Vascular image in autoimmune pancreatitis by contrast-enhanced color-Doppler endoscopic ultrasonography: Comparison with pancreatic cancer.

INTRODUCTION: In autoimmune pancreatitis (AIP), veins of various sizes are highly affected by obliterative phlebitis without damage to arteries, in contrast, the involvement of both arteries and veins is observed in the mass of pancreatic cancer. A vascular image without blooming artifact in the pancreas is clearly observed in the directional eFLOW (Prosound α10, Aloca Co., Tokyo, Japan) color mode using contrast-enhanced color-Doppler endoscopic ultrasonography (CC-EUS) despite perfusion of the contrast media. AIMS AND METHODS: The aim of this study was to compare the vascular structure of AIP with that of pancreatic cancer using CC-EUS. We evaluated the perfusion image and the vascular image of the mass in AIP patients (11) with an increase in serum IgG4 levels (477.3 ± 314.2 IU/mL) and in pancreatic cancer patients (11) with elevated serum CA19-9 levels (49839.0 ± 80061.6 mg/dl), on CC-EUS. Perfusion images were obtained at 20-30 s after injection of a contrast agent, Sonazoid (GE Healthcare AG, Oslo, Norway), by extended pure harmonic detection mode and were assessed as to homogeneity or heterogeneity (containing partial low echoic areas or multiple spotty low echoic areas) enhancement. The vascular image was assessed in the directional eFLOW color mode despite perfusion of the contrast media (40-50 s after injection of Sonazoid) as to the presence of a dendritic vessel network or only a few feeder vessels. The parameters for imaging were as follows: Mechanical index, 0.22-0.24; transmission frequency, 5.0 MHz; and receiving frequency, 5.0 MHz. The Chi-square test or Fisher's exact test was used for comparison of categorical data of the two groups when appropriate. This study was approved by the institutional review board of Sendai City Medical Center. All subjects gave informed consent. RESULTS: A homogenous pattern in perfusion imaging was seen in 73% of patients with AIP (8/11) and 55% of those with pancreatic cancer (6/11). The rates were not significantly different between the two groups (P = 0.33). In the other patients with a heterogenous pattern, multiple spotty low echoic areas were seen in 33% (1/3) and 80% (4/5) in each group, respectively. A dendritic vascular pattern in the eFLOW color mode was seen in 82% (9/11) of patients with AIP, but was not seen in any of patients with pancreatic cancer. The other patients with AIP (18%) and all patients with pancreatic cancer showed only a few feeder vessels in the mass on CC-EUS. CONCLUSION: The eFLOW color mode using Sonazoid may be useful for evaluating the vascular structure of AIP for differential diagnosis from pancreatic cancer.

Lower extremity biomechanics during kendo strike-thrust motion in healthy kendo athletes.

AIM: The aim of this study was to demonstrate the kinematics and kinetics of the lower extremity during the kendo strike-thrust motion in experienced kendo athletes. METHODS: Fifteen experienced kendo athletes (age 20.4±1.2 years; height 171.5±4 cm; weight 73.9±9.1 kg; the kendo experience 11.1±3.1 years) volunteered to participate in the study. The three-dimensional kinematic and kinetic data was collected by the motion analysis system with eight cameras and with a force platform. We instructed the participants to perform three sets of kendo motion at the distances of 1.8 m, 2 m, and 2.2 m to the target. We then obtained the joint kinematic and kinetic data of the ankle dorsiflexion-planterflexion, foot pronation-supination, knee flexion-extension, and hip flexion-extension during the single support phase. The peak foot pronation angle and the range of motion (ROM) of foot pronation were also calculated. RESULTS: The result demonstrated the high intra-subject repeatability of the joint angle and the torque curve of the left lower extremity during the single support phase in the kendo motion. Our result also showed that as for the peak foot pronation angle and the pronation ROM there was no significant difference between different distances to the target. CONCLUSION: We provided the basic biomechanical information during the kendo strike-thrust motion, and the result will help us to understand the Achilles tendon injury occurrence in kendo athletes.

The common mechanisms of anterior cruciate ligament injuries in judo: a retrospective analysis.

