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Physical therapies including photodynamic therapy (PDT) and photothermal therapy (PTT) can be effective against diseases that are resistant to chemotherapy and remain as incurable malignancies (for example, multiple myeloma). In this study, to enhance the treatment efficacy for multiple myeloma using the synergetic effect brought about by combining PDT and PTT, iodinated silica/porphyrin hybrid nanoparticles (ISP HNPs) with high photostability are developed. They can generate both 1O2 and heat with irradiation from a light-emitting diode (LED), acting as photosensitizers for PDT/PTT combination treatment. ISP HNPs exhibit the external heavy atom effect, which significantly improves both the quantum yield for 1O2 generation and the light-to-heat conversion efficiency. The in vivo fluorescence imaging demonstrates that ISP HNPs, modified with folic acid and polyethylene glycol (FA-PEG-ISP HNPs), locally accumulate in the tumor after 18 h of their intravenous injection into tumor-bearing mice. The LED irradiation on the tumor area of the mice injected with FA-PEG-ISP HNPs causes necrosis of the tumor tissues, resulting in the inhibition of tumor growth and an improvement in the survival rate.

This paper describes the potential of jack bean urease to behave as a catalyst, template and assembling agent by considering its enzymatic activity, steric structure and thermostability, respectively, for the synthesis of 2D structured iron oxide and the 3D hierarchical organization of the 2D structured iron oxide. Maintaining a mixture of JBU, urea and FeCl2 at 60 °C for several hours produced Fe3O4 plates; the use of denatured JBU or ammonia instead of JBU yielded spherical particles. Furthermore, the spherical particles synthesized using denatured JBU were akaganeite. These results suggest that JBU acts as both template and catalyst for the synthesis of Fe3O4 plates. In addition, the plates organize into flower-like hierarchical structures as the reaction time is lengthened, which aggregate to form spheres after 48 h. The self-assembly of the plates can be controlled by tuning the denaturation of JBU, which is controlled easily because JBU is thermostable and denatures slowly. The flower-like hierarchical structures possess a relatively high saturation magnetization making them potentially useful in environmental and biomedical applications.

Propidium iodide (PI) is a fluorescent nucleic acid dye that contains iodide molecules. Thiol-organosilica particles that were internally functionalized with PI (PI particles) were prepared for X-ray computed tomography (CT) and multicolor fluorescence imaging. PI particles of various sizes were synthesized in a one-pot process. The particles showed unique fluorescent signals and X-ray absorption, with enhancement of the fluorescence intensity of PI located inside organosilica particles. PI particles had multicolor fluorescence, including original fluorescence and near-infrared (NIR). Orally administered PI particles were observed in the gastrointestinal tract (GIT) using fluorescent imaging devices and X-ray CT. An in vivo fluorescence imaging system could detect the NIR fluorescence of PI particles in the GIT specifically. Multipurpose zoom fluorescence microscopy was used to noninvasively visualize the real-time passage of particles and the movement of the GIT. The passage and distribution of particles...

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.

Conifer foliage-like rutile TiO2 nanoparticles (CFR NPs), spherical anatase TiO2 NPs (SA NPs), and a mixture of CFR and SA NPs were synthesized in aqueous solution at low temperature. Furthermore, sea urchin-like nanostructures combining SA and CFR NPs, which were designated as anatase/rutile nanostructures (A/R NSs), were produced through the self-assembly of jack bean urease (JBU). The specific surface area of the A/R NSs was considerably larger than those of the CFR NPs, SA NPs, and commercial TiO2 (P25). In addition, the amount of reactive oxygen species (ROS) yielded from the A/R NSs was significantly higher than that yielded from CFR NPs, SA NPs, and P25 because of the large surface area of the A/R NSs and a synergistic effect caused by the integration of anatase and rutile phases. The A/R NSs showed no cytotoxicity at concentrations

Prostaglandin F2α is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F2α binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F2α synthesized in the neuronal somata and dendrites exert an autocrine action there.—Suzuki-Yamamoto, T., K. Toida, Y. Sugimoto, and K. Ishimura. Colocalization of prostaglandin F2α receptor FP and prostaglandin F synthase-I in the spinal cord.

