Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

Low programmed cell death-1 (PD-1) expression in peripheral CD4(+) T cells in Japanese patients with autoimmune type 1 diabetes.

Programmed cell death-1 (PD-1) is a co-stimulatory molecule that inhibits T cell proliferation. We aimed to clarify PD-1 expression in CD4(+) T cells and the association between PD-1 expression and the 7785C/T polymorphism of PDCD1, with a focus on the two subtypes of type 1 diabetes, type 1A diabetes (T1AD) and fulminant type 1 diabetes (FT1D), in the Japanese population. We examined 22 patients with T1AD, 15 with FT1D, 19 with type 2 diabetes (T2D) and 29 healthy control (HC) subjects. Fluorescence-activated cell sorting (FACS) and real-time PCR were utilized to analyse PD-1 expression quantitatively. Genotyping of 7785C/T in PDCD1 was performed using the TaqMan method in a total of 63 subjects (21 with T1AD, 15 with FT1D and 27 HC). FACS revealed a significant reduction in PD-1 expression in CD4(+) T cells in patients with T1AD (mean: 4.2 vs. 6.0% in FT1D, P=0.0450; vs. 5.8% in T2D, P=0.0098; vs. 6.0% in HC, P=0.0018). PD-1 mRNA expression in CD4(+) T cells was also significantly lower in patients with T1AD than in the HC subjects. Of the 63 subjects, PD-1 expression was significantly lower in individuals with the 7785C/C genotype than in those with the C/T and T/T genotypes (mean: 4.1 vs. 5.9%, P=0.0016). Our results indicate that lower PD-1 expression in CD4(+) T-cells might contribute to the development of T1AD through T cell activation.

Novel PAX9 mutations cause non-syndromic tooth agenesis.

PAX9 is a transcription factor expressed in the tooth mesenchyme during tooth morphogenesis. In Pax9-null mice, tooth development is arrested at the bud stage. In humans, heterozygous mutations in PAX9 have been associated with non-syndromic tooth agenesis, predominantly in the molars. Here, we report 2 novel mutations in the paired domain of PAX9, a three-nucleotide deletion (73-75 delATC) and a missense mutation (C146T), in two unrelated Japanese patients with non-syndromic tooth agenesis. The individual with the 73-75del ATC mutation was missing all maxillary molars and mandibular second and third molars. The individual with the C146T mutation was missing the mandibular central incisors, maxillary second premolars, and first molars, along with all second and third molars. Both mutations affected amino acids that are highly conserved among different species and are critical for DNA binding. When both mutants were transfected to COS7 cells, nuclear localization of PAX9 proteins was not affected. However, reduced expression of the mutant proteins and almost no transcriptional activity of the target BMP4 gene were observed, suggesting haploinsufficiency of PAX9 as the cause of non-syndromic tooth agenesis.

Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models.

Polyoxomolybdates (PMs) as discrete molybdenum-oxide cluster anions have been investigated in the course of study of their medical applications. Here, we show the significant antitumour potency of the polyoxomolybdate [Me(3)NH](6)[H(2)Mo(V)(12)O(28)(OH)(12)(Mo(VI)O(3))(4)].2H(2)O (PM-17), which is a photo-reduced compound of [NH(3)Pr(i)](6)[Mo(7)O(24)].3H(2)O. The effect of PM-17 on the growth of cancer cell lines and xenografts was assessed by a cell viability test and analysis of tumour expansion rate. Morphological analysis was carried out by Hoechst staining, flow-cytometric analysis of Annexin V staining, terminal deoxynucleotidyl transferase-mediated 'nick-end' labelling staining, and electron-microscopic analysis. Activation of autophagy was detected by western blotting and fluorescence-microscopic analysis of the localisation of GFP-LC3 in transfected tumour cells. PM-17 inhibited the growth of human pancreatic cancer (AsPC-1) xenografts in a nude mice model, and induced morphological alterations in tumour cells. Correspondingly, PM-17 repressed the proliferation of AsPC-1 cells and human gastric cancer cells (MKN45) depending on the dose in vitro. We observed apoptotic patterns as the formation of apoptotic small bodies and translocation of phosphatidylserine by Hoechst staining and flow-cytometric analysis following Annexin V staining, and in parallel, autophagic conformation by the formulation of autophagosomes and localisation of GFP-LC3 by electron- and fluorescence-microscopic analysis.

Antitumor activity of polyoxomolybdate, [NH3Pri]6[Mo7O24].3H2O, against, human gastric cancer model.

