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The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.

ABSTRACTNumerous cell adhesion molecules, extracellular matrix proteins and axon guidance molecules participate in neuronal network formation through local effects at axo-dendritic, axo-axonic or dendro-dendritic contact sites. In contrast, neurotrophins and their receptors play crucial roles in neural wiring by sending retrograde signals to remote cell bodies. Semaphorin 3A (Sema3A), a prototype of secreted type 3 semaphorins, is implicated in axon repulsion, dendritic branching and synapse formation via binding protein neuropilin-1 (NRP1) and the signal transducing protein PlexinAs (PlexAs) complex. This review focuses on Sema3A retrograde signaling that regulates dendritic localization of AMPA-type glutamate receptor GluA2 and dendritic patterning. This signaling is elicited by activation of NRP1 in growth cones and is propagated to cell bodies by dynein-dependent retrograde axonal transport of PlexAs. It also requires interaction between PlexAs and a high-affinity receptor for nerve growth factor, toropomyosin receptor kinase A. We propose a control mechanism by which retrograde Sema3A signaling regulates the glutamate receptor localization through trafficking of cis-interacting PlexAs with GluA2 along dendrites; this remote signaling may be an alternative mechanism to local adhesive contacts for neural network formation.

Introduction:The semaphorins were initially described as axon guidance molecules that play important roles in the development of nervous system. Recent studies suggest that semaphorins and their receptors also exert such diverse functions as immune response, control of vascular endothelial cell motility and invasion of many types of cancer cells.Areas covered:The available results concerning application of class 3 semaphorins and their inhibitors for the treatment in animal disease models.Expert opinion:Because semaphorins are now recognized as key players in immune, cardiovascular, bone metabolism and neurological system, semaphorins and their receptors are most promising therapeutic targets for various disease states. As semaphorins exert their diverse or even opposing activities in vivo,more elaborate studies on pathophysiology and signal transduction mechanisms of semaphorins are required.

Abstract: In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays.

We have developed a new methodology to fabricate high-resolution DNA microarrays (2500 dots/cm2) in combination with microcontact printing (CP) and surface plasmon resonance (SPR) imaging. A novel COOH-terminated PEG-disulfide with para-carborane (p-carborane) was synthesized as an initiator chemically bound to a gold substrate for surface coupling reactions with NH2-terminated DNA. The hybridization between target DNA immobilized on gold nanoparticles and probe DNA arrayed on flat gold substrates was successfully demonstrated by SPR imaging, where nonspecific adsorption was not observed at the array background. The gold nanoparticles on the array give quite high contrast even at low surface coverage with gold nanoparticles (∼10%) by the enhancement effect of optical signals based on nanoscale phenomena. This enhancement effect can be well described by the simulation based on the Maxwell-Garnett theory for the effective dielectric constants and Fresnel's equation.

The oriented structure of acridine orange (AO) in both monolayer and Langmuir–Blodgett (LB) film has been studied by optical waveguide (OWG) spectroscopy using polarized incident light. Mixed monolayer and LB films, consisting of octadecyl acridine orange (C18‐AO) incorporated in stacked base pairs of octadecyl adenine (C18‐Ade) and octadecyl thymine (C18‐Thy), were prepared on a quartz waveguide. Absorption of transverse electric field (TE) polarized light was about twice that of transverse magnetic field (TM) polarized light. Both OWG spectra have λmaxat 500 nm, which is characteristic of monomeric AO molecules. This result strongly suggests that C18‐AO molecules were dispersed uniformly in the mixed monolayer and were excited more effectively by the TE polarized light. Since the absorption moment of AO molecules is related to their long axis, it is proposed that C18‐AO molecules are incorporated in C18‐Ade/C18‐Thy pairs with the long axis parallel to the layer surface. The absorbance at 500 nm was proportional to the number of layers on the waveguide. The dichroic ratio of the absorbance at 500 nm for TE polarized light to that for TM polarized light was constant regardless of the number of layers. The C18‐AO molecules were uniformly incorporated in each layer with the long axis relatively parallel to the layer surface. Copyright © 2004 John Wiley & Sons, Ltd.

