Atrial Natriuretic Peptide Improves Neurite Outgrowth from Spiral Ganglion Neurons In Vitro through a cGMP-Dependent Manner.

The spiral ganglion neurons (SGNs) are the primary afferent neurons in the spiral ganglion (SG), while their degeneration or loss would cause sensorineural hearing loss. As a cardiac-derived hormone, atrial natriuretic peptide (ANP) plays a critical role in cardiovascular homeostasis through binding to its functional receptors (NPR-A and NPR-C). ANP and its receptors are widely expressed in the mammalian nervous system where they could be implicated in the regulation of multiple neural functions. Although previous studies have provided direct evidence for the presence of ANP and its functional receptors in the inner ear, their presence within the cochlear SG and their regulatory roles during auditory neurotransmission and development remain largely unknown. Based on our previous findings, we investigated the expression patterns of ANP and its receptors in the cochlear SG and dissociated SGNs and determined the influence of ANP on neurite outgrowth in vitro by using organotypic SG explants and dissociated SGN cultures from postnatal rats. We have demonstrated that ANP and its receptors are expressed in neurons within the cochlear SG of postnatal rat, while ANP may promote neurite outgrowth of SGNs via the NPR-A/cGMP/PKG pathway in a dose-dependent manner. These results indicate that ANP would play a role in normal neuritogenesis of SGN during cochlear development and represents a potential therapeutic candidate to enhance regeneration and regrowth of SGN neurites.

Electrical Properties of Ba0.96Ca0.04Ti0.90Sn0.10O3 Lead-Free Ceramics with the Addition of Nano-CeO2.

Well dispersed CeO2 nanoparticles are prepared by azeotropic co-precipitation method. (Ba0.96Ca0.04)(Ti0.90Sn0.10)O3 lead-free piezoelectric ceramics doped with nano-CeO2 (x =0 mol%, 0.03 mol%, and 0.07 mol%) and micro-CeO2 (x = 0.03 mol%) are prepared at 1430 °C for 2 h by the conventional solid state sintering method. XRD diffraction indicates that all components have typical perovskite structure. Both doping of nano-CeO2 and micro-CeO2 can inhibit grain growth. And the average grain size decreased apparently with the increase of nano-CeO2 amount. All the samples exhibit typical diffuse phase transition behavior. The optimized electrical performances are obtained at x = 0.03 mol% with d33 = 512 pC/N, kp = 41.5%, and Pr = 14.00 µC/cm².

De novo assembly and functional annotations of the transcriptome of Metorchis orientalis (trematoda: Opisthorchiidae).

Metorchis orientalis is a neglected zoonotic parasite, living in the gallbladder and bile duct of poultry and some mammals as well as humans. In spite of its economic and medical importance, the information known about the transcriptome and genome of M. orientalis is limited. In this study, we performed de novo sequencing, transcriptome assembly and functional annotations of the adult M. orientalis, obtained about 77.4 million high-quality clean reads, among which the length of the transcript contigs ranged from 100 to 11,249 nt with mean length of 373 nt and N50 length of 919 nt. We then assembled 31,943 unigenes, of which 20,009 (62.6%) were annotated by BLASTn and BLASTx searches against the available database. Among these unigenes, 19,795 (62.0%), 3407 (10.7%), 10,620 (33.2%) of them had significant similarity in the NR, NT and Swiss-Prot databases, respectively; 5744 (18.0%) and 4678 (14.6%) unigenes were assigned to GO and COG, respectively; and 9099 (28.5%) unigenes were identified and mapped onto 256 pathways in the KEGG Pathway database. Furthermore, we found that 98 (1.08%) unigenes were related to bile secretion and 5 (0.05%) to primary bile acid biosynthesis pathways category. The characterization of these transcriptomic data has implications for the better understanding of the biology of M. orientalis, and will facilitate the development of intervention agents for this and other pathogenic flukes of human and animal health significance.

Complete mitochondrial genome of parasitic nematode Cylicocyclus nassatus and comparative analyses with Cylicocyclus insigne.

