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To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2. To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2. Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse β-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.

Oxygen imaging of biological cells and tissues is becoming increasingly important in cell biology and in the pathophysiology of various hypoxia-related diseases. The optical oxygen-sensing method using luminescent probes provides very useful, high spatial resolution information regarding oxygen distribution in living cells and tissues. This review focuses on recent advances in biological oxygen measurements based on the phosphorescence quenching of probe molecules by oxygen, and on hypoxia-sensitive fluorescent probes. Special attention is devoted to metal complex probes, Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes. Current knowledge regarding the mechanism of phosphorescence quenching of metal complexes by oxygen is described in relation to the oxygen sensitivity of the probes, and recent advances in optical oxygen probes and detection techniques for intracellular and tissue oxygen measurements are reviewed, emphasizing the usefulness of chemical modifications for improving probe properties. Tissue oxygen imaging and hypoxic tumor imaging using these metal complex probes demonstrate the vast potential of optical oxygen-sensing methods using luminescent probes.

A cationic iridium(III) complex (btp)2Ir(phen-NH2) (btp = benzothienylpyridine; phen-NH2 = 5-amino-1,10-phenanthroline) and its analog with extended π-electronic structures (btq)2Ir(phen-NH2) (btq = benzothienylquinoline), (ttph)2Ir(phen-NH2) (ttph = thienothiophenylphenanthridine), (btph)2Ir(phen-NH2) (btph = benzothienylphenanthridine) have been designed and synthesized to develop new oxygen probes for living cells. The photophysical and cellular properties of these complexes were systematically investigated by using (btp)2Ir(acac) and its derivatives as reference compounds. The extension of the π-electronic systems of ligands resulted in remarkable red shifts in the absorption and phosphorescence spectra; the phosphorescence bands of (ttph)2Ir(phen-NH2) and (btph)2Ir(phen-NH2) appeared in the near-infrared region, maintaining the relatively high phosphorescence quantum yields ( Φ p 0 = 0.14–0.24). Dimethylation and diethylation of the amino group of the phen-NH2 ligand in (btp)2Ir(phen-NH2) and its analog significantly enhanced the cellular uptake efficiency as compared with their neutral analog with an acetylacetonate (acac) ligand. The cationic iridium(III) complex (btp)2Ir(phen-NH2) and its analog internalized into living HeLa cells showed selective distribution into mitochondria. Owing to the high cellular uptake, relatively high cytotoxicity was found for the cationic iridium complexes. The phosphorescence intensity of newly developed iridium complexes exhibited oxygen response in HeLa cells, demonstrating that these complexes have potential as mitochondria-selective oxygen sensors for biological cells and tissues.

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin–IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment–induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

Singlet oxygen sensitization of the water-soluble silylated tetraphenylporphyrin derivative has been studied. Quantum yield of singlet oxygen sensitization of the silylated compound was improved compared with that of the non-silylated one. To clarify the mechanism of the improvement, photophysical processes have been studied. The silylation increased the fraction of the triplet state quenched responsible for the formation of singlet oxygen, and resultantly, this effect improved the quantum yield of singlet oxygen sensitization. To demonstrate the silylated compound suitable for a photosensitizer in photodynamic therapy for cancer, a cell culture study was carried out with a human cancer cell line. We found that the silylated compound displayed much higher photodynamic activity than the non-silylated one. We conclude that this high activity was caused by the improvements of both quantum yield of singlet oxygen sensitization and cellular uptake efficiency. We emphasize that improved lipophilicity by silylation contributes much to the high cellar uptake efficiency of porphyrin derivatives.

We have synthesized novel perylene derivatives bearing a silyl function with modifiable functional groups as a feasible material for visualizing biological substances. The fluorescent perylene derivative was coupled to C-5 position of pyrimidine nucleotide and the nucleotide was subsequently incorporated to the middle position of oligoDNA using an automated DNA synthesizer. Also, the derivative was coupled with a cholesterol analog with different length of linking alkyl function. The resulting modified fluorescent oligoDNA exhibited a small change in its fluorescent signal upon hybridization with the complementary oligoDNA. While the cholesterol analogs bearing the silylated perylene exhibited marked fluorescent signal in living cells. The signal is stronger in the analog having longer linker function compared to the analog having shorter linker function. In each case, however, the signal was much prominent compared to that of dehydroergosterol (DHE), a fluorescent cholesterol analog widely used in biological study.

Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by β-hydroxy-β-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 β-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40–50 mol% is crucial for insulin secretion and SG formation functions.

Functional variations in the secretogranin III (SCG3) gene are associated with susceptibility to obesity. SCG3 forms secretory granules with orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), and POMC in the hypothalamus. In this study, we screened proteins for SCG3-binding activity and identified secretogranin II (SCG2) using a yeast two-hybrid system. Immunoprecipitation revealed that SCG2 interacts with SCG3. In situ hybridization and immunohistochemistry indicated that SCG2 was highly expressed in the lateral hypothalamic area, paraventricular nucleus, and arcuate nucleus of the hypothalamus. Double-labeling immunohistochemical analysis demonstrated that SCG2 was expressed in orexin-, MCH-, NPY-, and POMC-expressing neurons. SCG2 was also coexpressed with SCG3. Upon introduction into neuroblastoma cells, SCG2 was expressed in the cytosol and formed granule-like structures with SCG3, orexin, NPY, or POMC. SCG3 bound to POMC; however, it did not bind to orexin, MCH, or NPY. By contrast, SCG2 formed aggregates with orexin, MCH, NPY, and POMC. SCG2 may act as a hormone carrier for orexin, MCH, NPY, and POMC by binding with SCG3, which targets proteins to the secretory granules. SCG2 mRNA levels increased along with those of SCG3, orexin, MCH, and NPY after a 24-h fast, suggesting that the SCG2/SCG3 system may respond in an adaptive manner to acute body weight changes. However, this SCG2/SCG3 system appears to be unresponsive to chronic body weight changes, such as diet-induced obesity or obesity in ob/ob mice. We suggest that SCG2, as well as SCG3, may be a potential regulator of food intake based on its capacity to accumulate appetite-related hormones into secretory granules.

Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic β-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA . The open reading frame was predicted to encode a 66 kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA ( AtRadA ) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.

Although hydrophilic acrylic resins including LR White have been widely utilized as embedding media for immunocytochemical use, the constituents of tissues are often extracted by the resin monomer during the infiltration process of the embedment, resulting in a discernible impairment of the ultrastructure when the tissue is weakly fixed only with aldehydes. To minimize the extraction by the resin monomer, the embedding procedure with LR White resin was reexamined in the present study. Among the treatments tested, a partial dehydration with 70% ethanol containing 2% phosphotungstic acid (PTA) well preserved the ultrastructure of the pituitary tissue without spoiling the antigenicity of LHbeta and other representative markers for the Golgi apparatus. In addition, treatment with 1% tannic acid (TA) prior to the dehydration described above synergistically improved both the ultrastructure and antigenicity of the tissue so that the orientation of the Golgi apparatus could be determined by double immunogold labeling with commercially available anti-GM130 and anti-TGN38 antibodies. The ultrathin sections from the LR White-embedded tissue treated with TA and dehydrated in 70% ethanol containing 2% PTA also enhanced contrast without conventional heavy-metal staining with uranyl acetate and lead citrate. Our findings further suggest that the precipitation of TA and PTA protected the tissue from being extracted during the embedment, probably because an insoluble complex was transiently formed with the constituents of the tissue. This simple modification of the LR White embedment can extend the application of post-embedding immunocytochemistry as an alternative to pre-embedding immunolabeling with frozen ultrathin sections.

Small luminescent molecular probes based on the iridium(III) complex BTP, (btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone) have been developed for sensing intracellular and in vivo O2. These compounds are BTPSA (containing an anionic carboxyl group), BTPNH2 (containing a cationic amino group), and BTPDM1 (containing a cationic dimethylamino group); all substituents are incorporated into the ancillary acetylacetonato ligand of BTP. Introduction of the cationic dimethylamino group resulted in an almost 20-fold increase in cellular uptake efficiency of BTPDM1 by HeLa cells compared with BTP. The phosphorescence intensity of BTPDM1 internalized in living cells provided a visual representation of the O2 gradient produced by placing a coverslip over cultured monolayer cells. The intracellular O2 levels (pO2) inside and outside the edge of the coverslip could be evaluated by measuring the phosphorescence lifetime of BTPDM1. Furthermore, intravenous administration of 25 nmol BTPDM1 to tumor-bearing mice allowed the tumor region to be visualized by BTPDM1 phosphorescence. The lifetime of BTPDM1 phosphorescence from tumor regions was much longer than that from extratumor regions, thereby demonstrating tumor hypoxia (pO2 = 6.1 mmHg for tumor and 50 mmHg for extratumor epidermal tissue). Tissue distribution studies showed that 2 h after injection of BTPDM1 into a mouse, the highest distribution was in liver and kidney, while after 24 h, BTPDM1 was excreted in the feces. These results demonstrate that BTPDM1 can be used as a small molecular probe for measuring intracellular O2 levels in both cultured cells and specific tissues and organs.

