous space was about 25%, and that of VIP was about 40%. Vasoactive intestinal polypeptide may act as a coworker in these fibers both in cavernous trabeculae and around arteries, as about 40% of NOS-containing fibers also showed VIP immunoreactivity. The physiological significance of the colocalization of TH and NOS is unclear, and further studies are required to know the physiological significance of the colocalization of NOS and other neurotransmitters in penile tissue.

Thyroid glands of 7 to 21 day-old chick embryos were examined by electron microscopy using freeze-fracture and thin-section preparations. The primitive follicle lumen first appears between two adjacent epithelial cells in 8 day-old embryos, and is formed in the region of a focal tight junction (macula occludens). The focal tight junction develops into the zonula occludens when the primitive follicle lumen first forms.

Recently, nitric oxide (NO) has been believed to act as a neuronal messenger to mediate penile erection. In the present study using human penile tissue, we investigated the coexistence of neuronal NO synthase (NOS), tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP) by a triple staining method using NADPH diaphorase (ND) staining, a specific histochemical marker of neuronal NOS, and immunohistochemical staining for TH and VIP. Numerous ND-positive nerve fibers and TH-containing fibers were seen in axon bundles, but their distributions were different. Only a few axons in the bundles showed VIP immunoreactivity. Abundant fine varicose nerve terminals innervating cavernous smooth muscles and deep and helicine arteries were observed. The proportion of fibers showing TH-immunoreactivity in ND-positive terminals in the cavernous space was about 25%, and that of VIP was about 40%. Vasoactive intestinal polypeptide may act as a coworker in these fibers both in cavernous trabeculae and around arteries, as about 40% of NOS-containing fibers also showed VIP immunoreactivity. The physiological significance of the colocalization of TH and NOS is unclear, and further studies are required to know the physiological significance of the colocalization of NOS and other neurotransmitters in penile tissue.