Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3.

Skin lesions in a patient with IgG4-related disease.

IgG4-related disease (IgG4-RD) is a newly recognized condition that is characterized by raised levels of serum IgG4, tissue infiltration of IgG4-positive plasma cells and presence of fibrosis. It affects multiple organs, including the pancreas, bile duct, and lacrimal and salivary glands. Skin lesions have rarely been reported, and those that have were described as distributed mainly in the head and neck region. We report a case of IgG4-RD with autoimmune pancreatitis and skin lesions on the trunk and limbs. The skin lesions responded well to oral prednisolone (PSL); however, tapering of PSL to 5 mg/day resulted in recurrence. At present, the skin disease is well controlled at a dose of 7 mg/day. Interestingly, IgG4 levels fluctuated with changes in the PSL dose but did not coincide with the severity of the skin disease, implying that the raised levels of IgG4 did not directly influence the skin symptoms.

Immunohistochemical expression of heat shock proteins in the mouse periodontal tissues due to orthodontic mechanical stress.

The histopathology of periodontal ligament of the mouse subjected to mechanical stress was studied. Immunohistochemical expressions of HSP27 and p-HSP27 were examined. Experimental animals using the maxillary molars of ddY mouse by Waldo method were used in the study. A separator was inserted to induce mechanical stress. After 10 minutes, 20 minutes, 1 hour, 3 hours, 9 hours and 24 hours, the regional tissues were extracted, fixed in 4% paraformaldehyde and 0.05M phosphate-buffered fixative solution. Paraffin sections were made for immunohistochemistry using HSP27 and p-HSP27. In the control group, the periodontal ligament fibroblasts expressed low HSP27 and p-HSP27. However, in the experimental group, periodontal ligament fibroblasts expressed HSP27 10 minutes after mechanical load application in the tension side. The strongest expression was detected 9 hours after inducing mechanical load. p-HSP27 was also expressed in a time-dependent manner though weaker than HSP27. The findings suggest that HSP27 and p-HSP27 were expressed for the maintenance of homeostasis of periodontal ligament by the activation of periodontal ligament fibroblasts on the tension side. It also suggests that these proteins act as molecular chaperones for osteoblast activation and maintenance of homeostasis.

Two-dimensional gel electrophoresis analysis in simultaneous influenza pneumonia and bacterial infection in mice.

Severe pneumonia is found in simultaneous influenza pneumonia and bacterial infection, and suggests a relationship with immunological mechanisms. Here, we performed two-dimensional gel electrophoresis to detect immunological molecules related to the fulminant pneumonia caused by influenza virus and Streptococcus pneumoniae co-infection in mice. We found two spots that were expressed strongly in co-infected mouse lungs, compared with S. pneumoniae or influenza virus singly infected mouse lungs. The spots were analysed by mass spectrometry, and identified as alpha-1 anti-trypsin (A1AT), known as an anti-protease for neutrophil-derived proteolytic enzymes, and creatine kinase, which reflects a greater degree of lung damage and cell death. A1AT expression was increased significantly, and proteolytic enzymes from neutrophils, such as neutrophil elastase, myeloperoxidase and lysozyme, were also secreted abundantly in influenza virus and S. pneumoniae co-infected lungs compared with S. pneumoniae or influenza virus singly infected lungs. These data suggest that A1AT may play a central role as a molecule with broad anti-inflammatory properties, and regulation of the neutrophil-mediated severe lung inflammation is important in the pathogenesis of co-infection with influenza virus and bacteria.

Jagged1 peptide appearing in mandibular condylar cartilage development.

We investigated the expression pattern of Jagged1 peptide in mandibular condylar cartilage, as a type of secondary cartilage. Mandibular condyle of ddY mice were fixed from embryonic day 15 (E15) through just after birth (equivalent to E19). Serial sections were examined using histological immunohistochemical (IHC) techniques. At E15, the proliferating cells had positive products of Jagged1 in their cytoplasms and cell membrane of almost all coagulating cells. At E17, cytoplasmic and membranuous reactions of Jagged1 factors appeared strongly in the cells just inside the condylar cartilage sheath. At E18, Jagged1 positive products were observed in almost all cells of the layers, and they were mostly distinct in the sheath of the condyle. At just after birth, Jagged1 was observed in a portion of almost all layer cells in their cytoplasm and membrane. These results suggest that Jagged1 plays an essential role for mandibular condylar cartilage morphogenesis and development.