The calcineurin inhibitor cyclosporine A (CsA) has emerged as a major cause of secondary hypertension in humans, but the underlying pathogenetic mechanisms have remained enigmatic. Synapsins are a family of synaptic vesicle phosphoproteins that are essential for normal regulation of neurotransmitter release at synapses. In addition to synaptic vesicles, synapsins and other vesicle proteins are found on microvesicles in sensory nerve endings in peripheral tissues. However, the functions of the sensory microvesicles in general, and of synapsins in particular, are unknown. We now demonstrate in a mouse model that CsA raises blood pressure by stimulating renal sensory nerve endings that contain synapsin-positive microvesicles. In knockout mice lacking synapsin I and II, sensory nerve endings are normally developed but not stimulated by CsA whereas a control stimulus, capsaicin, is fully active. The reflex activation of efferent sympathetic nerve activity and the increase in blood pressure by CsA seen in control are greatly attenuated in synapsin-deficient mice. These results provide a mechanistic explanation for CsA-induced acute hypertension and suggest that synapsins could serve as a drug target in this refractory condition. Furthermore, these data establish evidence that synapsin-containing sensory microvesicles perform an essential role in sensory transduction and suggest a role for synapsin phosphorylation in this process.

Synapsins constitute a family of synaptic vesicle proteins essential for regulating neurotransmitter release. Only two domains are conserved in all synapsins: a short N-terminal A domain with a single phosphorylation site for cAMP-dependent protein kinase (PKA) and CaM Kinase I, and a large central C domain that binds ATP and may be enzymatic. We now demonstrate that synapsin phosphorylation in the A domain, at the only phosphorylation site shared by all synapsins, dissociates synapsins from synaptic vesicles. Furthermore, we show that the A domain binds phospholipids and is inhibited by phosphorylation. Our results suggest a novel mechanism by which proteins reversibly bind to membranes using a phosphorylation-dependent phospholipid-binding domain. The dynamic association of synapsins with synaptic vesicles correlates with their role in activity-dependent plasticity.

In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes. Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates. The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP. We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers. This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization. These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences. The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice.

Synapsins I and II are synaptic vesicle proteins essential for normal Ca2+ regulation of neurotransmitter release. Synapsins are composed of combinations of common and variable sequences, with the central C-domain as the largest conserved domain. The C-domain is structurally homologous to ATPases, suggesting that synapsins function as ATP-dependent phosphotransfer enzymes. We have now identified an unanticipated third synapsin gene that is also expressed in brain. The product of this gene, synapsin IIIa, shares with synapsins Ia and IIa three conserved domains that are connected by variable sequences: the phosphorylated A-domain at the amino terminus, the large ATP-binding C-domain in the center, and the E-domain at the carboxyl terminus. Like other synapsins, synapsin IIIa binds ATP with high affinity and ADP with a lower affinity, consistent with a cycle of ATP binding and hydrolysis. ATP binding to the different synapsins is directly regulated by Ca2+ in a dramatically different fashion: Ca2+activates ATP binding to synapsin I, has no effect on synapsin II, and inhibits synapsin III. Thus vertebrates express three distinct synapsins that utilize ATP but are specialized for different modes of direct Ca2+ regulation in synaptic function.

Synapsins are abundant synaptic vesicle proteins with an essential regulatory function in the nerve terminal. We determined the crystal structure of a fragment (synC) consisting of residues 110‐420 of bovine synapsin I; synC coincides with the large middle domain (C-domain), the most conserved domain of synapsins. SynC molecules are folded into compact domains and form closely associated dimers. SynC monomers are strikingly similar in structure to a family of ATPutilizing enzymes, which includes glutathione synthetase and D-alanine:D-alanine ligase. SynC binds ATP in a Ca 2F -dependent manner. The crystal structure of synC in complex with ATPgS and Ca 2F explains the preference of synC for Ca 2F over Mg 2F . Our results suggest that synapsins may also be ATP-utilizing enzymes.

