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This study was designed to determine the effects and underlying mechanism of honokiol (HNK) on traumatic brain injury (TBI). A rat TBI model was constructed using the modified Feeney free-fall percussion method and treatment with HNK via intraperitoneal injection. The brain tissues of the rats in each group were assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect the level of neuronal apoptosis. Western blots were used to detect the expression levels of apoptosis-related proteins (Bcl-2 and Bax), and ELISAs were used to measure the levels of pro-inflammatory cytokines (IL-18 and IL-1β) and the activity of caspase-1. In addition, the mitochondrial membrane potential, reactive oxygen species (ROS), and adenosine 5'-triphosphate (ATP) were also measured. Western blots and qRT-PCRs were used to determine the relative expression levels of the mitochondrial unfolded protein response (UPRmt)-related proteins and mRNAs. Based on the experimental results, treatment with HNK was associated with a decrease in the number of TUNEL-positive cells, downregulated Bax expression levels, elevated Bcl-2 expression levels, and inhibition of neuronal apoptosis in the brain tissue of TBI rats. HNK also suppressed neuroinflammation by decreasing IL-1β and IL-18 levels and caspase-1 activity. Additionally, HNK lowered the mitochondrial membrane potential and ROS levels, increased ATP levels, and improved mitochondrial dysfunction in neural cells. Furthermore, in the investigation of the mechanism of HNK on TBI, we observed that HNK could activate UPRmt by upregulating the mRNA and protein expression levels of HSPA9, CLPP, and HSP60 in the brain tissues of TBI rats. Collectively, HNK reduced mitochondrial dysfunction, inhibited the apoptosis of nerve cells, and attenuated inflammation in the brains of TBI rats. The protective effect of HNK may be achieved through the activation of UPRmt.

We designed, assembled, and tested a reliable laser system for 87Rb cold atom fountain clocks. The laser system is divided into four modules according to function, which are convenient for installing, adjusting, maintaining, and replacing of the modules. In each functional module, all optical components are fixed on a baseplate with glue and screws, ensuring the system’s structural stability. Mechanical stability was verified in a 6.11g RMS randomvibration test, where the change in output power before and after vibration was less than 5%. Thermal stability was realized by optimizing of the structure and appropriate selection of component materials of the modules through thermal simulation. In the laser splitting and output module, the change in laser power was less than 20% for each fiber in thermal cycles from 5 °C to 43 °C. Finally, the functionality of the laser system was verified for a rubidium fountain clock.

To analyze the endogenous metabolic biomarkers and pathways in serum involving acupuncture-induced improvement of symptoms of patients with rheumatoid arthritis (RA) by using metabolomics technique.A total of 30 RA patients who were treated in the Department of Acupuncture and Moxibustion of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (from January 2018 to October 2018) were selected in the present study. They were randomly and equally divided into acupuncture group and medication group. Acupuncture (lifting-thrusting reinforcing and uniform reducing-reinforcing methods) was applied to bilateral Fengchi (GB20), Yangchi (TE4), Quchi (LI11), Yingu (KI10), Taixi (KI3), Xuehai （SP10）, Guanyuan (CV4), Yanglingquan (GB34) and Ashi-points for 20 min every time, once daily for 3 months. Patients of the medication group were asked to take Tripterygium Wilfordii Polyglycoside tablets (a positive drug for RA, one tablet per time, 3 times a day) for 3 months. Other 10 healthy volunteers were selected as the normal control group. The tenderness scale (0-4 points) and swelling scale (0-3 points) and morning stiffness time were recorded, and serum rheumatoid factor (RF), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were detected for analyzing pharmacodynamic effects. Serum samples were captured for profiling and quantifying metabolite biomarkers by using GC-MS (gas chromatography-mass spectrometry) technique. The acquired metabolite profiles were processed (multivariable data analysis) by using ProteoWizard package, XCMS Online software and SIMCA 13.0 software, respectively, followed by screening differential metabolites according to variable importance projection (VIP) and by constructing metabolic pathways with MetaboAnalyst 4.0.After acupuncture treatment, the tenderness score, swelling score, morning stiffness time, serum RF and CRP contents were significantly decreased in both acupuncture and medication groups in comparison with their own pretreatment (P0.05). No significant differences were found between acupuncture and medication groups in the levels of tenderness score, swelling score, morning stiffness time, serum RF and CRP contents (P0.05). A total of 14 differential metabolites including citrate, creatine, 3-hydroxybutyrate arachidonic acid, arachidonic acid, valine, lactic acid and palmitic acid (up-regulated), and tryptophan, arginine, L-phenylalanine, glucose, glycine, glutamine, aspartic acid and (down-regulated) which mainly involve metabolic pathways of alanine, aspartic acid and glutamate; metabolism of phenylalanine, tyrosine and tryptophan; metabolism of glycine, serine and threonine; glyoxalic acid dicarboxylic acid metabolism; metabolism of starch and sucrose; and metabolism of phenylalanine and arachidonic acid, respectively.Citrate, creatine, arachidonic acid, valine and glucose was positively correlated with tenderness index, swelling index, morning stiff time, RF, CRF and ESR. Glycine, L-phenylalanine , glutamine was negatively correlated with efficacy indicators.Acupuncture can relieve symptoms of patients with RA, which may be related to its effects in improving amino acid metabolism and glucose metabolism.