Cylicocyclus nassatus is a common and important parasite in the large intestine of equine. In this study, the complete mitochondrial (mt) genome sequence of C. nassatus was determined and comparatively analyzed with Cylicocyclus insigne. The mt genome size of C. nassatus was 13,846 bp, 18 bp longer than that of C. insigne. The circular mt genome includes 12 protein-coding genes, two rRNA genes, 22 tRNA genes and two non-coding regions. All the genes are transcribed in the same direction and gene arrangement is consistent with that of gene arrangement 3 (GA3). The overall sequence difference between the two complete mt genomes was 10.7%. For the 12 protein-coding genes, the comparison between C. nassatus and C. insigne revealed sequence divergence at both the nucleotide (6.3-13.0%) and amino acid (0.8-6.6%) levels. The nucleotide lengths of the 12 protein-coding genes were the same, except for cox3 which was longer in C. insigne. Phylogenetic analysis based on the concatenated amino acid sequence of the 12 protein-coding genes was performed using all the Strongylidae nematodes of the horse available in the GenBank. Phylogenetic analysis showed that C. nassatus and C. insigne clustered together with very high nodal support and the genus Cylicocyclus was closer to the genus Triodontophorus than to genus Strongylus. The mtDNA data determined in this study provides novel genetic markers for further studies on the identification, population genetics and molecular epidemiology of the genus Cylicocyclus.

Metacercaria Infection Status of Fishborne Zoonotic Trematodes, Except for Clonorchis sinensis in Fish from the Heilongjiang Province, China.

To investigate the metacercarial infections of fishborne zoonotic trematodes (FZT), a total of 6815 freshwater fish (in representing 13 species of 5 families) were collected from Songhua river (n = 2636), Nenjiang river (n = 1935), Mudanjiang river (n = 301), and other lakes or ponds (n = 1943) in 36 representative regions in Heilongjiang Province, China, from August 2012 to December 2015. Metacercariae of four FZT species, that is, Clonorchis sinensis, Metorchis orientalis, Isthmiophora hortensis, and Metagonimus yokogawai, metacercariae were detected in the examination by the artificial digestion method. As the partial data for C. sinensis were previously reported, the remaining three FZT species are to be treated in this study. The overall prevalence of M. orientalis, I. hortensis, and M. yokogawai, metacercariae was 10.54%, 0.28%, and 1.35%, respectively. Metacercariae of M. orientalis were detected in seven fish species, that is, Pseudorasbora parva, Hemiculter leucisculus, Saurogobio dabryi, Rhynchocypris lagowskii, Carassius auratus, Rhodeus ocellatus and Perccottus glehnii. Their prevalences were the highest in false dace, P. parva (26.81%), and in fish from Songhua river (17.94%). Metacercariae of I. hortensis were detected in only one fish species, Misgurnus anguillicaudatus, from Nenjiang river only. Metacercariae of M. yokogawai were detected in three fish species, that is, P. parva, H. leucisculus and S. dabryi. Their prevalences were the highest in sharpbelly, H. leucisculus (6.05%), and in fish from Mudanjiang river (5.65%). This study first demonstrated the existence of M. orientalis, I. hortensis, and M. yokogawai in freshwater fish from Heilongjiang Province, posing a major public health concern. Eight fish species, namely M. anguillicaudatus, P. parva, H. leucisculus, S. dabryi, R. lagowskii, C. auratus, R. ocellatus, and P. glehnii, cannot be eaten raw. Moreover, the findings of this study not only extended the second intermediate host range of FZT, but also improve the information of the distribution of FZT in China.

Complete mitochondrial genomes of Dermacentor silvarum and comparative analyses with another hard tick Dermacentor nitens.