A simple conventional method of immunohistochemistry (i.e. fixing the frozen sections in cold methanol) was used to determine the immunolocalization of cellular prion protein (PrPc), with good results. In the rat cerebrum, the cytoplasm of neural cells in the cortex and corpus stratum, pia mater, membrane limitans gliae superficialis, choroid plexus and blood vessel wall were immunostained. The formation of network structures of immunostained neural and/or glial fibers in the cerebral cortex was also observed. These immunostained network structures of neural and/or glial fibers were also observed in cultured neural cells. The results suggest that fixation of frozen sections and cultured cells with cold methanol is a useful method for detecting the immunolocalization of PrPc and that PrPc exists in the various components of the central nervous system of the rat.

We prepared self-assembled monolayers (SAMs) composed of tetrathiafulvalene (TTF) derivative modified on gold substrate and investigate their electrochemical properties by cyclic voltammogram (CV). The first and second oxidation peaks were observed at E 1 1/2 =0.40 V and E 2 1/2 =0.81 V, respectively. The peak current was observed to be proportional to the scan rate, suggesting the feature of modified SAMs molecules. The result indicates that TTF-thiolate SAMs on gold electrode have electronic structure similar to bulk TTF molecules.

Dialkyl sulfide self-assembled monolayers on gold were characterized by Fourier transform infrared reflective absorption spectroscopy (FTIR-RAS) and scanning tunneling microscopy (STM). It was found that dialkyl sulfide SAMs have quite different surface characteristics compared to alkanethiol SAMs. Our STM result clearly elucidated that dialkyl sulfide SAMs are composed of the liquid-like disorder phase with many small nucleations, not the ordered phase.

To realize an efficient hybridization of polynucleotides, self-assembled monolayer (SAM) containing pyrene groups was prepared on a gold substrate because pyrene molecules have an affinity for nucleic acid bases through van der Waals interaction. The SAM was prepared from 11-(1-pyrene)-1-undeacathiol and 11-mercapto-1-undecanol mixed solution. The mixed SAM gave an efficient hybridization of polyadenylic acid and polyuridylic acid observed by surface plasmon resonance.

AbstractTwo types of multifunctional thin films based on DNA polyion complex intercalated with photofunctional chromophore were fabricated on the 2D-surface. One is monolayer of DNA polyion complex transferred to a glass substrate at the air/water interface, and is intercalated with acridine orange (AO). Another is DNA containing thiol moiety, and is intercalated with AO and formed polyion complex with lipid, and is fabricated on a gold substrate at the solid/liquid interface. Molecular orientation of AO was investigated by surface plasmon resonance, polarized UV and FT-IR spectroscopy. AO intercalated DNA polyion complex film (a) was aligned perpendicular to the DNA long axis. In the case of DNA polyion complex film (b), orientated DNA could be fabricated on the gold substrate through S-Au interaction, and the orientation of DNA was more stable by mixing with lipid to provide polyion complex. AO in DNA polyion complex film (b) was aligned parallel to the substrate.

AbstractWe synthesized an unsymmetric disulfide containing an anthryl group, which can form a self-assembled monolayer (SAM) on a gold surface, to immobilize DNA onto the gold surface through the intercalation between DNA and anthryl groups. A surface plasmon resonance (SPR) measurement indicates that the SAM can detect DNA efficiently at a liquid-solid interface.

AbstractSelf-assembled monolayer of mesoscopic size with Tetrathiafuluvalene (TTF) skeleton has attracted because it is expected to have quantum size effect for perpendicular to the substrate as well as two-dimensional π-electron interaction. Surface plasmon resonance (SPR) revealed that TTF-CH2SH monolayer is formed on Au (111) only at proper concentration. The presence of etch pits which is characteristic to chemisorbed SAMs was confirmed by scanning tunneling microscopy (STM). From those SPR and STM results, it has been suggested that the molecular long axis of TTF-CH2SH was aligned nearly perpendicular to the substrate. In addition, cyclic voltammogram (CV) result indicates that TTF-thiolate SAMs on gold electrode are stable in repeated scans.