The sigma-1 receptor, which is expressed throughout the brain, provides physiological benefits that include higher brain function. The sigma-1 receptor functions as a chaperone in the endoplasmic reticulum and may control cell death and regeneration within the central nervous system. Cutamesine (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine dihydrochloride) is a ligand selective for this receptor and may mediate neuroprotective effects in the context of neurodegenerative disease. We therefore assessed whether cutamesine protects the inner ear from noise-induced or aging-associated hearing loss. Immunohistochemistry and Western blotting revealed that the sigma-1 receptor is present in adult cochlea. We treated mice with 0, 3, or 30 mg/kg cutamesine from 10 days before noise exposure until the end of the study. All subjects were exposed to a 120-dB, 4-kHz octave-band noise for 2 hr. We assessed auditory thresholds by measuring the auditory-evoked brainstem responses at 4, 8, and 16 kHz, prior to and 1 week, 1 month, or 3 months following noise exposure. For the aging study, measurements were made before treatment was initiated and after 3 or 9 months of cutamesine treatment. Damage to fibrocytes within the cochlear spiral limbus was assessed by quantitative histology. Cutamesine significantly reduced threshold shifts and cell death within the spiral limbus in response to intense noise. These effects were not dose or time dependent. Conversely, cutamesine did not prevent aging-associated hearing loss. These results suggest that cutamesine reduces noise-induced hearing loss and cochlear damage during the acute phase that follows exposure to an intense noise.

Fibrocytes in the cochlear lateral wall and spiral limbus play an important role in transporting K(+) and have the capacity of self-renewal. We showed that acute energy failure in the rat cochlea induced by local administration of the mitochondrial toxin 3-nitropropionic acid (3NP) caused hearing loss in a concentration-dependent manner, mainly due to degeneration of cochlear fibrocytes. We produced long-lasting profound cochlear damage in this model by modifying the 3NP administration protocol and observed morphological changes at 16 weeks after the administration. In the spiral ligament, severe degeneration of fibrocytes was observed in the basal turn, and the levels of the Na,K-ATPase alpha and beta1 subunits and of NKCC1 were decreased in these cells, whereas connexin 26 (Cx26) level increased in the type 1 fibrocytes adjacent to the stria vascularis. In the stria vascularis, levels of Kir4.1 and L-PGDS decreased. In the spiral limbus, severe degeneration of fibrocytes was observed in the middle and basal turns, but NKCC1 and Cx26 were still found in the center of the limbus in the middle turn. These results indicate long-lasting changes in the cochlear lateral wall and spiral limbus, which may compensate for damaged K(+) recycling and protect cells from ATP shortage.

Spiral ligament fibrocytes (SLFs) play an important role in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Our previous study showed that transplantation of mesenchymal stem cells (MSCs) into the inner ear of rats with damaged SLFs significantly accelerates hearing recovery compared with rats without MSC transplantation. To elucidate this mechanism of SLF repair and to determine the contribution of transplanted MSCs in this model, we investigated the mutual effects on differentiation and proliferation between MSCs and SLFs in a coculture system. Factors secreted by SLFs had the ability to promote the transdifferentiation of MSCs into SLF-like cells, and the factors secreted by MSCs had a stimulatory effect on the proliferation of SLFs. Cytokine antibody array analysis revealed the involvement of transforming growth factor-β (TGF-β) in SLF proliferation induced by MSCs. In addition, a TGF-β inhibitor reduced SLF proliferation induced by MSC stimulation. Our results suggest that there are two mechanisms of hearing recovery following transplantation of MSCs into the inner ear: 1) MSCs transdifferentiate into SLF-like cells that compensate for lost SLFs, and 2) transplanted MSCs stimulate the regeneration of host SLFs. Both mechanisms contribute to the functional recovery of the damaged SLF network. © 2012 Wiley Periodicals, Inc.