Polyoxometalates are negatively charged inorganic compounds which contain metal ions such as tungsten, molybdenum, vanadium etc. and which make clusters with the surrounding oxygen atoms. [NH3Pri]6[Mo7O24].3H2O (PM-8) was found to be a significant antitumor polyoxomolybdates. It had already been reported that the PM-8 suppressed the growth of Co-4 human colon cancer, MX-1 human breast cancer and OAT human lung cancer xenografted in nude mice. However, the mechanism of the antitumor activity has not been clarified. In this study, the antitumor activity of one of the metal oxide clusters (polyoxometalates), hexabis(isopropylammonium) heptamolybdate trihydrate, [NH3Pri]6[Mo7O24].3H2O (PM-8) were shown in an MTS assay. DNA ladder formation and detection of apoptotic bodies in nuclei were revealed that antitumor activity of PM-8 in MKN45 cells was due to apoptosis. It is concluded that the observation of significant tumor growth suppression of PM-8 in MKN45-bearing mice results from the induction of apoptosis. PM-8 shows promise as a novel anti-cancer agent.

Anticancer activity of polyoxomolybdate.

Anticancer polyoxomolybdates have been investigated for medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH3Pri]6[Mo7O24].3H2O (PM-8) has been recognized as one of significant antitumoral polyoxomolybdates. PM-8 had shown the growth suppression against several tumors, for examples, Co-4, human colon cancer, MX-1, human breast cancer, and OAT, human lung cancer. PM-8 showed the tumor growth suppression for MKN-45 human gastric cancer in tumor bearing mice. PM-8 inhibited the cell growth of AsPC-1 which depended on the dose with showing DNA ladder formation and DNA fragmentation, and positive Hoechst staining indicating apoptosis. The ratio of apoptotic cells on flow cytometry analysis were 35%, and 57% with treatment of PM-8 after 48, and 72 h, respectively. One of the anti-tumor activity of PM-8 result from the activation of apoptotic pathway. It is thought that polyoxomolybdates will be applied as a novel anti-tumor agent especially against cancers which are difficult to be treated clinically.

A novel anti-tumor agent, polyoxomolybdate induces apoptotic cell death in AsPC-1 human pancreatic cancer cells.

Anti-tumoral polyoxomolybdates have been investigated in the course of study of the medical application of polyoxometalates as discrete cluster anions of metal oxides. [NH(3)Pr(i)](6)[Mo(7)O(24)].3H(2)O (PM-8) has been recognized as one of significantly anti-tumoral polyoxomolybdates. PM-8 inhibited the cell growth of human pancreatic cells (AsPC-1) depending on the dose. DNA ladder formation and DNA fragmentation were observed by Hoechst and TUNEL staining and flowcytometry analysis. The ratio of apoptotic cells were 29%, 35%, and 57% with treatment of PM-8 after 24, 48, and 72 h, respectively, which suggested that the anti-tumor activity of PM-8 results from the activation of the apoptotic pathway. Polyoxomolybdates provide promising, novel anti-tumor agent, especially for the treatment of cancers that are difficult to treat.

Percutaneous radiofrequency ablation for unresectable large hepatic tumours during hepatic blood flow occlusion in four patients.

AIM: To evaluate percutaneous radiofrequency (RF) ablation therapy for unresectable large hepatic tumours combined with regional interruption of hepatic blood flow, and to assess the safety and efficacy of this procedure. MATERIALS AND METHODS: Four patients with hepatic tumours were enrolled in this study. Patients were treated by a single session of RF ablation during occlusion of both hepatic artery and hepatic vein. Tumour size ranged from 45-57 mm (mean 50.2 mm). Initial therapeutic efficacy was evaluated with helical computed tomography (CT) performed within 9 days after the treatment. CT or magnetic resonance imaging (MRI) was performed every 2-3 months thereafter. RESULTS: The largest axis of coagulated lesions after the ablation was 50-60 mm (mean 56.5 mm) in diameter. The ablation therapy was considered complete in three patients; after a mean follow-up of 12.7 months, CT and MRI revealed complete destruction of their tumours. One patient required further treatment. No severe complications occurred. CONCLUSION: Although further studies are needed, in this limited clinical trial a local ablation area exceeding 50 mm in diameter was achieved safely.

Alternative splicing isoforms of hippostasin (PRSS20/KLK11) in prostate cancer cell lines.