Ticks are obligate blood-sucking ectoparasites of a wide range of vertebrates. They can transmit a range of pathogens that cause economic losses to livestock production as well as human disease. In the present study, the complete mitochondrial (mt) genome of Dermacentor silvarum was determined. The mt genome is 14,945 bp in length contains 37 genes, including 13 are protein-coding genes (cox1-3, nad1-6, nad4L, cytb, atp6 and atp8), two ribosomal RNA genes and 22 transfer RNA genes. The nucleotide composition of the D. silvarum mt genome was A + T biased at 78.78%; T was the most abundant nucleotide and G the least abundant. The mt genome of D. silvarum was 106 bp longer than that of Dermacentor nitens and the arrangements of two genomes were identical. For the 13 protein-coding genes, comparison between D. silvarum and D. nitens revealed sequence divergence at both the nucleotide (15.46-35.14%) and amino acid (6.05-48.98%) levels. Among them, cox1 was the most conserved gene, while atp8 was the least conserved. The lengths of the 13 protein-coding genes were the same or similar, except for cytb which was significantly longer in D. silvarum than in D. nitens. The mtDNA contained a variable repeat region consisting of a "similar to nad1" motif that was repeated three times, and the "Tick-box" motifs were also found. The overall difference between the nucleotide sequences of the two complete mt genomes was 21.4%. The mtDNA data presented in this study provide a rich resource for further studies on the phylogenetics, population genetics, and molecular epidemiology of ticks.

The complete mitochondrial genome of Strongylus equinus (Chromadorea: Strongylidae): Comparison with other closely related species and phylogenetic analyses.

The roundworms of genus Strongylus are the common parasitic nematodes in the large intestine of equine, causing significant economic losses to the livestock industries. In spite of its importance, the genetic data and epidemiology of this parasite are not entirely understood. In the present study, the complete S. equinus mitochondrial (mt) genome was determined. The length of S. equinus mt genome DNA sequence is 14,545 bp, containing 36 genes, of which 12 code for protein, 22 for transfer RNA, and two for ribosomal RNA, but lacks atp8 gene. All 36 genes are encoded in the same direction which is consistent with all other Chromadorea nematode mtDNAs published to date. Phylogenetic analysis based on concatenated amino acid sequence data of all 12 protein-coding genes showed that there were two large branches in the Strongyloidea nematodes, and S. equinus is genetically closer to S. vulgaris than to Cylicocyclus insignis in Strongylidae. This new mt genome provides a source of genetic markers for the molecular phylogeny and population genetics of equine strongyles.

Histone deacetylase inhibitor sodium butyrate attenuates gentamicin-induced hearing loss in vivo.

OBJECTIVE: Although histone deacetylase (HDAC) inhibition has been shown to protect against gentamicin (GM)-induced hearing loss in vitro, its protective effect has not been proven in vivo. In the present study, the aim was to investigate the protective effect of sodium butyrate (NaB), a specific HDAC inhibitor, on GM-induced ototoxicity in vivo. METHODS: Forty 8-week-old albino guinea pigs were divided into two experimental groups. Group 1 (n=10) underwent bilateral ear surgery to place sponges (0.3mm(3)) permeated with NaB (10µl, 100mg/ml) and physiological saline (10µl; control) in the right and left round window niches, respectively. The sponges were left in place for 15days to evaluate the effects of NaB at the applied concentration. Group 2 (n=30) underwent the same bilateral ear surgery described for Group 1, except three days after surgery, the animals received intramuscular GM injections (200mg/kg/day) for 5 consecutive days. Seven days after the final GM injection, the protective effects of NaB were examined. RESULTS: After 15days of NaB treatment (10µl, 100mg/ml), an increase in histone acetylation was detected in Corti organ samples. Auditory brainstem response (ABR) threshold shifts and hair cell loss were also reduced in NaB-treated ears after GM administration. Furthermore, GM treatment increased HDAC1 expression in outer hair cells (OHCs) in vivo, and NaB blocked this action. CONCLUSION: GM increases HDAC1 expression in OHCs, and NaB is able to block this action. Thus, it appears that the HDAC inhibitor, NaB, attenuates GM-induced hearing loss in guinea pigs.

Inhalation of hydrogen gas attenuates ouabain-induced auditory neuropathy in gerbils.