Previously, we noticed the presence of nerve fiber-like structures in a whole mount preparation of the rat cornea that had not been stained with the non-specific acetylcholinesterase (NsAchE) method. These nerve-like fibers were projected into the central area of the cornea, forming a mesh-like pattern. The aim of this study is to examine the properties of these mesh-like fibers using the following two methods : their sensitivity to capsaicin and the detection of isolectin B4 (IB4)- and capsaicin receptor TRPV1 (transient receptor potential vanilloid 1)-reactivities. The mean disappeared area of non-stained fibers after NsAchE treatment was 26% of the total areas in the neonatally capsaicin-treated cornea. Bunches composed of fine IB4-positive nerve fibers were seen in a whole mount preparation. There were connections between the bunches, producing a mesh-like pattern similar to that of the fibers that were not stained with NsAchE. Fine TRPV1-immunoreactive (ir) nerve fibers were also shown to form bunches, with connections between each bunch observed in whole mount preparations. Thus, TRPV1-ir nerve fibers seem to densely innervate the rat corneal subepithelial stroma and are distinct from the NsAchE-positive nerve fibers. The TRPV1-ir fine nerve fibers overlapped with the IB4-positive nerve fibers, suggesting that the mesh-like fibers that were not stained with NsAchE are fine nociceptive sensory nerve fibers because of their sensitivity to capsaicin and similar distribution pattern to IB4- and TRPV1-positive nerve fibers.

Using a one-pot synthesis, thiol-organosilica nanoparticles (NPs) made from (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, and (3-mercaptopropyl)methyldimethoxysilane have been successfully prepared. We compared the synthesis processes of thiol-organosilica NPs made of these three kinds of organosilicates, as well as particles made from tetraethoxysilicate (TEOS), at concentrations varying between 6.25 and 200 mM. We examined three types of synthetic conditions: the Stöber method, in which particles are prepared in 65% ethanol, and two entirely aqueous solvent syntheses, containing either 2% or 27% ammonium hydroxide. The synthetic mixtures were examined using transmission electron microscopy (TEM) to evaluate the as-prepared NPs. The formation trends and rates for these organosilica NPs vary with differing organosilicate precursors, concentrations, and synthetic conditions. The Stöber method is not suitable for formation of thiol-organosilica NPs as compared with the case of TEOS, but the conditions without ethanol and with 27% ammonium hydroxide are suitable for the formation of thiol-organosilica NPs. The size distributions of the formed NPs were evaluated using TEM and dynamic light scattering. The mean diameters of NPs increase with increasing concentrations of silicate, but the size distributions of NPs prepared under various conditions also differ between silicate sources. Thiol-organosilica NPs internally functionalized with fluorescent dye were also prepared using a one-pot synthesis and were characterized using fluorescence microscopy. The thiol-organosilica NPs retain fluorescent dye maleimide very well. In addition, rhodamine B-doped thiol-organosilica NPs show higher fluorescence than thiol-organosilica NPs prepared with rhodamine red maleimide. The surface of thiol-organosilica NPs contains exposed thiol residues, allowing the covalent attachment of fluorescent dye maleimide and protein maleimide. This is the first report describing the synthesis of thiol-organosilica NPs made of three kinds of thiol-organosilicates, differences in nanoparticle formation due to the kinds and concentrations of thiol-organosilicate and due to synthetic conditions, and the advantages of thiol-organosilica NPs due to the existence of both interior and exterior thiol residues.

Novel organosilica nanoparticles made of 3-mercaptopropyltrimethoxysilane as the single silica source (MPS NPs) have been synthesized successfully by using a Stober 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.

In order to promote better understanding of the physiological roles of prostaglandin F(2alpha) in the mouse testis, we investigated the protein expression and the cellular localization of the enzymes cyclooxygenase and prostaglandin F synthase that are essential for the production of prostaglandin F(2alpha), and the binding site, which is the prostaglandin F(2alpha )receptor (FP). Western blot exhibited the expression of FP protein in wild type mouse testis, and that of prostaglandin F synthase and cyclooxygenase-1 proteins in the both of wild type mouse and FP-deficient mouse testes. The expression of prostaglandin F synthase and cyclooxygenase-1 were detected intensely in Leydig cell-rich fraction, and that of FP was detected equally in Leydig cell-rich fraction and the other fraction. Immunohistochemistry for cyclooxygenase-1 and prostaglandin F synthase demonstrated their co-localization in mouse Leydig cells. Histochemistry for FP demonstrated the localization in Leydig cells and in spermatids of seminiferous tubules. Double histochemical staining confirmed the co-localization of cyclooxygenase-1, prostaglandin F synthase and FP in the Leydig cells. These findings indicate that prostaglandin F(2alpha) may have an effect on the functions of Leyding cells in an autocrine fashion. It implies that prostaglandin F synthase and FP are involved in the control of testosterone release from Leydig cells and in spermatogenesis via the local pathway and the hypothalamo-hypophysial-testis pathway, and affect the testicular function.

