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Diffuse-type gastric cancer (DGC) is a highly invasive subtype of gastric adenocarcinoma that frequently exhibits scattered peritoneal metastasis. Previous studies have shown that the genes of receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor 2 (FGFR2) or Met, are amplified in some DGC cell lines, leading to the constitutive activation of corresponding RTKs. In these cell lines, the survival of cancer cells appears to be dependent on the activation of RTKs. To gain novel insights into the downstream signaling pathways of RTKs specific to DGC, phosphotyrosine-containing proteins associated with activated FGFR2 were purified through two sequential rounds of immunoprecipitation from the lysates of two DGC cell lines. As a result, transferrin receptor 1 (TfR1) was identified as the binding partner of FGFR2. Biochemical analysis confirmed that TfR1 protein binds to FGFR2 and is phosphorylated at tyrosine 20 (Tyr20) in an FGFR2 kinase activity-dependent manner. The knockdown of TfR1 and treatment with an inhibitor of FGFR2 caused significant impairment in iron uptake and suppression of cellular proliferation in vitro. Moreover, the suppression of expression levels of TfR1 in the DGC cells significantly reduced their tumorigenicity and potency of peritoneal dissemination. It was indicated that TfR1, when phosphorylated by the binding partner FGFR2 in DGC cells, promotes proliferation and tumorigenicity of these cancer cells. These results suggest that the control of TfR1 function may serve as a therapeutic target in DGC with activated FGFR2.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actin-rich Triton X-100–insoluble pellets following platelet aggregation induced by thrombin receptor–activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin- and actin-related protein 2 and 3 (Arp2/3)–rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actin-rich Triton X-100–insoluble pellets following platelet aggregation induced by thrombin receptor–activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin- and actin-related protein 2 and 3 (Arp2/3)–rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets.

The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.

WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Δ basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Δ basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex.

Estimation of Dioxin Exposure Concentrations and Dioxin Intakes of Workers at Continuously Burning Municipal Waste Incinerators: Shinji Kumagai, et al.Department of Occupational Health, Osaka Prefectural Institute of Public Health—In order to examine the dioxin exposure level in incinerator workers, total dust concentration in the breathing zone and dioxin concentrations in fly ash, slag and deposited dust were determined at three municipal waste incineration plants A, B and C equipped with continuously burning incinerators of stoker type. Three incinerators were operated at plants A and B, and four at plant C. The incinerators of plants A and B were equipped with electrostatic precipitators for removing particulate matter from the flue gas stream. In plant C, three incinerators were equipped with electrostatic precipitators and one with a bag filter. Total dust exposure concentrations during daily operation of the incinerators were 0.11 to 1.50 mg/m3, and dioxin concentrations in the deposited dust were 1.0 to 6.4 ng toxicity equivalent (TEQ)/g. Dioxin exposure concentrations were estimated to be 0.5 to 7.1 pg TEQ/ m3. Total dust exposure concentrations during periodic cleaning of the inside of the incinerator were 30 to 780 mg/m3, and dioxin concentrations in the slag were 0.004 to 1.1 ng TEQ/g. Dioxin exposure concentrations were estimated to be 0.5 to 48 pg TEQ/m3. Total dust exposure concentrations during periodic cleaning of the inside of the electrostatic precipirator were 51 to 2,000 mg/m3, and dioxin concentrations in the fly ash were 7.3 to 64 ng TEQ/g. Dioxin exposure concentrations were estimated to be 370 to 92,000 pg TEQ/m3, which were 150 to 37,000 times as high as the dioxin administrative level (2.5 pg TEQ/m3). Total of occupational and environmental dioxin intakes for an incinerator worker is predicted to exceed the tolerable daily intake (TDI: 4 pg TEQ/kg/d), when dioxin concentration in fly ash is 1,000 ng TEQ/g, even if an airline mask is used during the periodic maintenance. Consequently, it is essential to decrease the dioxin concentration in fly ash. When the dioxin concentration in fly ash is no higher than 100 ng TEQ/g, if an airline mask is used during the periodic maintenance, the dioxin intake is predicted to be lower than the TDI. Thus, wearing an airline mask would be effective.