AIM: Auditory neuropathy (AN) is a hearing disorder characterized by abnormal auditory nerve function with preservation of normal cochlear hair cells. This study was designed to investigate whether treatment with molecular hydrogen (H(2)), which can remedy damage in various organs via reducing oxidative stress, inflammation and apoptosis, is beneficial to ouabain-induced AN in gerbils. METHODS: AN model was made by local application of ouabain (1 mmol/L, 20 mL) to the round window membrane in male Mongolian gerbils. H(2) treatment was given twice by exposing the animals to H(2) (1%, 2%, and 4%) for 60 min at 1 h and 6 h after ouabain application. Before and 7 d after ouabain application, the hearing status of the animals was evaluated using the auditory brainstem response (ABR) approach, the hear cell function was evaluated with distortion product otoacoustic emissions (DPOAE). Seven days after ouabain application, the changes in the cochleae, especially the spiral ganglion neurons (SGNs), were morphologically studied. TUNEL staining and immunofluorescent staining for activated caspase-3 were used to assess the apoptosis of SGNs. RESULTS: Treatment with H(2) (2% and 4%) markedly attenuated the click and tone burst-evoked ABR threshold shift at 4, 8, and 16 kHz in ouabain-exposed animals. Neither local ouabain application, nor H(2) treatment changed the amplitude of DPOAE at 4, 8, and 16 kHz. Morphological study showed that treatment with H(2) (2%) significantly alleviated SGN damage and attenuated the loss of SGN density for each turn of cochlea in ouabain-exposed animals. Furthermore, ouabain caused significantly higher numbers of apoptotic SGNs in the cochlea, which was significantly attenuated by the H(2) treatment. However, ouabain did not change the morphology of cochlear hair cells. CONCLUSION: The results demonstrate that H(2) treatment is beneficial to ouabain-induced AN via reducing apoptosis. Thus, H(2) might be a potential agent for treating hearing impairment in AN patients.

Seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in northeast China.

The seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in Heilongjiang Province, northeast China, was surveyed between April 2009 and May 2011. A total of 1803 (693 beef cattle and 1110 dairy cattle) serum samples were collected from 10 administrative regions rearing beef cattle and dairy cattle, and antibodies to T. gondii were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of T. gondii in beef cattle and dairy cattle was 2.6% (46/1803), and the prevalence in beef cattle (3.0%) was slightly higher than that in dairy cattle (2.3%). The prevalence of antibodies in adult animals was higher than that in calves, but the differences among the age groups were not significant (p>0.05). The seroprevalence in female (3.4%) and male (2.5%) beef cattle was not statistically significant (p>0.05). Though the prevalence in intensively reared beef cattle and dairy cattle was lower than that in semi-intensively reared animals, the difference was not statistically significant (p>0.05). The results of this survey indicated the presence of T. gondii infection in beef cattle and dairy cattle in Heilongjiang Province, the coldest province in China, which may cause economic losses to the local livestock industry, and may be a source of T. gondii infection for humans in this region.

[Corrigendum] miR­29b overexpression induces cochlear hair cell apoptosis through the regulation of SIRT1/PGC­1α signaling: Implications for age­related hearing loss.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that the bar charts shown in Fig. 4A and B, which were intending to have shown the RT­qPCR and western blot analyses of SIRT1 and PGC­1α in HEI­OC1 cells, respectively, under different experimental conditions were apparently identical. Similarly, in Fig. 5, the histograms shown in Fig. 5C and D, which were intending to have shown the RT­qPCR and western blot analyses, respectively, of SIRT1 and PGC­1α in HEI­OC1 cells subjected to different treatments were also apparently identical. The authors have re­examined their data, and realize that the data properly belonging to the protein expression levels had been wrongly used to show the mRNA levels, and therefore Figs. 4A and 5C were presented incorrectly in these figures. The revised versions of Figs. 4 and 5, containing the correct data for the RT­qPCR experiments in Figs. 4A and 5C, are shown on the next page. These errors did not affect the major conclusions reported in the paper. All the authors have agreed to this corrigendum, and thank the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this. The authors regret these errors went unnoticed during the compilation of the figures in question, and apologize to the readership for any confusion that this may have caused. [the original article was published in International Journal of Molecular Medicine 38: 1387­1394, 20186 DOI: 10.3892/ijmm.2016.2735].