The effects of silyl and hydrophilic groups on the photodynamic properties of tetraphenylporphyrin (TPP) derivatives have been studied in vitro and in vivo. Silylation led to an improvement in the quantum yield of singlet oxygen sensitization for both sulfo and carboxy derivatives, although the silylation did not affect other photophysical properties. Silylation also improved the cellular uptake efficiency for both sulfo and carboxy derivatives, enhancing the in vitro photodynamic activity of the photosensitizer in U251 human glioma cells. The carboxy derivative (SiTPPC4 ) was found to show higher cellular uptake efficiency and in vitro photodynamic activity than the corresponding sulfo derivative (SiTPPS4 ), which indicates that the carboxy group is a more promising hydrophilic group than the sulfo group in the silylated porphyrin. SiTPPC4 was found to show high selective accumulation efficiency in tumors, although almost no tumor selectivity was observed for the nonsilylated porphyrin. The concentration of SiTPPC4 in tumors was 13 times higher than that in muscle 12 h after drug administration. We also studied tumor response after treatment and found that silylation enhanced in vivo photodynamic activity significantly. SiTPPC4 shows higher photodynamic activity than NPe6 with white light irradiation.

Quartz crystals synthesized hydrothermally above the high-low transition temperature showed various hexagonal dipyramidal morphologies mostly with and rarely without prism faces, which are sometimes curved leading to a spindle-shape. The development of prism faces is distinctly different from simple hexagonal dipyramidal morphology without prism faces commonly exhibited by natural high quartz crystals. Thus the hitherto believed representative morphology of natural high quartz is a habit appearing when high quartz crystals grew in silicate systems.

We have recently reported that a red-emitting iridium complex (btp) 2 Ir (acac) (BTP) serves as a hypoxia-sensing probe for tumor imaging in living mice. BTP exhibits oxygen-sensitive phosphorescence that can be utilized to monitor oxygen levels in living cells and to visualize hypoxic tissues. To improve the tissue penetrance of BTP, we designed and synthesized near-IR emitting iridium complexes by two different approaches: extension of the π- conjugated system of benzothienyl-pyridinato ligand in BTP and introduction of substituents into suitable positions of ligands. The former approach was successful, and near-IR emitting iridium complexes were obtained without reduction in the emission quantum yield. Cellular uptake of BTP was greatly improved by introducing a hydrophilic group into the acetylacetonato ligand. Using these improved probes, in-vivo lifetime measurements were made to substantiate the hypoxia of tumor tissues in SCC-7 tumor-bearing mice. The second-harmonic (532 nm) of Nd 3+ :YAG laser was used to excite iridium complexes in tissues, and the phosphorescence lifetime was measured using the time-correlated single photon counting technique. The phosphorescence emitted from the tumor region actually gave longer lifetimes compared to those emitted from the normal tissues, demonstrating the hypoxic nature of tumor tissues.

We describe here the isolation and characterization of cDNAs from mouse brain coding for the precursor of substance P and neurokinin A, preprotachykinin A (PPT-A), and for the precursor of neurokinin B, preprotachykinin B (PPT-B). Two types of mouse PPT-A cDNA encoding β-PPT-A and γ-PPT-A were obtained. The latter lacked the coding region corresponding to the exon 4 of the PPT-A gene, as is observed in other species. Mouse β-PPT-A deduced from the cDNA sequence consisted of 130 amino acids, and showed extensive homology to rat, human, and bovine β-PPT-As, with 99%, 95%, 92% amino acid identities, respectively. The substance P and neurokinin A sequences were the same among these species

Iridium complex is a promising organic light–emitting diode material for next generation video displays that emits phosphorescence quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light–emitting Ir(btp)2(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor-1α images. BTP was further applied to imaging five nude mouse transplanted with tumors. All tumors presented a bright BTP-emitting image even 5 minutes after injection. The minimal image recognition size was ∼2 mm in diameter. By morphologic examination and phosphorescence lifetime measurement, BTP appeared to localize to the tumor cells. Because BTP is easily modifiable, we synthesized BTP analogues with a longer excitation/emission wavelength. One of them, BTPHSA, depicted clear imaging from tumors transplanted 6 to 7 mm deep from the skin surface. We suggest that iridium complex materials have a vast potential for imaging hypoxic lesions such as tumor tissues. Cancer Res; 70(11); 4490–8. ©2010 AACR.