Multifluorescent silica nanoparticles were synthesized by the Stöber method using conjugates of (3-aminopropyl)triethoxysilane and fluorescent dye-N-hydroxysuccinimide esters. The nanoparticles containing the fluorescent dyes were well dispersed and showed high, stable, and tunable fluorescence intensities. In addition, we prepared multifluorescent silica nanoparticles containing two kinds of fluorescent dyes and used the nanoparticles in biological applications. Flow cytometry analysis showed high and tuned fluorescence and multiple fluorescences from single nanoparticles with diameters of approximately 400 nm. Fluorescence microscopy analysis also showed high and tuned fluorescence, as well as multiple fluorescences from single nanoparticles and from cells labeled with multifluorescent silica nanoparticles. The intracellular distribution of nanoparticles was evaluated by confocal microscopy and electron microscopy. We discuss the advantages and demonstrate the usefulness of our nanoparticles in relation to commercially available fluorescent nanoparticles including quantum dots.

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.

We have established a method for selecting binding phages from a phage immunoglobulin heavy chain variable region (VH) library by panning with nitrocellulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clones that bind to hen egg white lysozyme (HEL) was used for panning against HEL. The efficiency of our method was as high as that of panning with magnetic beads. In addition, we performed membrane panning against target proteins and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to target proteins was confirmed by dot blotting. When applied to disease-associated antibodies, these methods will likely benefit clinical research. In addition, these techniques may be applicable to systematic analysis in proteome studies.

The effect of a synthetic steroidal anti−androgen, chlormadinone acetate(CMA), on spontaneous benign prostatic hyperplasia(BPH)in dogs was investigated. Male beagle dogs(5−8 years old) were divided into four experimental groups. Group 1 consisted of untreated controls. Groups 2 to 4 received CMA 0.03, 0.1, and 0.3mg/kg/day, p.o., respectively, for 6 months. In group 1, glandular hyperplasia of the prostate was clearly detected. The glandular epithelial cells showed uniformly intense nuclear staining for androgen receptor(AR). AR was also localized in the nuclei of the fibro−muscular stromal cells. Immunolocalization of 5 alpha−reductase type II was clearly detected in the interacinar stromal cells, but not in the glandular epithelial cells. In groups 2 to 4, CMA produced marked atrophy of the glandular epithelium. The interacinar fibro−muscular stroma was prominent. The intensity of the nuclear staining for AR in both epithelial and stromal cells was negative or very weak. Furthermore, the immunoreaction for 5 alpha−reductase type II in the interacinar stromal cells was negative or very weak. The decreased AR−immunostaining may be explained by the decrease in the number of AR and it may play a key role in the down−regulation of 5 alpha−reductase type II gene expression by CMA. The atrophy after treatment with CMA may be due to shrinkage of both glandular and stromal compartments in the prostate tissue.

12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid, and produces 12S-hydroperoxy-arachidonic acid. When mouse gastrointestinal tract was immunostained with an antiserum against human platelet 12-lipoxygenase and examined by light microscopy, positively-stained cells were found in the epithelium of stomach and small and large intestines. Electron microscopic immunohistochemistry revealed that the positively-stained cells were open-type enteroendocrine cells. The endocrine cells had many granules of heterogeneous sizes, forms and internal structures, and most of the granules had electron lucent area. 12-Lipoxygenase was localized in the cytoplasm, but not in the nucleus, plasma membranes and other subcellular organelles. Double immunostaining revealed that all 12-lipoxygenasepositive cells contained serotonin. With an antiserum against 12-lipoxygenase isozyme of porcine leukocytes, no positively-stained cells were observed in gastrointestinal epithelium.

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/Δ5-4 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.

Owing to the surface plasmon resonance-enhanced electromagnetic field, clustered gold nanoparticles–fluorescent silica core–shell nanoparticles became excited within the therapeutic window and fluoresced strongly in this window. The nanoparticles enabled tumor detection using fluorescence imaging and X-ray computed tomography.