Gene expression of Jagged2 in mandibular condylar cartilage development.

Expression pattern of Jagged2 gene in mandibular condylar cartilage was examined by means of in situ hybridization (ISH) technique. At E14, Jagged2 mRNA signals appeared in cytoplasm of proliferating chondrocytes. From E15 to E19, Jagged2 mRNA was detected throughout almost all cytoplasm in all layers. However, the distribution pattern was not uniform. These results suggest that Jagged2 plays an essential role for mandibular condylar cartilage morphogenesis and development.

Role of Msx2 as a promoting factor for Runx2 at the periodontal tension sides elicited by mechanical stress.

Early changes of Runx2 and Msx2 expressions were examined by immunohistochemistry in mouse periodontal ligament exposed to mechanical stress. 8-week-old ddY mouse was used as experimental animal. To provide a continuous mechanical stress on periodontal ligament, rubber dam sheet was placed between upper molars of the mouse. At 20 minutes, 1 hour, 3 hours, 9 hours and 24 hours after insertion of the sheet, relevant parts of the mouse tissues were excised and fixed in 4% paraformaldehyde/0.05M phosphate buffered fixative solution. Then serial paraffin sections were prepared and histopathological evaluation as well as examination of Runx2, Msx2 and alkaline phosphatase (ALP) expressions by immunohistochemistry were performed. Control animals were not subjected to mechanical stress. In the experimental group, strong expressions of Runx2 and Msx2 were seen in periodontal fibroblasts of the tension side at 20 minutes after mechanical stress. Expressions of Runx2 and Msx2 became stronger in parallel with time, and at 24 hours after mechanical stress, the periodontal fibroblasts, cementoblasts as well as osteoblasts showed strong expression. Moreover, ALP has also demonstrated similar strong expression. On the other hand, in the control group, although expressions of Runx2, Msx2 and ALP were detected at all the experiment times, the expressions were weak. All these results strongly suggested that Runx2 promoted differentiation of osteoblasts at early stage and Msx2 worked as an activator of Runx2 function.

Gabexate mesilate suppresses influenza pneumonia in mice through inhibition of cytokines.

Gabexate mesilate is a synthetic protease inhibitor that is effective for acute pancreatitis. The effect of gabexate mesilate in influenza pneumonia in mice was investigated by examining the changes in pulmonary inflammatory cytokines and chemokines. Pathological changes in the lungs of treated mice were extremely mild, compared with changes in infected, untreated mice. Intrapulmonary levels of interleukin-6 and macrophage inflammatory protein-2 decreased in treated mice compared with untreated mice, despite similar viral titres in the lungs. Survival terms for treated and untreated groups were similar. These data indicate that gabexate mesilate has beneficial effects on influenza pneumonia, which may be due to the modulation of inflammatory cytokine/chemokine responses.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

Notch signaling in mandibular condylar cartilage development.

OBJECTIVE: The purpose of this study was to investigate the expression pattern of Notch signaling in mandibular condylar cartilage, as a type of secondary cartilage. METHODS: Mandibular condyle of ddY mice were fixed from embryonic day 14 (E14) through just after birth (equivalent to E19). Samples were cut into 4 mum serial sections through the central area of the mandibular condyle at the sagittal plane. Serial sections were examined using histological, immunohistochemical (IHC) and in situ hybridization (ISH) techniques. RESULTS: At E14, there were no developmental features of mandibular condyle. At the distal upper portion of developmental mandibular bone, mesenchymal cell proliferation and condensation without metacholomatic reaction to toluidine blue (TB) were seen. At E15, mandibular condylar cartilage was clearly evident, as TB metacholomasia. In IHC specimens at E14, expression of Notch1 intracellular domain (NICD) was observed in the nuclei of coagulating mesenchymal cells. After E15, NICD appeared in the nuclei and the cytoplasms of cells. In ISH examination at E14, expressions of Notch1 mRNA appeared in cytoplasm of proliferating chondrocytes. From E15 to E19, Notch1 mRNA was detected throughout almost all cytoplasm in all layers. CONCLUSION: These IHC and ISH results suggest that Notch signaling plays an essential role for mandibular condylar cartilage morphogenesis and development.