Iridium complex, a promising organic light-emitting diode material for next generation television and computer displays, emits phosphorescence. Phosphorescence is quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting iridium complex Ir(btp)2(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor (HIF)-1 expression. BTP was further applied to imaging five nude mouse-transplanted tumors. All tumors presented a bright BTP-emitting image as early as 5 min after the injection. The BTP-dependent tumor image peaked at 1 to 2 h after the injection, and was then removed from tumors within 24 h. The minimal BTP image recognition size was at least 2 mm in diameter. By morphological examination and phosphorescence lifetime measurement, BTP is presumed to localize to the tumor cells, not to stay in the tumor microvessels by binding to albumin. The primary problem on suse of luminescent probe for tumor imaging is its weak penetrance to deep tissues from the skin surface. Since BTP is easily modifiable, we made BTP analogues with a longer excitation/emission wavelength to improve the tissue penetrance. One of them, BTPHSA, displayed 560/720 wavelength, and depicted its clear imaging from tumors transplanted over 6-7 mm deep from the skin surface. We suggest that BTP analogues have a vast potential for imaging hypoxic lesions such as tumor tissues.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Iridium complexes exhibit highly-efficient phosphorescence that is quenched notably by ambient molecular oxygen. We utilized the oxygen-sensitive properties of the phosphorescence to visualize tumors in vivo because the levels of oxygen in tumors may be significantly below those of normal tissue. We used (btp) 2 Ir(acac) (BTP) as an oxygen probe because the phosphorescence of BTP appears in the red to near-infrared region and has a relatively long lifetime (5.8μs) and high quantum yield (0.32) in solution. The oxygen-quenching rate constants were determined to be 5.7 x 10 4 and 1.2 x 10 4 mmHg -1 s -1 in n -hexane and in lipid bilayer of DMPC in Tris-HCl buffer solution, respectively, at 35°C. We took the phosphorescence image of HeLa cells that had been incubated under 5% and 20% O 2 conditions. In 20% O 2 culture condition, HeLa cells did not exhibit notable phosphorescence, while in 5% O 2 culture condition, they emitted bright red phosphorescence due to BTP. Then we tested this probe to image tumors transplanted in nude mice. After 5 minutes of the BTP injection, tumor moieties began to emit red phosphorescence and after one hour each tumor was visualized very clearly by the BTP phosphorescence that could be seen only in a low oxygen state.

Raman spectra of the SiO 2 -NaOH-H 2 O system solution that was prepared by a reaction of 1 NaOH solution with SiO 2 under the conditions of hydrothermal growth of Δ-quartz were measured in the range of room temperature to 320°C for the spectral region of about 50–4000 cm -1 by use of an autoclave ( V =0.50 cm 3 ) with sapphire windows. The solution, with a mole ratio of Na 2 O to SiO 2 of 0.56, contains monomers, dimers, trimers and polysilicic ions, etc., and has at Raman spectrum peaks at 175, 430 b , 600 b , 780, 1035, 1645, 3200 and 3400 cm -1 at room temperature. The Raman spectrum peaks 780 and 1035 cm -1 were observed at 300°C.

Iridium complex, a promising organic light-emitting diode for next generation television displays, emits phosphorescence. Phosphorescence is quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting iridium complex Ir(btp)~2~(acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with HIF-1[alpha] expression. BTP was further applied to imaging five tumors (four from human origin and one from mouse origin) transplanted in athymic mice. All tumors presented a bright BTP-emitting image even 5 min after the injection. The BTP-dependent tumor image peaked at 1 to 2 h after the injection, and was then cleared from tumors within 24 h. The minimal BTP image recognition size was 3 to 4 mm in diameter. Compared with ^18^F-FDG/PET images, BTP delineated a clearer image for a tumor profile. We suggest that iridium complex has a vast potential for imaging hypoxic lesions such as tumor tissues.

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy.

Novel fluorescent cholesterol derivatives bearing dimethylsilylated fluorescent molecules were prepared. The derivatives exhibit markedly enhanced fluorescence compared to that of an analogous deri...

Micro α-quartz crystals were synthesized in 1N NaOH, 1N Na 2 CO 3 or sodium formate at a temperature of 300°C by hydrothermal hot-press method.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.