BACKGROUND: Although high prevalence of anterior cruciate ligament injuries (ACL) in judokas has been reported, there has been very little research concerning events preceding the injury. OBJECTIVE: To determine the common situations and mechanisms of ACL injury in judo. METHODS: A total of 43 cases of ACL injuries that had occurred during judo competition or practice were investigated, using questionnaires with interviews conducted by a single certified athletic trainer who has 20 years of judo experience to obtain information regarding the situation and mechanism in which the ACL injury occurred. RESULTS: The number of ACL injuries when the participant's grip style was different from the style of the opponent (ie, kenka-yotsu style) (28 cases) was significantly greater than when the participant's grip style was the same as that of the opponent (ie, ai-yotsu style) (15 cases; p<0.001). The number of ACL injuries was significantly higher when the participant was attacked by the opponent than when counterattacked or when attempting the attack (p<0.001). In addition, being attacked with osoto-gari was revealed as the leading cause of ACL injury incidence among the participants (16.8%). CONCLUSIONS: Grip style may be associated with ACL injury occurrence in judo. In addition, direct contact due to the opponent's attack may be a common mechanism for ACL injuries in judo.

Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos.

The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organised into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signalling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signalling is still unclear, owing to overlapping functions of Fgf family genes. To address this, we have examined, in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signalling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signalling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. We then examine the function of Fgf signalling in telencephalic development by use of several inhibitors to Fgf signalling cascade, including dominant-negative forms of Ras (Ras(N17)) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeded but the development of the subpallial telencephalon was suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development.

Synthesis of heparin partial structures and their binding activities to platelets.

A synthetic pentasaccharide corresponding to the antithrombin III-binding region in heparin was also found to bind to human platelets. To identify the platelet-binding site in the pentasaccharide which is expected to be a novel sequence in heparin responsible for its platelet-binding, five partial structures of this particular pentasaccharide were synthesized. In a competitive assay using [3H]-heparin, a trisaccharide, O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glucopyranosyl)-1--> 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranose, was concluded to be a high-affinity site for heparin's binding to platelets.

Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF.

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface.

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C-protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C-proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C-protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue-specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP-C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity.

This study analyzed the spatial and temporal expression pattern of zebrafish wnt11 and the regulation of the expression during zebrafish early development, focusing on the interaction with the no tail (ntl) gene, a zebrafish orthologue of mouse Brachyury (T). Zygotic expression of wnt11 was first detected at the late blastula stage in the blastoderm margin, a presumptive mesoderm region. wnt11 expression coincided with mesoderm induction, and the expression was induced by mesoderm inducers such as the yolk cell (Mizuno, T., Yamaha, E., Wakahara, M., Kuroiwa, A., Takeda, H., 1996. Mesoderm induction in zebrafish. Nature 383, 131-132) or FGFs, indicating that, like ntl, wnt11 is one of the immediate-early genes in mesoderm induction. Initial expression domains of wnt11 and ntl overlapped, and these genes showed a similar response to mesoderm inducers. However, analysis of the ntl mutant embryos suggested that wnt11 and ntl are placed in distinct genetic pathways; the ntl mutation had no effect on wnt11 expression in the blastoderm margin. This was further supported by the result of RNA injection experiments showing that overexpression of Wnt11 did not affect ntl expression in the margin. Thus, wnt11 and ntl expression are induced and maintained independently in their initial phase of expression. In later stages, wnt11 was expressed in various organs, such as the somites, particularly in the developing notochord. Since no wnt gene has been reported to be expressed in the axial mesoderm, which is known to act as a signaling source that patterns the neural tube and somites, zebrafish wnt11 is the first wnt gene expressed in the notochord. Furthermore, in contrast to early expression, wnt11 expression in the notochord depended on Ntl activity. In the ntl mutant in which somite patterning is severely affected, wnt11 expression was completely lost, while another signaling molecule, sonic hedgehog is expressed in the mutant notochord precursor cells (Krauss, S., Concordet, J.-P., Ingham, P.W., 1993. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444). wnt11 expression in the somite also shows a characteristic pattern, correlated with the migration and differentiation of slow muscle precursors. These observations suggest a role for wnt11 in patterning the somites.

Initial anteroposterior pattern of the zebrafish central nervous system is determined by differential competence of the epiblast.

Analyses using amphibian embryos proposed that induction and anteroposterior patterning of the central nervous system is initiated by signals that are produced by the organizer and organizer-derived axial mesoderm. However, we show here that the initial anteroposterior pattern of the zebrafish central nervous system depends on the differential competence of the epiblast and is not imposed by organizer-derived signals. This anteroposterior information is present throughout the epiblast in ectodermal cells that normally give rise both to neural and non-neural derivatives. Because of this information, organizer tissues transplanted to the ventral side of the embryo induce neural tissue but the anteroposterior identity of the induced neural tissue is dependent upon the position of the induced tissue within the epiblast. Thus, otx2, an anterior neural marker, was only ever induced in anterior regions of the embryo, irrespective of the position of the grafts. Similarly, hoxa-1, a posterior neural marker was induced only in the posterior regions. Furthermore, the boundary of each ectopic expression domain on the ventral side was always at an equivalent latitude to that of the endogenous expression of the dorsal side of the embryo. The anteroposterior specification of the epiblast is independent of the dorsoventral specification of the embryo because neural tissues induced in the ventralized embryos also showed anteroposterior polarity. Cell transplantation and RNA injection experiments showed that non-axial marginal mesoderm and FGF signalling is required for anteroposterior specification of the epiblast. However, the requirement for FGF signalling is indirect in that cells with compromised ability to respond to FGF can still respond to anteroposterior positional information.