BACKGROUND: Hippostasin is a kallikrein-like protease (PRSS20/KLK11), which is expressed preferentially in the hippocampus and prostate. We have reported that alternative splicing variants of human hippostasin are regulated in a tissue-specific manner. Brain-type hippostasin consists of 250 amino acids including a typical signal sequence, and is expressed in the brain and prostate. The prostate-type hippostasin, which has 32 extra amino acids at the N-terminal end, is expressed only in the prostate. METHODS: We analyzed the expression and localization of hippostasin in normal prostate tissue, BPH tissue, and prostate cancer cell lines. We performed northern blotting, in situ hybridization, immunohistochemistry, and RT-PCR. RESULTS: Hippostasin mRNA is expressed preferentially in the normal prostate and weakly in the testis. It was detected in prostate secretory epithelium. Hippostasin protein was localized in the prostate secretory epithelium, and western blotting showed that hippostasin was present in semen. All tested prostate cancer cell lines, including PSA-negative cell lines, expressed hippostasin. Interestingly, all the prostate cancer cell lines expressed only brain-type but not prostate-type hippostasin, while normal prostate and BPH expressed both types of hippostasin CONCLUSIONS: Our results suggest the possibility that hippostasin may be a useful marker by which prostate cancer and BPH can be distinguished.

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding.

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.

Characterization of a brain-related serine protease, neurosin (human kaillikrein 6), in human cerebrospinal fluid.

Neurosin (also known as zyme or protease M) is a trypsin-like serine protease dominantly expressed in the human brain. According to the official nomenclature, this gene is now designated as human kallikrein 6 (KLK6) and the protein is designated hK6. To investigate the metabolism of neurosin in human brain, neurosin contained in the human cerebrospinal fluid (CSF) was analyzed. Neurosin was detected in the all CSFs tested by Western blot analysis using an anti-neurosin monoclonal antibody. We purified neurosin from CSF (CSF-neurosin) using an immunoaffinity chromatography and an anion-exchange chromatography. SDS-PAGE revealed that the purified protein has a relative mol. mass (Mr) of 25,000 Da. The observed sequence of the N-terminal amino acids, Glu-Glu-Gln-Asn-Lys, of the purified CSF-neurosin was identical to the sequence of N-terminal of the pro-enzyme form, which is presumed to have no enzyme activity. CSF-neurosin neither showed any enzyme activity to Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide, which is known to be degraded by the mature neurosin, nor cleaved gelatin. To confirm that the major portion of CSF-neurosin is present in the pro-enzyme form, Western blot analysis using antibodies specific to the pro- or mature enzyme was carried out. The antibody against the mature neurosin fragment did not react with CSF-neurosin. Only the antibody against the pro-enzyme fragment detected CSF-neurosin. Thus, our results suggest that neurosin is present as an inactive pro-enzyme in the human CSF.

Molecular cloning of bovine zona pellucida glycoproteins ZPA and ZPB and analysis for sperm-binding component of the zona.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N-acetyllactosamine at the nonreducing portion of sugar chains with endo-beta-galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta 1201, 7-14]. The N-terminal amino-acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino-acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino-acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala-Asp-Asp/Glu. We purified bovine zona components and examined their sperm-binding activity with an in vitro competition assay and sperm-bead-binding assay. As a result, ZPB showed the strongest sperm-binding activity among the components. ZPC also showed sperm-binding activity and the activity per molecule was about one-sixth that of ZPB according to the result of the sperm-bead-binding assay. We could not determine if ZPA has significant sperm-binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.

Antagonistic role of E4BP4 and PAR proteins in the circadian oscillatory mechanism.

E4BP4, a basic leucine zipper transcription factor, contains a DNA-binding domain closely related to DBP, HLF, and TEF, which are PAR proteins. Here, we show that the phase of e4bp4 mRNA rhythm is opposite to that of the dbp, hlf, and tef rhythms in the suprachiasmatic nucleus (SCN), the mammalian circadian center, and the liver. The protein levels of E4BP4 and DBP also fluctuate in almost the opposite phase. Moreover, all PAR proteins activate, whereas E4BP4 suppresses, the transcriptional activity of the reporter gene containing a common binding sequence in transcriptional assays in vitro. An electrophoretic mobility shift assay demonstrated that E4BP4 is not able to dimerize with the PAR proteins, but is able to compete for the same binding sites with them. Furthermore, we showed sustained low e4bp4 and high dbp mRNA levels in mCry-deficient mice. These results indicate that the E4BP4 and PAR proteins are paired components of a reciprocating mechanism wherein E4BP4 suppresses the transcription of target genes during the time of day when E4BP4 is abundant, and the PAR proteins activate them at another time of day. E4BP4 and the PAR proteins may switch back and forth between the on-off conditions of the target genes.

Hepatocyte growth factor (HGF) activator expressed in hair follicles is involved in in vitro HGF-dependent hair follicle elongation.