We have cloned and sequenced a mouse cDNA encoding the 793-residue amino acid sequence of furin, which is a protein homologous to the yeast Kex2 protease. The entire sequence is 94% identical to that of human furin, and it contains the 289-residue sequence of the subtilisin-like catalytic domain. Within this region, 99, 64, and 53% of the amino acids are identical to those of human furin, human PC2 (the other mammalian Kex2-like protein), and yeast Kex2, respectively. It has been proposed that furin is a mammalian prohormone processing enzyme which cleaves precursors at paired basic amino acids, based on the fact that the Kex2 protease is responsible for processing of alpha-mating factor and killer toxin precursors at dibasic sites. However, Northern blot analysis has revealed that a furin mRNA transcript is present in all tested mouse tissues and culture cell lines, including those known not to process prohormones. Moreover, when furin and a prohormone, prorenin, have been coexpressed in mammalian cells by DNA transfection, no processing has been observed. These observations fail to show a role for furin, a Kex2-like mammalian protease, in prohormone processing.

Under low degree of filling of 50% or less, quartz crystals could be grown hydrothermally in NaCl and KCl solutions by a reversed gradient method whereby the nutrient was placed at the low temperature side of 440°C and the seed crystals were at the high temperature side of 500°C. The growth rate for each seed was the fastest at 30% fill, but was extremely slow at filling of 50 and 10%.

It has been shown that quartz crystals can be grown in NaCl solution by the hydrothermal temperature gradient method, and it was possible to produce large crystals after a few months. The effect of the various experimental conditions on the growth rates was investigated and observations of morphology and infrared absorption spectra were made. Two of the outstanding differences between quartz crystals grown in NaCl solution and those grown in alkaline solutions were that contrary to NaOH in NaCl striations were observed on the prism face and that the relative growth rates for the R and r faces were R > r.

Natural quartz grows from solutions containing mainly Na+, K+ and Cl− ions. In this experiment, the growth of synthetic quartz under the same growth conditions as those of natural quartz was attempted using NaCl and KCl solutions as a growth solution. As a result, colored quartz equivalent in quality and color tone to natural amethyst and citrine was obtained. The quartz grown from r-cut and R-cut plate seeds with iron added to the growth solution was colorless but γ-ray irradiation produced a purplish tint. The quartz grown from X-cut plate and Y-bar seeds had a yellowish tint. Although the depth of color increased in proportion to the amount of iron, it reached close to saturation with an iron concentration of approximately 90 μg/ml solvent.

Vapor phase reactions in the systems In-InCl3 and Ga-GaCl3 have been investigated by means of vapor pressure measurments and a high temperature spectroscopic technique. It was found that the vapor phases of indium and gallium dichloride existed in the forms of In2Cl4 (g) and Ga2Cl4 (g) respectively which had been overlooked in the previous theoretical studies of vapor phase chemical transport. These dichlorides are disproportionate to trichloride and monochloride according to the following reactions: In2Cl4 (g) ⇆ ⇆InCl3 (g)+InCl (g) and Ga2Cl4 (g) ⇆ GaCl3(g)+GaCl(g). From the present work, the temperature dependences of the equilibrium constants for the above reactions are shown to be log K p atm = -7.463×10 3 T +6.78 and log K p atm = -5.100×10 3 T+6.30 . In the absorption spectra for the disproportionation of GaCl (g), absorption bands for Ga2Cl4 (g) were observed besides the bands for GaCl3 (g) and GaCl (g). Therefore, considering the disproportionation of Ga2Cl4 (g), the temperature dependence of equilibrium constants for the reaction, 2 Ga (1)+GaCl3 (g)⇆3 GaCl (g), comes out to be log K p atm = -10.541×10 3 T +12.49 .

The process of variation of growth patterns on the prism (m) faces of synthetic quartz from striations to pyramid-shaped overgrowth was studied by changing the concentration of the NaOh and Na2CO3 solutions respectively in a NaCl solution or in pure H2O. When the concentrations of the added alkaline solutions were extremely low, the striations were obtained on the m face. These striations were of the same type, which can be seen on crystals grown in a NaCl solution or in pure H2O. It is considered that the anisotropy due to the PBC vectors affects the growth of striations and causes faster growth in the X[11.0] direction in comparison with the c[00.1] direction.

Semi-transparent rose-colored quartz crystal could be obtained successfully by heating a quartz crystal grown hydrothermally with titanium added.