Superparamagnetic nanoparticles (SPIONs) could enable cancer theranostics if magnetic resonance imaging (MRI) and magnetic hyperthermia treatment (MHT) were combined. However, the particle size of SPIONs is smaller than the pores of fenestrated capillaries in normal tissues because superparamagnetism is expressed only at a particle size

The retinoic acid (RA, a vitamin A metabolite) receptor (RAR) is a transcription factor. Vitamin A/RA administration improves the Alzheimer's disease (AD)- and age-related attenuation of memory/learning in mouse models. Recently, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was identified as a key molecule in RA-mediated anti-AD mechanisms. We investigated the effect of chronic administration of the RAR agonist Am80 (tamibarotene) on ADAM10 expression in senescence-accelerated mice (SAMP8). Moreover, we estimated changes in the expression of the amyloid precursor protein (APP), amyloid beta (Aβ), and hairy/enhancer of split (Hes), which are mediated by ADAM10. Spatial working memory and the levels of a hippocampal proliferation marker (Ki67) were also assessed in these mice. ADAM10 mRNA and protein expression was significantly reduced in the hippocampus of 13-month-old SAMP8 mice; their expression improved significantly after Am80 administration. Further, after Am80 administration, the expression levels of Hes5 and Ki67 were restored and the deterioration of working memory was suppressed, whereas APP and Aβ levels remained unchanged. Our results suggest that Am80 administration effectively improves dementia by activating the hippocampal ADAM10-Notch-Hes5 proliferative pathway.

The localization of prostaglandin (PG) endoperoxide synthase in bovine intestine was examined immunocytochemically with polyclonal antibody raised against PG endoperoxide synthase purified from bovine seminal glands. The most intense positive staining reaction for the enzyme was present in mast cells. Mast cells were found to be widely distributed in the intestinal wall, and were particularly numerous in the lamina propria. Most of the mast cells in the lamina propria of the intestinal villi were elongated and oriented with their long axis parallel to the plane of the absorptive epithelium. In whole mount preparations of jejunal villi, mast cells were seen to form a two-dimensional network in the lamina propria. In addition to mast cells, smooth muscle cells of the inner circular muscle layer and muscularis mucosae, nerve cells and fibers, endothelial cells of arterioles, and serosal epithelial cells also showed faint to moderate staining for the enzyme. These results suggested that mast cells are the major source of PGs in the bovine intestinal wall. The characteristic arrangement of mast cells in the intestinal villi may be related to their functions in this portion of the bovine intestine.

Nociceptin (NOC), an endogenous ligand of the opioid receptor-like 1 receptor, is thought to be involved in learning and memory processes. Since acetylcholine (ACh) is involved in hippocampal function, and the hippocampus plays a critical role on the learning and memory function, hippocampal ACh release in NOC-receptor knockout mice was examined using an in vivo microdialysis method. The release of hippocampal ACh was largely increased in the knockout mice. Furthermore, in the knockout mice, an enhanced hippocampal theta rhythm, which is known to be linked to hippocampal memory function, was also observed. Immunohistochemically, in septum, co-existence of NOC receptor with cholinergic, but not with GABAergic neurons, was verified. The findings demonstrate that the NOC receptor is involved in hippocampal cholinergic function.

Prostaglandin F synthase has at least two isozymes, i.e. prostaglandin F synthase I and II. Recently, we demonstrated immunocytochemically that prostaglandin F synthase I was localized in neuronal dendrites and somata, and in endothelial cells of blood vessels in the whole area of rat spinal cord. In the present study, we immunocytochemically localized prostaglandin F synthase II in ependymal cells and tanycytes surrounding the central canal and in endothelial cells of blood vessels, but not in any neuronal elements at all segmental levels of the rat spinal cord. Immunoelectron microscopy and confocal laser scanning microscopy confirmed these findings and further revealed that strong immunoreactivity was found in the basal processes of the tanycytes. Our present and recent studies using antibodies against the two isozymes of prostaglandin F synthase clearly indicated that they were localized differentially in ependymal (prostaglandin F synthase II) and neuronal elements (prostaglandin F synthase I), but were co-localized in blood vessels in the rat spinal cord. The distinct localization of the two isozymes suggests that prostaglandin F2 has different transcellular biological actions via different cell groups.