Epimorphin functions as a key morphoregulator for mammary epithelial cells.

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

Identification of cellular recognition sequence of epimorphin and critical role of cell/epimorphin interaction in lung branching morphogenesis.

A mesenchymal protein, epimorphin, is known to bind directly to the cell surface through its central portion and to act as a signaling molecule for epithelial morphogenesis. Utilizing several recombinant polypeptides and synthetic peptides, we identified the cellular recognition sequence of epimorphin in the central portion of this molecule (amino acids 105-123, NGNRTSVDLRIRRTQHSVL; termed NL-peptide sequence). Interestingly, although a model cell type bound to the NL-peptide as strong as to the full-length epimorphin, this peptide itself didn't induce the cellular functional responses so far tested. We found that the NL-peptide behaved as an antagonist for the endogenous epimorphin and severely perturbed lung branching morphogenesis in organ culture. These results not only revealed a part of the functional mechanism of epimorphin but also demonstrated that cell/ epimorphin interaction through the NL-peptide sequence is a critical step for lung epithelial morphogenesis.

Complete primary structure of chicken cardiac C-protein (MyBP-C) and its expression in developing striated muscles.

C-protein (MyBP-C) is a myosin binding protein of about 140 kDa which is known to modulate myosin assembly in striated muscles. A cardiac-type isoform of C-protein appears not only in cardiac muscle but also in skeletal muscle before skeletal muscle-type isoforms become detectable during myogenesis, suggesting that the cardiac isoform is involved in the early phase of myofibrillogenesis (Bähler et al., 1985; Kawashima et al., 1986). In this study, in order to understand the structure and functional domains of the cardiac-type C-protein, we cloned and sequenced full-length cDNAs encoding chicken cardiac C-protein from lambda gt11 cDNA libraries which were prepared with poly (A)+ RNA from embryonic chicken cardiac muscle as well as embryonic chicken skeletal muscle by using antibodies specific for cardiac C-protein. Two cDNA variants, probably generated by alternative RNA splicing and encoding different C-protein isoforms, were detected. As judged by the cDNA sequences determined, overall homology of the peptide sequence between cardiac and skeletal muscle C-proteins (Einheber et al., 1990; Fürst et al., 1992, Weber et al., 1994) was about 50-55%. Like other myosin binding proteins, skeletal C-proteins, 86 kDa protein and M-protein, cardiac C-protein contains several copies of fibronectin type III motifs and immunoglobulin C2 motifs in the molecule, but their number and arrangements differed somewhat from those in the other proteins. Northern blot analysis with the cloned cDNA as a probe demonstrated that mRNA of 5.0 kb is transcribed in both cardiac and embryonic skeletal muscle, and that it is specifically expressed in cardiac muscle among adult tissues.

Assembly of cardiac C-protein during myofibrillogenesis in myogenic cells in culture.

To examine the function of C-protein, a thick filament-associated protein of vertebrate striated muscles, during myofibrillogenesis, the cDNA encoding chicken cardiac C-protein and the truncated cDNA were subcloned into a expression vector and introduced into mouse C2 myogenic cells. The expression and assembly of the C-protein was investigated by immunofluorescence methods. When the cDNA containing the entire open reading frame was introduced, in C2 myoblasts, the transiently expressed exogenous cardiac C-protein existed only diffusely in the cytoplasm, but it became localized in striated structures together with sarcomeric myosin heavy chains (MHC) in myotubes. To clarify the functional domains of C-protein, the cDNA constructs that lack the regions encoding the C-terminal immunoglobulin (Ig) C2 motif or the N-terminal Ig C2 motif were introduced into C2 cells to produce mutant proteins. The truncated chicken cardiac C-protein, which lacked the C-terminal Ig C2 motif, apparently lost the ability to bind to myosin filaments; the protein was not assembled into myofibrils but diffused in the cytoplasm even in the myotubes. The protein without N-terminal Ig C2 motif, however, was assembled into sarcomeric structures just as complete protein molecules. From these results, we conclude that 1) the assembly of sarcomeric MHC into myofibrils in myotubes is accompanied with that of cardiac C-protein, and 2) the C-terminal Ig C2 motif is necessary for assembly of cardiac C-protein in sarcomeric structures in the cytoplasm.