Effects of MgADP on length dependence of tension generation in skinned rat cardiac muscle.

The effect of MgADP on the sarcomere length (SL) dependence of tension generation was investigated using skinned rat ventricular trabeculae. Increasing SL from 1.9 to 2.3 microm decreased the muscle width by approximately 11% and shifted the midpoint of the pCa-tension relationship (pCa(50)) leftward by about 0.2 pCa units. MgADP (0.1, 1, and 5 mmol/L) augmented maximal and submaximal Ca(2+)-activated tension and concomitantly diminished the SL-dependent shift of pCa(50) in a concentration-dependent manner. In contrast, pimobendan, a Ca(2+) sensitizer, which promotes Ca(2+) binding to troponin C (TnC), exhibited no effect on the SL-dependent shift of pCa(50), suggesting that TnC does not participate in the modulation of SL-dependent tension generation by MgADP. At a SL of 1. 9 microm, osmotic compression, produced by 5% wt/vol dextran (molecular weight approximately 464 000), reduced the muscle width by approximately 13% and shifted pCa(50) leftward to a similar degree as that observed when increasing SL to 2.3 microm. This favors the idea that a decrease in the interfilament lattice spacing is the primary mechanism for SL-dependent tension generation. MgADP (5 mmol/L) markedly attenuated the dextran-induced shift of pCa(50), and the degree of attenuation was similar to that observed in a study of varying SL. The actomyosin-ADP complex (AM.ADP) induced by exogenous MgADP has been reported to cooperatively promote myosin attachment to the thin filament. We hereby conclude that the increase in the number of force-generating crossbridges on a decrease in the lattice spacing is masked by the cooperative effect of AM.ADP, resulting in depressed SL-dependent tension generation.

Tissue reaction to poly (lactic-co-glycolic acid) copolymer membrane in rhBMP used rabbit experimental mandibular reconstruction.

A rabbit experimental mandibular defect was reconstructed with 1% atelocollagen gel including rhBMP-2 10microg and a covering a poly (lactic-co-glycolic acid) copolymer (PLGA) membrane. For this experiment, eight male rabbits were used and a histological study was conducted. Our study purpose was to examine the effects and fate of PLGA membrane during bone reconstruction. PLGA membrane was phagocytized by foreign body giant cells and macrophages in the healing course of reconstruction osteogenesis. These histological data suggest that the PLGA membrane was gradually absorbed and replaced by fibrous connective tissue or bone tissue. In the osteogenesis course, the outer periphery of the new bone was maintained by PLGA membrane without expansion.

Three-dimensional observation of reconstruction course of rabbit experimental mandibular defect with rhBMP-2 and atelocollagen gel.

For the experimental animals, eight rabbits were chosen. A bone defect was made and was filled with 1% atelocollagen gel including rhBMP-2 10 microg. The reconstruction course was observed using micro-computed tomography (muCT) in vivo. In muCT observation, the density was slightly elevated at the bone marrow side at day 7, and the phenomenon gradually expanded during the course of this experiment which lasted for 28 days. By utilized muCT, we could construct 3D images, and that process enabled us to visualize bone formation more closely. These data suggest that the experimental animal model muCT and 3D image are extremely useful for follow-up of reconstruction of animal bone defects and that the atelocollagen gel is effective as a carrier of rhBMP-2.

Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes.

The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported. These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components. At the end of the first 4 months in culture 2 sublines with different morphologies were identified. In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments. The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis. However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated. The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively. The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product. The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular.