Estimation of Dioxin Exposure Concentrations and Dioxin Intakes of Workers at Continuously Burning Municipal Waste Incinerators: Shinji Kumagai, et al. Department of Occupational Health, Osaka Prefectural Institute of Public Health—In order to examine the dioxin exposure level in incinerator workers, total dust concentration in the breathing zone and dioxin concentrations in fly ash, slag and deposited dust were determined at three municipal waste incineration plants A, B and C equipped with continuously burning incinerators of stoker type. Three incinerators were operated at plants A and B, and four at plant C. The incinerators of plants A and B were equipped with electrostatic precipitators for removing particulate matter from the flue gas stream. In plant C, three incinerators were equipped with electrostatic precipitators and one with a bag filter. Total dust exposure concentrations during daily operation of the incinerators were 0.11 to 1.50 mg/m3, and dioxin concentrations in the deposited dust were 1.0 to 6.4 ng toxicity equivalent (TEQ)/g. Dioxin exposure concentrations were estimated to be 0.5 to 7.1 pg TEQ/ m3. Total dust exposure concentrations during periodic cleaning of the inside of the incinerator were 30 to 780 mg/m3, and dioxin concentrations in the slag were 0.004 to 1.1 ng TEQ/g. Dioxin exposure concentrations were estimated to be 0.5 to 48 pg TEQ/m3. Total dust exposure concentrations during periodic cleaning of the inside of the electrostatic precipirator were 51 to 2,000 mg/m3, and dioxin concentrations in the fly ash were 7.3 to 64 ng TEQ/g. Dioxin exposure concentrations were estimated to be 370 to 92,000 pg TEQ/m3, which were 150 to 37,000 times as high as the dioxin administrative level (2.5 pg TEQ/m3). Total of occupational and environmental dioxin intakes for an incinerator worker is predicted to exceed the tolerable daily intake (TDI: 4 pg TEQ/kg/d), when dioxin concentration in fly ash is 1,000 ng TEQ/g, even if an airline mask is used during the periodic maintenance. Consequently, it is essential to decrease the dioxin concentration in fly ash. When the dioxin concentration in fly ash is no higher than 100 ng TEQ/g, if an airline mask is used during the periodic maintenance, the dioxin intake is predicted to be lower than the TDI. Thus, wearing an airline mask would be effective.

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology andacidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (Δcof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of Δcof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of Δcof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-Δcof double mutant has no significant dominant negative effect. Finally, the expression of the Δcof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.

An acrylate monomer with a pendant biguanide, N'-4-(2-acryloyl- oxyethyl)phenyl-N5-4-chlorophenylbiguanide hydrochloride (2), when incor porated covalently into cross-linked polyacrylamide matrices was found to hydrolyzed in water to release N1-4-(2-hydroxyethyl)phenyl-N5-chlorophenylbi guanide hydrochloride (1). When the degree of cross-linking was high, the release was approximately zero-order at least over a period of one week. In con trast to 2, methacrylate and acrylamide monomers with pendant biguanide groups were strongly resistant to hydrolysis. Detailed release profiles from various cross-linked polyacrylamide films containing covalently or non- covalently bound biguanide compounds indicate that the rate of release can be tailored to specific delivery needs.

Uroguanylin, a 16-amino acid peptide, is an endogenous activator of intestinal and possibly renal guanylate cyclase C (GC-C). Using two synthetic topological isoforms of human uroguanylin, one bioactive, the other inactive, we prepared two antisera specific for the individual isoforms and developed sensitive radioimmunoassays (RIAs). The respective plasma concentrations of the bioactive and inactive uroguanylins in the normal individuals tested were 5.0 ± 0.3 fmol/ml (mean ± SE) and 1.6 ± 0.1 fmol/ml. These concentrations increased in chronic renal failure (CRF). The major endogenous uroguanylin molecule in normal human urine was 16 amino acids long, whereas in the plasma and urine of CRF patients the major molecule was 10-kDa prouroguanylin. The RIAs established are promising tools for clarifying the physiological functions and pathophysiological implications of uroguanylin in water and electrolyte homeostasis.