AbstractWe attempted the immobilization of DNA onto self-assembled monolayer (SAM) which contains the intercalated for DNA. Anthryl moieties were attached to the terminal OH groups in SAM of 1-Mercapt-11-undecanol (MU-SAM) by esterification. In the case of the SAM containing anthryl moieties (Anth-SAM), surface plasmon resonance measurement indicates that the adsorption of DNA onto the SAM was observed in an aqueous DNA solution, and the DNA remained on the surface even after rinsing. Although the adsorption of DNA onto MU-SAM was also observed, little DNA remained on the SAM after rinsing. These results indicate that the anthracene which was attached to the SAM can interact with DNA because the binding ability was increased by introduction of anthracene moieties to the SAM.

The semaphorin family of proteins constitute one of the major cues for axonal guidance. The prototypic member of this family is Sema3A, previously designated semD/III or collapsin-1. Sema3A acts as a diffusible, repulsive guidance cue in vivo for the peripheral projections of embryonic dorsal root ganglion neurons. Sema3A binds with high affinity to neuropilin-1 on growth cone filopodial tips. Although neuropilin-1 is required for Sema3A action, it is incapable of transmitting a Sema3A signal to the growth cone interior. Instead, the Sema3A/neuropilin-1 complex interacts with another transmembrane protein, plexin, on the surface of growth cones. Certain semaphorins, other than Sema3A, can bind directly to plexins. The intracellular domain of plexin is responsible for initiating the signal transduction cascade leading to growth cone collapse, axon repulsion, or growth cone turning. This intracellular cascade involves the monomeric G-protein, Rac1, and a family of neuronal proteins, the CRMPs. Rac1 is likely to be involved in semaphorin-induced rearrangements of the actin cytoskeleton, but how plexin controls Rac1 activity is not known. Vertebrate CRMPs are homologous to the Caenorhabditis elegans unc-33 protein, which is required for proper axon morphology in worms. CRMPs are essential for Sema3A-induced, neuropilin–plexin-mediated growth cone collapse, but the molecular interactions of growth cone CRMPs are not well defined. Mechanistic aspects of plexin-based signaling for semaphorin guidance cues may have implications for other axon guidance events and for the basis of growth cone motility. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 219–229, 2000

Toward the functional application of DNA as novel molecular devices, we have immobilized DNA as two-dimensional molecular assemblies by means of specific intermolecular interaction at the air-water interface. Preparation of DNA-mimetics at the air-water interface is briefly shown in the second part of this paper. The final part describes micropattern formation of DNA assemblies by using a novel lithography-free technique based on a general physical phenomenon. Mesoscopic two-dimensional patterns of DNA assemblies are prepared by freezing of dissipative structures formed in casting processes of DNA solution.

Collapsin-1/SemaIII, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cell-surface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose–response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1–alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 579–589, 1999

Summary: Hemipituitary glands from 30 rats were isotransplanted under renal capsules. At 1, 2, 4, 7 and 20 days after transplantation, the grafts were examined by light and electron microscopy. Two days after transplantation, the central area of graft showed necrosis; however, the peripheral area, where marginal cells of Rathke's cleft were ingesting the remnants of necrotic glandular cells, survived. Four days after transplantation, mitotic figures of marginal cells were observed. Seven days after transplantation, the damaged area of the graft disappeared, and Rathke's cleft was completely lined by marginal cells. Remnants of necrotic glandular cells were not seen in intercellular spaces or in the cytoplasm of marginal cells. Cystlike structures formed in the grafts; some were connected to Rathke's cleft by narrow cavities. The cavity-lining cells of the cysts were agranular and similar to those that lined Rathke's cleft. At 20 days after transplantation, granular cavity-lining cells appeared. It is suggested that marginal cells of immature rats can differentiate into granular cells.

The α subunits of Gqfamily G proteins, GL1α(G14α), GL2α(G11α), and Gqα were expressed with G protein β1and γ2subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1(GL1αβγ), GL2(GL2αβγ), and Gq(Gqαβγ), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gqactivated phospholipase C-β purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding to GL1, GL2, and Gqin the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-β were reconstituted in lipid vesicles, each subtype of Gqfamily G proteins mediated the activation of phospholipase C-β by carbamylcholine in the presence of either 1 μM GTPγS or 1 mM GTP. Phospholipase C-β stimulated the GTPase activity of GL1, GL2, and Gqin the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of Go. Protein kinase C phosphorylated m1 receptor and phospholipase C-β, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-β in the reconstitution system of purified proteins.