The effects of flavonoids on NADPH diaphorase activity were studied in vitro, and we found that the enzyme activity was markedly inhibited by quercetin. This inhibitory action was shown to be accompanied by an increase in the apparent Km value of the enzyme for the cofactor NADPH, with a decrease in the Vmax, and an increase in the apparent Km for the substrate nitro blue tetrazolium, without any significant change in the Vmax. These results indicate that quercetin may directly inhibit NADPH diaphorase, thus suggesting the possibility that this compound may be able to inhibit the production of nitric oxide in the brain.

Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin-fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin-biotin technology.

Prostaglandin F synthase (PGFS) is a multi-functional enzyme, and catalyzes the reduction of prostaglandin (PG)D2to 9a, 11f -PGF2and that of PGH2to PGF2aon the same molecule. PGFS has at least two isoformes, the lung-type enzyme’ (Km value of 120 µM for PGD2) and the liver-type one2(Km value of 10 µM for PGD2). The bovine lung-type PGFS is a:monomeric protein with a Mr of 36,666 consisting of 323 amino acids, and its amino acid sequence shows high homology compared with those of other aldo-keto reductase family (AKR family) members. However, the liver-type PGFS cDNA has not been isolated, and the primary structure has not been known. In the present study, we decided the primary structure of bovine liver-type PGFS and deduced the amino acids related the affinity for PGD2by the mutagenesis study.3Furthermore, our immunohistochemical study revealed PGF-producing cells in the liver.4

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.

Prostaglandin (PG) F2 is widely distributed in various organs, and exhibits various biological actions such as smooth muscle contraction of uterus [1], bronchus [2], and trachea [3], the initiation of parturition [4], and pain transmission [5]. PGF synthase (PGFS) is a dual function enzyme, which catalyzes the reduction of not only PGD2 but also PGH2 on the same molecule [6]. PGFS exhibits reductase activities toward various carbonyl compounds [6], such as phenanthrenequinone (PQ) in addition to PGD2 and PGH2. PGFS belongs to the aldo-keto reductase (AKR) superfamily based on substrate specificity, molecular weight, and the amino acid sequence [7–10]. Recently, we isolated the clones of four human enzymes [9,10], including PGFS, which belong to the AKR family. Although the amino acid sequences of these enzymes show high homology (83–98%), only PGFS, which was classified to AKR1 C3 [ 10], catalyzed the reduction of PGD2 and PGH2, and the oxidation of 9α, 11β-PGF2 [9, 10]. Moreover, we developed a high-stringency PCR technique to discriminate fine sequence differences using each specific primer, and examined RT-PCR analysis to estimate expression of AKR mRNAs [10]. The tissue distribution of these four enzymes was different, and human PGFS was localized in lung, liver, kidney, muscle, and peripheral blood lymphocyte. The expression in lymphocytes was markedly suppressed by the T cell mitogen concanavalin A. Moreover, PGFS was abundant in the contractile interstitial cells (CIC) in the alveolar septum of bovine lung, and was diffusely present in the cytoplasm of the endothelial cells of bovine liver. These results suggest that PGFS plays important roles in both the physiological and pathophysiological processes.

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.

A lipoxygenase is an enzyme which incorporates one molecule of oxygen into arachidonic acid or other polyunsaturated fatty acids producing hydroperoxy acids with a conjugated diene system. Enzymes oxygenating at the positions 5, 8, 12 and 15 of arachidonic acid and producing the corresponding hydroperoxy eicosatetraenoic acid (HpETE) have been isolated from mammalian tissues (Figure 1). All enzymes except for the 8-lipoxygenase have been highly purified, and their cDNAs and genomic DNAs have been already cloned (1,2).

The biosynthesis of prostaglandin (PG) and thromboxane (TX) is initiated by a bifunctional enzyme with oxygenase and peroxidase activities, and PGH2 is produced from arachidonic acid via PGG21. This bifunctional enzyme (prostaglandin endoperoxide synthase, E.C. 1.14.99.1) will be referred to briefly as cyclooxygenase in this chapter. There are two cyclooxygenase isozymes (COX-1 and COX-2), and COX-1 is generally known to be present in human platelets and involved in the synthesis of proaggregatory and vasoconstrictive TXA2. Since platelets have no nucleus and can not synthesize mRNA, the COX-1 protein is derived presumably from megakaryocytes which are precursor cells of platelets.