Here we report that we could obtain a highly differentiated smooth muscle cell line by screening the expression of a-smooth muscle actin from p53 knock out mice aorta. This cell revealed extended bipolar shape and expressed h-caldesmon and calponin as well as a-smooth muscle actin as protein markers of differentiated smooth muscle. Further intracellular calcium increase was induced by application of noradrenaline in a dose dependent manner and calcium oscillation was also observed in a higher dose (100μM). Appropriate application of 5-azacytidine enhanced these tendencies and induced slow contraction by endothelin-1 and phenylephrine.

Uroguanylin, a member of the guanylin peptide family, is an endogenous activator of intestinal guanylate cyclase (GC-C). A cDNA encoding a precursor for human uroguanylin was cloned from a human colon cDNA library and sequenced. The precursor was 112 amino acids long and included a signal peptide at the N-terminus and the human uroguanylin sequence at the C-terminus. RNA blot analysis and the reverse transcription-polymerase chain reaction (RT-PCR) showed that human uroguanylin mRNA is expressed in the stomach and intestine. Uroguanylin, as well as guanylin, may be a potent physiological regulator of intestinal fluid and electrolyte transport.

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.

The cross-linked polyacrylamide films containing covalently-bonded N'-4- (2-acryloyloxyethyl)phenyl-N5-4-chlorophenylbiguanide hydrochloride (CPF-2) exhibit antibacterial activity at their surfaces and successfully act as self- sterilizing materials. Scanning electron microscopy studies indicate that inoc ulated cells of E. coliand S. aureusonto CPF-2 films undergo morphological changes, such as shrinkage and deformation, while those inoculated on blank cross-linked films remain intact. These morphological changes were ascribed to the collapse of the bacterial cells due to the action of released N1-4-(2-hydroxy ethyl)phenyl-N5-4-chlorophenylbiguanide hydrochloride from CPF-2by hy drolysis.

New oligomers of in-chain quaternary ammonium salts as well as polymers were examined for antibacterial activity with special attention to the effect of their molecular weights. Two sets of homologs with different spacer structures were prepared: one with a rigid p-xylylene spacer and the other with a flexible hexamethylene spacer. It was found that the activity depends strongly on the molecular weight and that both bacteriostatic and bacteri cidal activity increase in the order of monomer — dimer < trimer < tetramer < polymer.

Induced circular dichroism (ICD) in the mixed bilayer of azobenzene amphiphiles with dipalmitoyl-l-α-phosphatidylcholine was closely related to membrane fluidity controlled by trans–cis photoisomerization of the chromophore. Degree of the photochemical perturbation on membrane properties was the strongest when the azobenzene moiety was located in the middle of the long alkyl chain.

The potential of chitosan granules as an oral sustained-release dosage form of indomethacin has been compared with conventional capsules in beagle dogs. When a commercial capsule was administered orally, the plasma concentrations reached the maximum level in 30 min. The granules did not give a sharp peak to the plasma concentration, but produced a sustained plateau of the drug. This may be due to the slow rate of release and a longer residence time in the stomach. Thus, in terms of reducing the peak in plasma concentration and maintenance of drug concentration in plasma, the chitosan granules were superior to conventional capsules.

We measured force-distance curves of polystyrene (PS)/poly (vinyl methyl ether) (PVME) blend thin films using atomic force microscopy (AFM) in order to pursue the possible usage of AFM as a tool for detecting viscoelastic properties of polymeric materials from a nanoscopic point of view. In quasi-static measurements of force-distance curves for a sample whose PS content equals 100%, both adhesive force and capillary force were measured separately. A phenomenon possibly assigned to pulling off of polymer chains by an AFM tip could also be observed for a sample whose PS content equals 60%. By changing the velocity of the AFM tip acting on a blend sample whose PS content equals 40%, we confirmed that the law of time-temperature reducibility holds even on such a nanoscopic scale. This blend sample behaved as a viscous fluid at room temperature, while its behavior became glassy when faster movements of the AFM tip were applied. A discussion on the future development of a new field of research which should be called "nanorheology" was also presented.