Propolis, a traditional medicine, has been widely used for a thousand years as an anti-inflammatory and antioxidant drug. The flavonoid fraction is the main active component of propolis, which possesses a wide range of biological activities, including activities related to heart disease. However, the role of the flavonoids extraction from propolis (FP) in heart disease remains unknown. This study shows that FP could attenuate ISO-induced pathological cardiac hypertrophy (PCH) and heart failure in mice. The effect of the two fetal cardiac genes, atrial natriuretic factor (ANF) andβ-myosin heavy chain (β-MHC), on PCH was reversed by FP. Echocardiography analysis revealed cardiac ventricular dilation and contractile dysfunction in ISO-treated mice. This finding is consistent with the increased heart weight and cardiac ANF protein levels, massive replacement fibrosis, and myocardial apoptosis. However, pretreatment of mice with FP could attenuate cardiac dysfunction and hypertrophyin vivo. Furthermore, the cardiac protection of FP was suppressed by the pan-PI3K inhibitor wortmannin. FP is a novel cardioprotective agent that can attenuate adverse cardiac dysfunction, hypertrophy, and associated disorder, such as fibrosis. The effects may be closely correlated with PI3K/AKT signaling. FP may be clinically used to inhibit PCH progression and heart failure.

The regeneration of lens tissue from the iris of newts has become a classical model of developmental plasticity, although little is known about the corresponding plasticity of the mammalian iris. We here demonstrate and characterize multipotent cells within the iris pigment epithelium (IPE) of postnatal and adult rodents. Acutely-isolated IPE cells were morphologically homogeneous and highly pigmented, but some produced neurospheres which expressed markers characteristic of neural stem/progenitor cells. Stem/progenitor cell markers were also expressed in the IPE in vivo both neonatally and into adulthood. Inner and outer IPE layers differentially expressed Nestin (Nes) in a manner suggesting that they respectively shared origins with neural retina (NR) and pigmented epithelial (RPE) layers. Transgenic marking enabled the enrichment of Nes-expressing IPE cells ex vivo, revealing a pronounced capacity to form neurospheres and differentiate into photoreceptor cells. IPE cells that did not express Nes were less able to form neurospheres, but a subset initiated the expression of pan-neural markers in primary adherent culture. These data collectively suggest that discrete populations of highly-pigmented cells with heterogeneous developmental potencies exist postnatally within the IPE, and that some of them are able to differentiate into multiple neuronal cell types.

FSH and IGF-I are both important determinants of follicle development and the process of cumulus cell-oocyte complex expansion. FSH stimulates the phosphorylation of Akt by mechanisms involving phosphatidylinositol 3-kinase (PI3-K), a pattern of response mimicking that of IGF-I. Cartilage link protein (Crtl1) is confined to the cartilaginous lineage and is assembled into a macroaggregate complex essential for hyaluronan-rich matrix stabilization. The present studies were performed to determine the actions of FSH and IGF-I on Crtl1 production in rat granulosa cells. Primary cultures of granulosa cells were prepared from 24-d-old rats. After treatments, cell extracts and media were prepared, and the Crtl1 level was determined by immunoblotting analysis using anti-Crtl1 antibodies. Here we showed that 1) treatment with FSH (> or = 25 ng/ml) or IGF-I (> or = 25 ng/ml) for 4 h increased Crtl1 production; 2) maximal stimulatory effects of FSH or IGF-I were observed at 100 or 50 ng/ml, respectively; 3) FSH caused a concentration-dependent increase in IGF-I-induced Crtl1 production and vice versa; 4) FSH and IGF-I also up-regulate the expression of Crtl1 mRNA; 5) FSH- and IGF-I-dependent Crtl1 production were abrogated by PI3-K inhibitors (LY294002 and wortmannin), and inhibition of Crtl1 production by p38 mitogen-activated protein kinase inhibitor (SB202190) was partial (approximately 30%), suggesting that PI3-K and, to a lesser extent, p38 mitogen-activated protein kinase are critical for the response. Our study represents the first report that FSH amplifies IGF-I-mediated Crtl1 production, possibly via PI3-K-Akt signaling cascades in rat granulosa cells.