Synergistic effect of Mn doping and hollow structure boosting Mn-CoP/Co2P nanotubes as efficient bifunctional electrocatalyst for overall water splitting.

The sluggish kinetic of hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) severely hampers the commercial application of electrochemical water splitting, promoting the urgent exploration of high-efficient bifunctional electrocatalysts. Heteroatom doping and structure engineering have been identified as the most effective strategies to boost the catalytic activity of electrocatalysts. Herein, Mn doping and hollow structure were integrated in the design of Co-based transition metal phosphide catalyst to prepare Mn-CoP/Co2P nanotubes (denoted as Mn-CP NTs) by a facile template-free method. Confirmed by characterization analysis, the introduced Mn species were in high dispersion in the regular CoP/Co2P hollow tubular framework. Such a favorable design in composition and structure effectively boosted the catalytic activity of Mn-CP NTs toward electrochemical water splitting. The Mn-CP NTs showed superior HER and OER activity demonstrated by the low overpotentials of 82 mV (vs HER) and 309 mV (vs OER) at the current density of 10 mA cm-2, as well as the satisfactory durability. When used as both cathode and anode in electrolyzer for overall water splitting, only a low cell voltage of 1.67 V was required for the Mn-CP NTs to drive 10 mA cm-2, accompanied with excellent stability confirmed by over 50 h test.

Atrial Natriuretic Peptide Promotes Neurite Outgrowth and Survival of Cochlear Spiral Ganglion Neurons in vitro Through NPR-A/cGMP/PKG Signaling.

Sensorineural hearing loss (SNHL) is a dominant public health issue affecting millions of people around the globe, which is correlated with the irreversible deterioration of the hair cells and spiral ganglion neurons (SGNs) within the cochlea. Strategies using bioactive molecules that regulate neurite regeneration and neuronal survival to reestablish connections between auditory epithelium or implanted electrodes and SGN neurites would become attractive therapeutic candidates for SNHL. As an intracellular second messenger, cyclic guanosine-3',5'-monophosphate (cGMP) can be synthesized through activation of particulate guanylate cyclase-coupled natriuretic peptide receptors (NPRs) by natriuretic peptides, which in turn modulates multiple aspects of neuronal functions including neuronal development and neuronal survival. As a cardiac-derived hormone, atrial natriuretic peptide (ANP), and its specific receptors (NPR-A and NPR-C) are broadly expressed in the nervous system where they might be involved in the maintenance of diverse neural functions. Despite former literatures and our reports indicating the existence of ANP and its receptors within the inner ear, particularly in the spiral ganglion, their potential regulatory mechanisms underlying functional properties of auditory neurons are still incompletely understood. Our recently published investigation revealed that ANP could promote the neurite outgrowth of SGNs by activating NPR-A/cGMP/PKG cascade in a dose-dependent manner. In the present research, the influence of ANP and its receptor-mediated downstream signaling pathways on neurite outgrowth, neurite attraction, and neuronal survival of SGNs in vitro was evaluated by employing cultures of organotypic explant and dissociated neuron from postnatal rats. Our data indicated that ANP could support and attract neurite outgrowth of SGNs and possess a high capacity to improve neuronal survival of SGNs against glutamate-induced excitotoxicity by triggering the NPR-A/cGMP/PKG pathway. The neuroregenerative and neuroprotective effects of ANP/NPRA/cGMP/PKG-dependent signaling on SGNs would represent an attractive therapeutic candidate for hearing impairment.

Primary myxofibrosarcoma of the parotid: case report.