Steroid hydroxylase cytochrome P450c17 has been previously purified from microsomal fractions of immature rat livers. In this study, we investigated the expression of P450c17 in rat livers to understand a role of steroidogenesis in the extrasteroidogenic tissue. Upon immunoblot analysis utilizing liver microsomes from rats, P450c17 was detected in 1 and 3 week old rats but not in adult rats. Data from immunohistochemical studies also showed a similar age-dependent expression of P450c17 and indicated that P450c17 detected in immature rat livers is localized in cells surrounding interlobular veins. This age-dependent expression of P450c17 in rat livers was observed in both sexes. Upon enzymatic analysis utilizing microsomal fractions from livers, levels of 17alpha-hydroxylase and 17,20-lyase activity for pregnenolone and progesterone increased by 3 weeks and dramatically reduced at 7 weeks, which is consistent with the expression level of P450c17. These data clearly indicate that P450c17 is expressed in immature rat liver to produce 17alpha-hydroxysteroids and C19-steroids. Based upon immunoblot analysis, the expression level of P450c17 in immature rat livers was approximately one third of that in testis. Compared expression level of P450c17 and total volume of organs between liver and testis, the total amount of steroid metabolites produced by liver P450c17 could be greater than that produced by gonadal P450c17. Because of the absence of P450c17 in rat adrenal glands, rat liver could be the major site for producing 17alpha-hydroxysteroids and C19-steroids in this particular period of life. Although physiological products formed by P450c17 in liver and their roles remain to be elucidated, this study suggests a large capacity of prepubertal rat liver for participating the production of steroid hormones and a putative importance of 17alpha-hydroxysteroids and C19-steroids, such as cortisol and androstendione, which are generally believed to be minor components of steroid hormones in rodents.

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.

Since arachidonate 12-lipoxygenase was earlier found in platelets as the first mammalian lipoxygenase, the enzyme has been found in a variety of tissues of a number of animal species. On the basis of our studies in the last decade, we have demonstrated that there are two isoforms of 12-lipoxygenase (1).

Objective: We examined the coexistence of nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP) in the efferent pathway to the rat corpus cavernosum. Methods: We used NADPH diaphorase (ND) histochemical staining. a specific marker of neuronal NOS in combination with retrograde axonal transport ofTrue Blue (TB) and VIP immunohistochemistry. Results: Many neurons were labeled by TB in the cavernous nerve and the body of the major pelvic ganglion (MPG). and fewer neurons in the pelvic nerve. In the cavernous nerve, about 90% of TB-labeled efferent neurons were ND positive, and about 80% in the body of the MPG and pelvic nerve. Besides, 60-80% of TB-labeled efferent neurons projecting to the corpus cavernosum showed VIP immunoreactivity. VIP was colocalized with NOS in 70-80% of neurons. Conclusions: These findings suggest that most of the ND-positive nerves and terminals in the corpus cavernosum come from the MPG via the cavernous nerve and that NO plays an important role: it may act in combination with NO because most NOS-containing neurons simultaneously showed VIP immunoreactivity.

Arachidonate 12-lipoxygenase introduces one oxygen molecule regiospecifically and stereoselectively into the carbon 12 of arachidonic acid. Our group has demonstrated the occurrence of two isoforms of 12-lipoxygenase (platelet-type and leukocyte-type) which were distinguished in terms of substrate specificity, the cross-reactivity of their antibodies and the homology of their amino acid sequences.1,2 The leukocyte-type 12-lipoxy-genases have been detected not only in leukocytes, but also in several non-hematopoietic cell types, including bovine tracheas,3 porcine anterior pituitary glands,4 canine cerebra5 and rat pineal glands.6 The platelet-type 12-lipoxygenases were found in platelets of many animal species and also in human and mouse skin cells.7–9 For a better understanding of the physiological role of the platelet-type 12-lipoxygenase, we attempted to further investigate the expression and the distribution of the enzyme by immunohistochemical study. However, the purification of platelet-type 12-lipoxygenase as antigen was difficult, and no polyclonal antibody with a high affinity for platelet-type enzyme was available until recently. Therefore, in this study we attempted to purify a recombinant enzyme as an antigen.