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). Here, semD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus– (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, we show that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1, the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, μ) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein that in turn transduces a semD signal into axon repulsion.

Summary: A certain kind of cell in the pituitary gland exhibited immunoreactive keratin and dome formations in vitro. We obtained epithelial cells, which were able to subculture, from the outgrowth of anterior pituitary organ cultures. These cells lacked hormone secretory granules and exhibited immunoreactive keratin. Furthermore, they produced dome formations or cystic structures in monolayer culture and under three-dimensional culture condition using type I collagen gel. Dome formation was stimulated by dibutyryl cyclic AMP (dbcAMP, 10−3 to 10−5 M). Their responsiveness to dbcAMP is similar to that of several other epithelial cells that possess transport functions in vivo and in vitro. Although the origin of our cultured cells is unknown, these cells formed dome formations that possessed transport function and were related to cystic structures in the pituitary gland in vivo.

An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's “corticotrophs” of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100–200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140–260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus.

In the present hearing tests in which a tuning fork or an audiometer is used, the response of the patient is the only indicator and there is no alternative but to depend upon his answer for the decision of confidence limit. Thus these hearing tests can be used only for those who are old enough to answer accurately and have normal mentality and intellect. In our daily hearing tests we experience the grestest difficulty in the case of very young children, who cannot make their sound perception known by answering questions accurately. Even some adults are not intelligent enough to answer correctly or, for some reason, try to feign illness. Under these circumstances it becomes necessary to use so-called objective audiometry. If the tester could eliminate the element of subjectivity in the response of the patient, and if he could obtain exact results by using as indicators reflexes and movements

Pulmonary hypertension (PH) is a severe and progressive disease that causes elevated right ventricular systolic pressure, right ventricular hypertrophy and ultimately right heart failure. However, the underlying pathophysiologic mechanisms are poorly understood. We previously showed that 3,4-L-dihydroxylphenyalanine (DOPA) sensitizes vasomotor response to sympathetic tone via coupling between the adrenergic receptor alpha1 (ADRA1) and a G protein-coupled receptor 143 (GPR143), a DOPA receptor. We investigated whether DOPA similarly enhances ADRA1-mediated contraction in pulmonary arteries isolated from rats, and whether GPR143 is involved in the PH pathogenesis. Pretreating the isolated pulmonary arteries with DOPA 1 μM enhanced vasoconstriction in response to phenylephrine, an ADRA1 agonist, but not to U-46619, a thromboxane A2 agonist or endothelin-1. We generated Gpr143gene-deficient (Gpr143-/y) rats, and confirmed that DOPA did not augment phenylephrine-induced contractile response in Gpr143-/yrat pulmonary arteries. We generated a rat model of monocrotaline (MCT)-induced PH. In the MCT model, the right ventricular systolic pressure was attenuated in the Gpr143-/yrats than in WT rats. Phenylephrine-induced cell migration and proliferation were also suppressed in Gpr143-/ypulmonary artery smooth muscle cells than in WT cells. Our result suggests that GPR143 is involved in the PH pathogenesis in the rat models of PH.

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre(KC-Crmp4wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/−(KC-Crmp4knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4WT mice was higher than that in KC-Crmp4KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.

Novel disulfide-derivatized stable p-radical was prepared and it was found to form a dense self-assembled monolayer of p-radical thiols on a gold surface through a reductive cleavage of the S-S bond.

Repulsive guidance signaling triggers the collapse of neuronal growth cones through a mechanism involving oxidation and phosphorylation.

The present study was performed to analyse the structure of non-digested and digested collagen type I molecules by atomic force microscopy (AFM). Collagen type I molecules from the bovine skin were diluted with 0.05 N acetic acid, spread on a mica plate, air-dried and observed by non-contact mode AFM in air. Collagen molecules digested with Clostridium histolyticum collagenase were also examined by AFM. Intact collagen type I molecules were observed as twisted threads ranging mainly between 280 and 310 nm in length. The surface of the molecules was uneven and both ends usually slightly bulged like a globule. Depressions on the molecules were found throughout the length, and were most prominent −70 nm from one end of the molecules. The collagenase-treated collagen molecules were degraded into fragments with various lengths, which corresponded to the data from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The end of these fragments often appeared like a tuft, suggesting that the triple-helix unraveled at these regions.