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

Hepatocellular carcinoma (HCC) was the most common primary liver cancer, and its resistance to anti-tumor drugs often caused the death of patients suffering with HCC. Matrix stiffness was reported to be closely related to tumor chemoresistance; however, the relationship between HCC drug resistance and three-dimensional (3D) matrix stiffness is still unclear at present. In this study, alginate gel (ALG) beads with controllable matrix stiffness were used to mimic tumor tissue rigidity, and the role of 3D matrix stiffness in regulating the chemoresistance of HCC cells was investigated by using these ALG beads. It was found that HCC cells in ALG beads with 105 kPa stiffness had highest resistance to paclitaxel, 5-FU, and cisplatin. Although the mechanism was still uncovered, ABC transporters and endoplasmic reticulum stress-related molecules were highly expressed in ALG bead-encapsulated HCC cells compared with two-dimensional-cultured cells, which suggested a very complex mechanism underlying HCC drug resistance in 3D culture conditions. In addition, to mimic the specific stiffness of HCC tumor tissue, or other tumor tissues in vivo, response surface methodology (RSM) was used to build up a prediction mathematical model so that ALG beads with desired matrix stiffness could be prepared by simply changing three factors: molecular weight, G content, and alginate concentration.

The sigma-1 receptor, which is expressed throughout the brain, provides physiological benefits that include higher brain function. The sigma-1 receptor functions as a chaperone in the endoplasmic reticulum and may control cell death and regeneration within the central nervous system. Cutamesine (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl) piperazine dihydrochloride) is a ligand selective for this receptor and may mediate neuroprotective effects in the context of neurodegenerative disease. We therefore assessed whether cutamesine protects the inner ear from noise-induced or aging-associated hearing loss. Immunohistochemistry and Western blotting revealed that the sigma-1 receptor is present in adult cochlea. We treated mice with 0, 3, or 30 mg/kg cutamesine from 10 days before noise exposure until the end of the study. All subjects were exposed to a 120-dB, 4-kHz octave-band noise for 2 hr. We assessed auditory thresholds by measuring the auditory-evoked brainstem responses at 4, 8, and 16 kHz, prior to and 1 week, 1 month, or 3 months following noise exposure. For the aging study, measurements were made before treatment was initiated and after 3 or 9 months of cutamesine treatment. Damage to fibrocytes within the cochlear spiral limbus was assessed by quantitative histology. Cutamesine significantly reduced threshold shifts and cell death within the spiral limbus in response to intense noise. These effects were not dose or time dependent. Conversely, cutamesine did not prevent aging-associated hearing loss. These results suggest that cutamesine reduces noise-induced hearing loss and cochlear damage during the acute phase that follows exposure to an intense noise.

We postulated that urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, may inhibit low molecular weight hyaluronic acid (HA) fragment-induced prostanoid release and de-novo expression of the inducible cyclo-oxygenase-2 (COX-2) isoform in human term amnion cells. Purified amnion cultures were obtained from human fetal membranes and were exposed to a HA fragment (molecular weight 35 kDa) in the presence or absence of UTI (0-5.0 micromol/l). Amnion cells treated with the HA fragment (100 nmol/l) released significantly more prostanoids (PGE2 and PGF2alpha) than controls (PGE2: 2.1 +/- 0.13 pg/10(6) cells/24 h compared with 0.42 +/- 0.01, P < 0.05; PGF2alpha: 1.0 +/- 0.17 pg/10(6) cells/24 h compared with 0.13 +/- 0.01, P < 0.05). UTI inhibited HA fragment-induced prostanoid release in a dose-dependent manner, with 50% inhibitory concentration values of 0.8 micromol/l for PGE2 and 1.9 micromol/l for PGF2alpha. Western blot analyses demonstrated that protein levels of COX-2 were substantially increased in amnion cells treated with HA fragment. HA fragment-mediated COX-2 production was markedly diminished by pretreatment with UTI (1.0 micromol/l). These results are the first to demonstrate that UTI is a potent inhibitor of HA fragment-induced arachidonic acid metabolism.

Proteins of the inter-α-trypsin inhibitor (|α|) family are reported to stabilize the expanding cumulus extracellular matrix by direct binding with hyaluronic acid in mouse. The aim of this study is to compare the expression and distribution of hyaluronic acid and |α| in the rat ovaries during gonadotropin-induced follicle development. Immuno-and affinity histochemical staining was performed in rat ovary using a polyclonal antibody raised against purified human |α| and a hyaluronic acid-specific probe (biotinylated hyaluronic acid-binding protein). During a preovulatory stage, relatively large amounts of hyaluronic acid were present in the follicular fluid as well as on the surface of expanding cumulus cells and theca/stromal cells. The area of |α| reactivity in stimulated preovulatory follicles was almost the same as that of diffuse deposition of hyaluronic acid. In an in vivo rat model, inhibition of hyaluronic acid synthesis resulted in inhibition of cumulus oocyte complex (COC) expansion accompanied by reduced staining for |α| and hyaluronic acid. Western blot analysis of |α| and immunoassay for hyaluronic acid in tissue extracts from hCG-stimulated ovaries indicated marked increase crease in |α| and hyaluronic acid expression. These findings support the hypothesis that the co-distribution of |α| and hyaluronic acid may serve to organize the formation of the cumulus extracellular matrix in the rat ovary.