The localization of 5 alpha-reductase was immunohistochemically studied in the anterior pituitary of male rats, using a polyclonal antibody against 5 alpha-reductase rat type 1. The immunoreactive cells were concentrated in the central region and on the border of the intermediate lobe in the anterior pituitary, but not in the intermediate or posterior lobe. The immunoreaction was located mostly in the cytoplasm and occasionally in the cell nuclei. The immunoreactive cells showed alterations in size and number and in the intensity of the immunoreaction after gonadectomy. One week after castration, the cells became larger and the immunoreactivity increased. Two weeks after castration, the number of immunoreactive cells increased. Double immunostaining using anti-luteinizing hormone beta-subunit or anti-follicle stimulating hormone beta-subunit antibody revealed that most of the cells containing 5 alpha-reductase were gonadotrophs. Electron microscopically, the immunoreactive cells showed lamelliform rough endoplasmic reticulum and a depletion of secretory granules 1 week after castration. One week later, the rough endoplasmic reticulum was developed and dilated and the number of secretory granules increased. These results suggest that 5 alpha-reductase is located in the gonadotrophs of rat anterior pituitary and that it is involved in the feedback regulation of gonadotropin secretion by androgens.

We immunohistochemically studied the localization of 5 alpha-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, 5 alpha-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 5 alpha-reductase. AR was present in the nuclei of all epithelial and stromal cells throughout the prostate. The number of 5 alpha-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 5 alpha-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 5 alpha-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.

In human platelets arachidonic acid is released from membrane phospholipids by the catalysis of phosopholipase A2, and then oxygenated either by cyclooxygenase or by 12-lipoxygenase. It is well known that the cyclooxygenase pathway leads to the synthesis of pro-aggregatory and vasoconstrictive thromboxane A2. 12-Lipoxygenase introduces one oxygen molecule regiospecifically and stereoselectively into the carbon 12 of arachidonic acid. The primary product is 12S-hydroperoxy-5, 8, 14-cis-l0-trans-eicosatetraenoic acid (12-HPETE), which is reduced to 12S-hydroxy acid (12-HETE) in a whole cell preparation. A number of papers have reported biological activities of either 12-HPETE or 12-HETE1. However, most of them could not be generalized in terms of the cell type and animal species, and the general biological significance of the enzyme has not been well understood1,2. Recently two groups of bioactive arachidonate metabolites were identified as the products of the 12-lipoxygenase pathway. Namely, transcellular synthesis of lipoxins via leukotriene A4 requires the participation of platelet 12-lipoxygenase3, and 12-HPETE is enzymatically transformed to hepoxilins4. Our group has demonstrated the occurrence of two distinct isoforms of l2-lipoxygenase in mammalian tissues1,5. The leukocyte-type 12-lipoxygenase has a broad substrate specificity reacting with linoleic and linolenic acids with 18 carbon atoms as well as arachidonic acid with 20 carbon atoms. In contrast, the platelet-type enzyme is much less active with the C18 fatty acids. The leukocyte-type enzyme is also found in other tissues in addition to leukocytes1. The platelet-type enzyme was found earlier in platelets of various animal species, and found recently in epidermis6–8. For a better understanding of the physiological role of the platelet-type 12-lipoxygenase we planned immunohistochemical studies of the enzyme. However, purification of this enzyme from platelets was difficult, and no polyclonal antibody with a high affinity for the enzyme had not been available until recently. Therefore, we attempted to purify a recombinant enzyme as antigen.

Mature adult rat hepatocytes were cultured as monolayers in serum-free Williams medium E containing 10(-7) M each of insulin (Ins), dexamethasone (Dex) and triiodothyronine (T3) and 30 mM pyruvate. The hepatocytes remained morphologically intact for at least 14 days, during which period they maintained normal liver functions such as the expressions of cytochrome P-450 mRNA and glucokinase and secretion of albumin. They also retained the ability to resume proliferation. Cells cultured with pyruvate had a much higher ATP level than those without pyruvate, suggesting that pyruvate can sustain functional hepatocytes for a long period in culture in the presence of Ins, Dex and T3, probably by producing enough energy for their maintenance.

Arachidonate 12-lipoxygenase is an enzyme that oxygenates the 12 position of arachidonic acid to produce its 12-hydroperoxy derivative. We were interested in the tissue distribution and subcellular localization of the platelet-type 12-lipoxygenase, which is distinguished from the leukocyte type by several criteria. Antiserum was raised in rabbits against purified recombinant arachidonate 12-lipoxygenase of human platelets. When mouse bone marrow cells and lung were immunostained and observed by light and electron microscopy, the positively stained cells were platelets, megakaryocytes, and eosinophils. 12-Lipoxygenase was localized in the cytoplasm of platelets but was hardly detectable in the plasma membrane and intracellular organelles. The enzyme was found in the cytoplasm of immature megakaryocytes with kidney-shaped nuclei and a few profiles of demarcation membranes, as well as in the mature form with well-developed demarcation membranes. These results indicated the expression of 12-lipoxygenase at an early stage in the course of megakaryocytopoiesis.