BACKGROUND: Myxofibrosarcoma is common in the extremities of elderly people and is characterized by a high frequency of local recurrence. CASE PRESENTATION: We report a 37 year old female who presented with a 4-month history of facial pain and a 3-month history of painful progressive swelling in the preauricular area. She underwent a total parotidectomy. The tumor was histopathologically and immunohistochemically diagnosed as a low-grade myxofibrosarcoma. The patient was free of disease 9 months after surgery with uneventful post-operative clinical course. CONCLUSIONS: Parotid area swelling should always alert doctors. To our knowledge, this is the first case of parotid myxofibrosarcoma. It should be added to the differential diagnosis of diseases of the parotid. We have to recognize this disease and seek adequate treatment for it.

HDAC inhibitor sodium butyrate prevents allergic rhinitis and alters lncRNA and mRNA expression profiles in the nasal mucosa of mice.

Our previous study demonstrated that intranasal administration of histone deacetylase inhibitor sodium butyrate (NaB) exhibits therapeutic effects on a mouse model of allergic rhinitis (AR). However, whether NaB is effective on AR when administered orally and prophylactically, as well as its potential effects on gene expression, remained unknown. The present study aimed to investigate the preventive effect of NaB on AR when added to the diet of newly weaned mice and to evaluate the changes in long non­coding (lnc)RNA and mRNA expression profiles in the nasal mucosa. Mice were randomly divided into three groups as follows: i) Control (C) group, (no treatment); ii) AR group [treated with ovalbumin (OVA)]; and iii) NaB + AR group (treated with OVA and NaB). The NaB + AR group was administered NaB in their feed (30 g/kg chow), whereas the other two groups were fed normal feed between 3 and 6 weeks of age. At 7 weeks of age, OVA administration was initiated to induce AR in the AR and NaB + AR groups. Following model establishment, behavioral assessments, western blotting and gene expression analysis were performed. NaB exhibited a preventive effect in the murine AR model, diminished the increases in histone deacetylase 1 (HDAC1) and HDAC8 expression and increased OVA­induced acetylation of histone H3 at lysine 9. In addition, NaB increased the AR­associated low expression of interleukin 2 (IL­2), interferon Î³ and IL­17 and decreased the expression of IL­4, IL­5 and transforming growth factor ß1. Gene Ontology and pathway analyses revealed the top 10 pathways among the groups. Octamer­binding transcription factor 1, ecotropic viral integration site 1 and paired box 4 were predicted to be target genes of lncRNA (NONMMUT057309). Thus, NaB may exhibit a preventive effect on AR. Additionally, the lncRNA and mRNA expression profiles in the nasal mucosa of mice with AR differed significantly following NaB treatment. These results may provide insights into the pathogenesis of AR and suggest new treatment targets.

Photovoltaic Effect in BiFeO3 Film Deposited by RF Magnetron Sputtering.

A polycrystalline BiFeO3 film was deposited on ITO substrate by RF magnetron sputtering method. Small crystallite size and compact structure are obtained for BiFeO3 film which has the excellent ferroelectric properties. The measured photovoltaic response reveals an open-circuit voltage of ~0.52 V and a short-circuit current density of ~10 µA/cm² under the illumination of 100 mW/cm² irradiance. Moreover, a tunable photovoltaic effect with light illumination is observed under different voltage sweep mode. High initial sweep voltage can enhance the photovoltaic effect largely, however, the photovoltaic response decreases with the increase of voltage sweep interval. The results indicate the ferroelectric polarization plays an important role in the photovoltaic effect.

Improved Photovoltaic Effect of p-i-n Structured BiFeO3 Film Deposited by Radio-Frequency Magnetron Sputtering.