Establishment of a human leiomyosarcoma cell line.

A cell line designated SKN was established from the human uterine leiomyosarcoma of a 52-year-old female. The cell line has grown well and the serial passages were successively carried out 82 times within 12 months. The monolayer cultured cells revealed anaplastic and pleomorphic features, and they multipled rapidly without contact inhibition. Electron microscope studies revealed myoibrils but no virus-like particles, while chromosomal studies showed that all cultured cells were hyperploid, the modal number was 112, and the marker chromosome was present. The cells were transplanted into an immune-depressed hamster cheek pouch and produced a histological leiomyosarcoma resembling the original tumor.

Length dependence of tension generation in rat skinned cardiac muscle: role of titin in the Frank-Starling mechanism of the heart.

BACKGROUND: At the basis of the Frank-Starling mechanism is the intrinsic ability of cardiac muscle to produce active tension in response to stretch. Titin, a giant filamentous molecule involved in passive tension development, is intimately associated with the thick filament in the sarcomere. Titin may therefore contribute to active tension development by modulating the thick filament structure when the muscle is elongated. METHODS AND RESULTS: Rat skinned right ventricular trabeculae were used. Passive tension at a sarcomere length (SL) of 2.0 to 2.4 micrometer was decreased after treatment of the preparation with trypsin (0.25 microgram/mL) for 13 minutes in the relaxed state at 20 degrees C. This mild trypsin treatment degraded titin without affecting other major contractile proteins. The sarcomere structure was little affected by brief contractions in the trypsin-treated preparations. When SL was adjusted to the slack SL (1.9 micrometer), active tension was unaffected by trypsin under partial (pCa 5.55) and maximal (pCa 4.8) activation. At longer SLs, however, active tension was significantly (P<0.01) decreased after trypsin treatment at either pCa. The increase in active tension on reduction of interfilament lattice spacing, produced by dextran T-500 (molecular weight approximately 500 000), was not influenced by trypsin (SL 1.9 micrometer). In trypsin-treated preparations, the increase in active tension as a function of muscle diameter was nearly the same for lengthening and osmotic compression at the slack SL. CONCLUSIONS: The length-dependent activation in cardiac muscle, an underlying mechanism of the Frank-Starling law of the heart, is at the myofilament level, predominantly modulated by titin and interfilament lattice spacing changes.

Evidence for association between the class I subset of the insulin gene minisatellite (IDDM2 locus) and IDDM in the Japanese population.

Although the shortest (class I) minisatellite (i.e., variable number of tandem repeats [VNTR]) alleles in the 5' region of the insulin gene are positively associated with IDDM in Caucasians, the majority of Japanese are homozygous for class I alleles. Here, we determined the exact length, in number of repeat units (RUs), of class I alleles in Japanese subjects. The distribution of class I alleles in Japanese was trimodal, with peaks located at 32/33, 41, and 44 RUs. The shortest component (i.e., 1S [25-38 RUs]) alleles were significantly increased in the IDDM group compared with the control group (54 vs. 46%; P = 0.040). The 1S/1S genotype was significantly increased in the IDDM patients (34 vs. 20%; P = 0.005; relative risk 2.1). Furthermore, the transmission disequilibrium test of Japanese families with 1S/1M or 1S/1L heterozygous parents confirmed the association of 1S alleles; 17 alleles of 1S and 6 alleles of 1M (39-41 RUs) or 1L (42-44 RUs) were transmitted to affected offspring (P = 0.022). In addition, we found tight linkage of 1S with allele 9 of the tyrosine hydroxylase gene microsatellite and allele (-) of the IGF-II gene Apa I polymorphism, but neither 9 nor (-) alleles were significantly associated with IDDM. The present study suggests that a class I subset may have a role in IDDM susceptibility in Japan. It was revealed that the difference between 1S alleles and 1M or 1L alleles is almost consistently characterized by a sequence variation generated by deletion of two copies of an ACAGGGGTCC CGGGG repeat element, implying that sequence variation of class I alleles may influence disease susceptibility.

Left ventricular regional function after acute anterior myocardial infarction in diabetic patients.