Increased concentrations of hyaluronic acid (HA) have been found in serum and at uterine cervix at term. In its native form, HA exists as a high molecular weight (MW) polymer, but during parturition a lower MW HA fragment accumulates. The aim of this study was to investigate the regulatory mechanisms responsible for increased amnion prostanoid production and cyclo-oxygenase (COX) expression in response to HA. Human term amnion cells in culture were exposed to native HA polymer (MW 2.2x106) and its fragment (MW 3.5x104). We have determined levels of prostanoids, prostaglandins E2 and F2alpha, in conditioned media using specific immunoassays. Expression of COX-1 and COX-2 was examined with Western blot. Results were analyzed for statistical significance with Mann-Whitney U-test. Human amnion cells treated with HA fragment (100 nmol/l) produced significantly more PGE2 (2.3+/-0.21 (mean+/-S.D.) pg/106 cells/24 h) than controls (0.34+/-0.03) or high MW HA-treated cells (1.2+/-0.21). Protein levels of COX-2, but not COX-1, were substantially increased in amnion cells treated with HA fragment. HA fragment-mediated prostanoid production is markedly diminished by pretreatment with indomethacin. Our results indicate that HA fragment, rather than physiologic native HA polymer, induces amnion cell-derived prostanoid production via increased COX-2 expression. COX-2-mediated prostanoid production is likely a key physiologic event in HA fragment-mediated cervical ripening and the labor onset.

We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of 125I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca2+, Mg2+-sensitive manner and another is via LP.

Fibrocytes of the spiral limbus are thought to play a significant role in maintaining ion homeostasis in the cochlea. The present study measured physiological and morphological changes in spiral limbus of mice in response to noise exposure. 6-week-old male C3H/HeJJcl mice were exposed to octave-band noise (120 dB SPL) for 2 h and evaluated at a series of times thereafter, up to 8 weeks. Permanent hearing loss resulted in the mice, as assessed by auditory brainstem response (ABR) recordings. The fibrocytes loss was found in the spiral limbus of the apical turn, which has been proved to be induced by apoptosis. These results suggest that noise exposure might result in apoptosis of fibrocytes in spiral limbus, which suggest a mechanism for noise-induced hearing loss.

The discovery of adult stem cells indicated a previously unrecognized degree of plasticity in stem cell function (1-3). Recent extensive studies have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs (4-6). However, more recent reports raised questions about some of the earlier results, proposing that transdifferentiation consequent to cell fusion could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells (7,8). Thus, cell transdifferentiation is of great interest, albeit a poorly understood process invoked to explain how tissue-specific adult stem cells can lose their properties and generate new cells of other tissues.

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP(40)) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP(40) was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP(40) were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP(40) displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP(40) and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH(2)-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH(2)-terminal subdomain of the LP molecule, and that LP and UTI-BP(40) exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP(40) is identical to LP and that the NH(2)-terminal domain of UTI is involved in the interaction with the NH(2)-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

The direct interaction of hyaluronic acid (HA) and heavy chain (HC) of the inter-alpha-trypsin inhibitor (IalphaI) family plays a critical role in the organization and stabilization of the extracellular matrix. The aim of the present investigation was to elucidate the distribution of the IalphaI HC and HA in adult mouse tissues. An immunohistochemical method using a rabbit polyclonal antibody raised against mouse IalphaI heavy-chain peptide and a specific probe for HA (biotinylated HA-binding protein) was used to demonstrate an immunolocalization of IalphaI HC and HA. Distribution and localization of HA was of three types, namely, colocalization with IalphaI HC itself (cartilaginous tissue and ovary), localization around IalphaI HC immunostaining (lung, intestine and skeletal muscle), and localization at a small distance from IalphaI HC or a different distribution pattern (brain, liver, skin and kidney). These results indicate that IalphaI HC could function as an HA-rich matrix stabilizer on the cells of cartilage and maturing ovary, in which IalphaI HC shows colocalization with its predominant ligand, HA.

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20 642–20 647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH 2 -terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan–hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.