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.

Near-infrared fluorescent silica-porphyrin hybrid nanotubes (HNTs) were successfully synthesized by π-π stacking, electrostatic interaction and a sol-gel reaction. The HNTs-labeled macrophages were detected in vivo, and the minimum detectable number of cells was 200. Furthermore, the biodistribution of HNTs-labeled macrophages was tracked by fluorescence imaging.

Recently, nitric oxide (NO) has been thought to be a neuronal messenger to evoke penile erection. NO synthase (NOS)-containing nerve fibers were identified and localized in human penile tissue, but detail distribution of NOS-containing nerve fibers in the human penis was unclear. In the present study we examined their distribution using histochemical staining of NADPH diaphorase (ND), which is a specific marker of neuronal NOS. In the crura penis some various sizes of ND-positive nerve bundles were observed in the cavernous spaces. In the penile shaft large bundles (> 50 microns) decreased in number and were observed only near deep artery. There were abundant ND-positive nerve terminals with fine varicosity innervating both corpus cavernous smooth muscles and deep, dorsal and helicine arteries. In the wall of deep dorsal vein there were also many groups of ND-positive fibers. Endothelium of deep artery and its large branches was clearly ND-positive, but parts of endothelium of deep dorsal vein or corpus cavernous sinus were only faintly stained. Some of the dorsal penile nerve fibers were ND-positive. In corpus spongiosum many ND-positive nerve fibers were observed and urethelium was clearly ND-positive. In conclusion, NO may have an important role in the function of both corpus cavernosum and corpus spongiosum because NOS-containing nerve fibers were widely distributed in both cavernous tissues.

When a crude extract of rat pineal glands (the 1000 × g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-lipoxygenase of rat pineal gland also reacted with linoleic and α-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E. coli . The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75 305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.

A multifunctional nanoparticle for biological applications has been developed by combining the useful functions of quantum dots (QDs), fluorescent dyes, and organosilica particles. Dual fluorescent thiol-organosilica nanoparticles composed of QDs and a fluorescent dye in a thiol-organosilica layer were synthesized in a one-pot process. The thiol-organosilica layer encapsulating the QDs could be tuned in thickness and functionalized. The QDs encapsulated with thiol-organosilica layer (thiol-OS-QDs) were well dispersed, and exhibited unique photophysical properties upon encapsulation within the thiol-organosilica layer. The thiol-OS-QDs could preserve their fluorescent properties under severe acid and alkali conditions as compared with those of bare QDs. The dual fluorescent thiol-OS-QDs containing QD605 and rhodamine B showed strong fluorescence, high photostability, and reduced photoblinking. Cells labeled with dual fluorescent thiol-OS-QDs were detected clearly in vivo, and their functionalization with antibody using the thiol residue on the surface was useful for molecular tracing of tumor antigen. The dual fluorescent thiol-OS-QDs showed high potential as non-photoblinking QDs.

In contrast to 5-lipoxygenase which is involved in the biosynthesis of bioactive leukotrienes, a general physiological role of 12-lipoxygenase has not yet been established. Although the enzyme was earlier found in platelets as the first mammalian lipoxygenase and has been found in various tissues of many animal species, most investigators have ignored the 12-lipoxygenase until very recently.

Rats were treated with heat-killed Propionibacterium acnes and subsequent injection of a small amount of lipopolysaccharide after 7 days. After 24 hr most of the rats died of massive liver cell necrosis. Nonparenchymal liver cells were isolated from this liver injury model and incubated with arachidonic acid. Reverse-phase high-pressure liquid chromatography detected the 5-lipoxygenase metabolites (leukotriene B4 and 5-hydroxy-arachidonic acid), whereas these compounds were produced in negligible amounts when the rats were treated with P. acnes only. Immunohistochemical studies with 5-lipoxygenase antiserum revealed that the injured livers contained a large number of positively stained round cells with segmented nuclei, which were rarely found in the livers treated with P. acnes only. These positively stained cells were histologically identified as neutrophils. The results suggested that the increased 5-lipoxygenase activity in the injured rat liver is attributable to the infiltrating neutrophils rather than to nonparenchymal hepatic cells. (HEPATOLOGY 1992;16:462–468.)