To elucidate the pathophysiological role of diabetes mellitus in determining the left ventricular regional function of the noninfarcted area, 55 patients with acute Q wave anterior myocardial infarction (MI) were studied. The regional ejection fraction of the noninfarcted area was obtained by radionuclide angiocardiography and was used to estimate the left ventricular regional function of the noninfarcted area. Multiple regression analysis was performed to determine the important variables contributing to the regional ejection fraction based on 10 clinical variables: age, sex, QRS score, diabetes mellitus, hypertension, smoking, postinfarction angina, body mass index, serum cholesterol, and coronary atherosclerosis. A high QRS score (P less than .001) and the association of diabetes mellitus (P less than .05) were the important factors contributing to regional left ventricular dysfunction. The regional ejection fraction and QRS score had an inverse linear relationship in the diabetic and nondiabetic groups, and the regional ejection fraction was significantly lower in diabetic patients at every QRS score (P less than .05). The association of hypertension, severity of coronary atherosclerosis, serum cholesterol level, age, and body mass index did not differ between diabetic and nondiabetic patients, which indicates that diabetes mellitus was not mediated through these atherogenic traits. Thus, diabetes mellitus is another discrete cause of regional left ventricular dysfunction of the noninfarcted area after acute MI.

Effect of developed tension on the time courses of Ca2+ transients and tension in twitch contraction in ferret myocardium.

OBJECTIVES: The aim of the study is to test the hypothesis that tension-dependent change in the affinity of cardiac troponin-C influences the time courses of Ca2+ transients and tension in twitch contraction. METHODS: The Ca(2+)-sensitive photoprotein, aequorin, was microinjected into superficial cells of ferret papillary muscles and the Ca2+ transients and tension were simultaneously measured. The peak of developed tension was altered by changing the extracellular Ca2+ concentration, initial muscle length, and the application of 2,3-butanedione monoxime. RESULTS: In each maneuver, the decay time of Ca2+ transients was prolonged and the tension relaxation time was shortened when the peak of developed tension was decreased. In contrast, when the peak of developed tension was increased, the decay time of Ca2+ transients was shortened and the tension relaxation time was prolonged. The decay time of Ca2+ transients measured with different maneuvers was negatively correlated with the peak tension and the tension relaxation time was positively correlated with the tension peak. CONCLUSIONS: The changes in the decay time of Ca2+ transients and the tension relaxation time indicate that developed tension modulates the affinity of troponin-C for Ca2+ in normal twitch contraction.

Effects of thapsigargin on aequorin-injected and skinned preparations of ferret ventricular muscles.

OBJECTIVE: We investigated the effects of thapsigargin (TG) (0.1-1 microM) on the relation between intracellular Ca2+ concentration and tension in ferret papillary muscles using aequorin-injected and skinned preparations. METHODS: Aequorin was injected into the superficial cells of ferret papillary muscles; the Ca2+ signals of aequorin and tension in twitch and those with the application of 15 mM caffeine were simultaneously measured. The alteration of Ca2+ sensitivity of the contractile elements was examined by measuring the pCa-tension relation in Triton-X-treated skinned preparations. RESULTS: TG decreased the peak of the Ca2+ signal accompanied by a prolonged decay time. However, the tension was scarcely altered even at 1 microM TG. TG inhibited the caffeine-induced Ca2+ signal. Prolongation of decay of the Ca2+ signal by TG in twitch was further enhanced by isoprenaline (10 nM). The pCa-tension relation of the skinned preparation was slightly but significantly shifted to the right by TG. CONCLUSIONS: The apparent dissociation of the effects of TG on the Ca2+ signal and tension in intact preparations is not a result of alteration of the Ca2+ sensitivity of the myofilaments. The effects of TG in multicellular preparations are probably limited to the outer layer of the preparation. The slower time course of the Ca2+ signal induced by TG is due to the inhibition of Ca2+ uptake by sarcoplasmic reticulum, which is more significantly observed when the intracellular Ca2+ transient is increased by isoprenaline.