Hepatocyte growth factor (HGF), a paracrine factor secreted by follicular papilla cells, acts on neighboring follicular epithelial cells to promote follicular growth, while HGF activator is a serine proteinase, which converts inactive single-chain HGF to the active heterodimeric form. In this study, using 3' rapid amplification of cDNA end/nested polymerase chain reaction (3' RACE/nested PCR) and immunoblotting, we confirmed the expression of HGF activator in both cultured human follicular papilla cells and outer root sheath cells. HGF activator mRNA was expressed in all of the isolated 15 anagen hair follicles taken from the scalps of seven individuals. In an organ culture system, single-chain HGF stimulated hair follicle elongation, which was partially inhibited by aprotinin, a serine proteinase inhibitor (P<0.01). These results suggest that single-chain HGF secreted from follicular papilla cells is converted to an active heterodimeric form by intrinsic HGF activator and that the resultant active form of HGF stimulates hair growth.

Cyclin-dependent kinase inhibitors, p21(waf1/cip1) and p27(kip1), are expressed site- and hair cycle-dependently in rat hair follicles.

In order to investigate the role of cyclin-dependent kinase (CDK) inhibitors in hair growth, we analyzed the expressions of p21(waf1/cip1) and p27(kip1) during the synchronized hair cycle of rat coat. The mRNAs of both p21(waf1/cip1) and p27(kip1) were detected in anagen hair follicles by reverse transcription and polymerase chain reaction and their localization was clearly demonstrated in the upper half portion of the hair bulb and the cortex by in situ hybridization. The dermal tissue containing hair follicles was then excised from the anterior dorsal skin of the 5-12-week-old rats at 0.5 week intervals and the expressions of p21(waf1/cip1) and p27(kip1) were analyzed by northern blot hybridization. The mRNA of both CDK inhibitors was expressed at relatively high levels during anagen than during telogen, a fact which correlated with the mRNA expression levels of hair differentiation markers, type I hair keratin (Ha3) and high sulfur protein B2. These results imply that CDK inhibitors, p21(waf1/cip1) and p27(kip1), are involved in the differentiation of follicular epithelial cells.

cDNA cloning and tissue-specific splicing variants of mouse hippostasin/TLSP (PRSS20).

Two splicing variants of mouse hippostasin/TLSP (PRSS20) were identified and termed brain-type and prostate-type, respectively. Mouse hippostasin/TLSP showed 76.8% identity to the human homologue. Transient expression showed that both translational products were secreted into the conditioned medium. Mouse hippostasin/TLSP was expressed preferentially in the fetal brain and the prostate, but not in the neonatal brain. The brain expressed only brain-type hippostasin/TLSP, while the prostate expressed both types.

Localization of a novel type trypsin-like serine protease, neurosin, in brain tissues of Alzheimer's disease and Parkinson's disease.

Neurosin, a novel type of trypsin-like serine protease, has been shown to be preferentially expressed in human brain by northern blotting. We examined neurosin immunolabeling in the brains of neurologically normal persons and patients with Alzheimer's disease (AD) and with Parkinson's disease. We also identified the expression of the mRNA for neurosin by in situ hybridization histochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The neurosin antibody stained all of the nuclei of various cell types. In neurons, there was also staining of neuronal cytoplasm, nucleoli and their processes. In AD, staining of neurons with processes was rare in the damaged areas. Some senile plaques, extracellular tangles and Lewy bodies were also positive for neurosin. Expression of the mRNA for neurosin was seen in neurons in the gray matter, and in microglial cells in the white matter. In AD, the intensity of the signal for neurosin mRNA in the gray matter was decreased compared with normal control brains. The relative levels of neurosin mRNA in AD brains, measured by RT-PCR, were lower than those in controls. These results suggest that in human brain neurosin plays various physiological roles, and that in AD this molecule, like other serine proteases, may have a role in the degradation of such substances as beta-amyloid protein.

Two mirror-image sensory maps with domain organization in the mouse main olfactory bulb.

The glomerular sheet in the olfactory bulb (OB) provides an olfactory sensory map identifying which odorant receptors (ORs) in the nose are activated by inhaled odorants. How are the glomeruli spatially arranged in the OB? Using OCAM and neuropilin-1 (NP1) as molecular markers for target glomeruli of distinct subsets of olfactory axons, we demonstrate here that glomeruli are parceled into topographically distinct domains. Spatial arrangement of these domains suggests that each OB contains two mirror-image maps of the glomeruli. In situ hybridization shows that the glomeruli representing the same OR are symmetrically arranged; one in a domain in the lateral hemisphere and the other in a corresponding domain in the medial hemisphere of the OB. These results suggest that OB contains two symmetrical OR maps with similar domain organization.