Pure phase polycrystalline BiFeO3 film was deposited onto FTO substrate by RF magnetron sputtering method. SEM result shows that BiFeO3 film has the obvious porosity and large clusters which lead to the poor ferroelectric and photovoltaic properties in FTO/BiFeO3/Ag device. However, these properties are improved in p-i-n structured FTO/TiO2/BiFeO3/HTM/Ag device by incorporating the electron and hole transport materials. The hysteresis loop measurement demonstrates the excellent ferroelectric property with large remnant polarization (2Pr = 180 µC/cm²) and low leakage current. The J-V curve shows the short-circuit current density is dozens of times larger than that of FTO/BiFeO3/Ag device. Moreover, the photovoltaic output depends on the poling field where the positive poling improves the short-circuit current density to -85 µA/cm2 and the negative poling reduces both the photocurrent and photovoltage. It is believed that the ferroelectric polarization plays a dominant role in the photovoltaic effect.

c-Met-targeted RNA interference inhibits growth and metastasis of glioma U251 cells in vitro.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, invasion, and angiogenesis. The present study was designed to determine the role of c-Met in growth and metastasis of glioma U251 cells using RNA interference (RNAi) technology in vitro. We constructed three kinds of shRNA expression vectors aiming at the c-Met gene, then transfected them into glioma U251 cells by lipofectamine(TM) 2000. The level of c-Met mRNA was investigated by real-time polymerse chain reaction (RT-PCR). The protein expression of c-Met was observed by immunofluoresence staining and western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. We got three kinds of c-Met specific shRNA expression vectors which could efficiently inhibit the growth and metastasis of U251 cells and the expression of c-Met in U251 cells. RT-PCR, immunofluoresence staining and western blotting showed that inhibition rate for c-Met expression was up to 90%, 79% and 85%, respectively. The expression of c-Met can be inhibited by RNA interference in U251 cells, which can inhibit the growth and metastasis of U251 cell and induce cell apoptosis. These results indicate that RNAi of c-Met can be an effective antiangiogenic strategy for glioma.

Stabilization of hepatocyte growth factor mRNA by hypoxia-inducible factor 1.

Hypoxia regulates expression of hepatocyte growth factor (HGF) by increasing its transcription and by stabilizing its mRNA. Despite the pivotal role of hypoxia-inducible factor 1 (HIF-1) in transcriptional activation of hypoxia-responsive genes, it is not known whether HIF-1 mediates hypoxia-induced stabilization of HGF mRNA. We constructed adenoviral vectors expressing either the wild-type HIF-1alpha (Ad2/HIF-1alpha/FL), a constitutively stable hybrid form of HIF-1alpha (Ad2/HIF-1alpha/VP16), or no transgene (Ad2/CMVEV). In rat glioma (C6) cells, human glioma (U251) cells human cardiac, vascular smooth muscle, and endothelial cells, infection with Ad2/HIF-1alpha/VP16 or Ad2/HIF-1alpha/FL increased HGF expression at both the mRNA and protein levels. Under normoxic conditions, the half-life of HGF mRNA was 43 min in C6 and U251 cells. Hypoxia and Ad2/HIF-1alpha/VP16 increased the half-life of HGF mRNA to 3.2 and 2.8 h, respectively, while Ad2/CMVEV had no effect. These studies are the first to demonstrate that overexpression of HIF-1alpha increases HGF mRNA stability. Our results also suggest that stabilization of HGF mRNA by hypoxia is mediated, at least in part, by HIF-1.

Polyclonal antibodies to LIM proteins CRP2 and CRIP2 reveal their subcellular localizations in olfactory precursor cells.

In this study, we describe the presence of CRP2 (cysteine- and glycine-rich protein 2) and CRIP2 (cysteine-rich intestinal protein 2), which are members of group 2 LIM proteins, in rat olfactory precursor cells by reverse transcription polymerase chain reaction. We have developed polyclonal antibodies against CRP2 and CRIP2 individually. Specificity of the antibodies was demonstrated by Western blot analysis, using CRP2 and CRIP2 transfected cells. No cross-reactivity was observed between the antibodies. Furthermore, we used the antibodies to determine the expression and localization of CRP2 and CRIP2 in olfactory precursor cells by Western blot analysis and immunofluorescence staining. Our results demonstrated that in undifferentiated olfactory precursor cells CRP2 was distributed both in the nucleus and the cytoplasm, whereas CRIP2 was predominantly localized in the cytoplasm. While the olfactory precursor cells differentiated into end cells, only the expression of CRIP2 would be detected. The function of these LIM proteins in olfactory precursor cells warrants further study.