Treatment with YIGSR peptide ameliorates mouse tail lymphedema by 67 kDa laminin receptor (67LR)-dependent cell-cell adhesion.

Impaired microcirculation can cause lymphatic leakage which leads to a chronic swelling in the tissues of the body. However, no successful treatment gives any protection against lymphedema due to the lack of well-revealed pathophysiology of secondary lymphedema. Binary image of laminin immunohistochemical expression revealed that distribution of laminin expression localized during surgically induced lymphedema. 67 kDa laminin receptor (67LR) mRNA expression showed a peak at during lymphedema exacerbation. Since the response of 67LR molecules may affect the prevention of inflammation and edema, here we have hypothesized that 67LR ligand of YIGSR peptide could permit reconstructive environment for amelioration of lymphedema and evaluated the effect of YIGSR in a mouse tail model of lymphedema. Indeed, intra-abdominal injections of YIGSR for the first 3 days after inducing lymphedema in the mouse tail model reduced the tail lymphedema on day 14 by 27% (P = 0.035). Histology showed that YIGSR treatment protected lymphedema impairment in epidermis and dermis, and it also inhibited the expansion of intercellular spaces and enhanced especially cell adhesion in the basement membrane as revealed by transmission electron microscopy. Interestingly, the treatment also reduced the local expression of transforming growth factor (TGF)ß. Further elucidation of the mechanisms of 67LR-facilitated lymphangiogenesis contributes to find potential targets for the treatment of lymphedema.

Synovial mesenchymal stem cells promote healing after meniscal repair in microminipigs.

OBJECTIVE: The induction of synovial tissue to the meniscal lesion is crucial for meniscal healing. Synovial Mesenchymal stem cells (MSCs) are an attractive cell source because of their high proliferative and chondrogenic potentials. We examined whether transplantation of synovial MSCs promoted healing after meniscal repair of extended longitudinal tear of avascular area in a microminipig model. DESIGN: Longitudinal tear lesion was made in medial menisci and sutured in both knees, and then a synovial MSC suspension was administered for 10 min only in unilateral knee. The sutured meniscus was evaluated morphologically and biomechanically at 2, 4, and 12 weeks. The behavior of transplanted MSCs was also examined. RESULTS: The meniscal healing at 12 weeks was significantly better in the MSC group than in the control group; macroscopically, histologically and by T1rho mapping analysis. Transmission electron microscopic analysis demonstrated that the meniscus lesion was occupied by dense collagen fibrils only in the MSC group. Biomechanical analysis revealed that the tensile strength to failure of the meniscus higher in the MSC group than in the control group in each microminipig. Synovial tissue covered better along the superficial layer from the outer zone into the lesion of the meniscus in the MSC group at 2 and 4 weeks in each microminipig. Synovial MSCs labeled with ferucarbotran were detected in the meniscus lesion and adjacent synovium by MRI at 2 weeks. CONCLUSION: Transplantation of synovial MSCs promoted healing after meniscal repair with induction of synovium into the longitudinal tear in the avascular zone of meniscus in pigs.

Initial fibroblast attachment to Erbium:YAG laser-irradiated dentine.

AIMS: To evaluate the effects of Erbium (Er):YAG laser irradiation on the morphology of resected dentine surfaces, and to investigate fibroblast attachment to laser-irradiated dentine surfaces. METHODOLOGY: Dentine blocks obtained from single-rooted human teeth were divided into the following groups after sterilization in an autoclave: (i) Laser group treated with Er:YAG laser irradiation (30 mJ per pulse, 10 pps, 60 s); (ii) L-MTAD group treated with laser irradiation as in (i) plus a mixture of doxycycline, tetracycline isomer and citric acid; (iii) RC-Prep group treated with EDTA gel or cream (RC-Prep) and (iv) Control group left untreated. After each treatment, the dentine blocks were incubated with NIH/3T3 fibroblasts cultured to subconfluency in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum and antibiotics. The number of attached cells amongst the groups was analysed statistically at the 5% significance level. The dentine surface morphologies and cell attachments were evaluated by counting assays, histological observations and scanning electron microscopy (SEM). RESULTS: The number of attached cells was significantly higher (P < 0.05) in the Laser group than in the RC-Prep and Control groups at 16 h. Dendritic cell extension of the fibroblasts was only observed in the Laser group at 8 h by SEM. In the histological analyses, significantly more attached cells were found on the dentine surfaces treated with laser irradiation. CONCLUSIONS: Er:YAG laser irradiation induced morphological alterations in dentine surfaces, which may improve the attachment of fibroblasts to dentine.

Magnesium enhances adherence and cartilage formation of synovial mesenchymal stem cells through integrins.

OBJECTIVE: We previously reported that more than 60% of synovial mesenchymal stem cells (MSCs) placed on osteochondral defects adhered to the defect within 10 min and promoted cartilage regeneration. The efficiency of adherence is considered to depend on the interaction between cells and extracellular matrix (ECM), in which integrins may play some important roles. Divalent cations such as calcium, magnesium, and manganese may affect functions of integrins, and the integrins may be involved in differentiation of MSCs. Among divalent cations, magnesium is used in clinical practice as a therapeutic agent and increases the affinity of integrin to ECM. In this study, we investigated whether magnesium enhanced adherence and chondrogenesis of synovial MSC through integrins. METHODS: We performed assays for adherence of human synovial MSCs to collagen-coated slides, in vitro chondrogenesis, ex vivo assays for adherence of human synovial MSCs to osteochondral defect, and in vivo assays for adherence and cartilage formation of synovial MSCs in a rabbit osteochondral defect model. RESULTS: Magnesium increased adhesion of human synovial MSCs to collagen, and this effect was inhibited by neutralizing antibodies for integrin α3 and ß1. Magnesium also promoted synthesis of cartilage matrix during in vitro chondrogenesis of synovial MSCs, which was diminished by neutralizing antibodies for integrin ß1 but not for integrin α3. Ex vivo analyses demonstrated that magnesium enhanced adherence of human synovial MSCs to osteochondral defects. In vivo studies in rabbits showed that magnesium promoted adherence at 1 day and cartilage formation of synovial MSCs at 2 weeks. CONCLUSION: Magnesium enhanced adherence of synovial MSCs through integrins, which promoted synthesis of cartilage matrix at an early phase.

Validity of mid-arm muscular area measured by anthropometry in nonobese patients with increased muscle atrophy and variation of subcutaneous fat thickness.

BACKGROUND: The anthropometric measurement of mid-arm muscular area (MAMA) involves overestimation because of various assumptions, this overestimation being progressive with increasing adiposity. However, the effects of muscle atrophy and variation of the subcutaneous fat thickness have remained uncertain. OBJECTIVES: The validity of MAMA estimated by anthropometry was examined by comparing with MAMA measured by computed tomography (CT) in a nonobese population. The effects of muscle atrophy and variation of the subcutaneous fat thickness on the validity of MAMA were examined by new indices. SUBJECTS/METHODS: The relative MAMA was compared between the anthropometric and CT methods in 45 patients. New indices were introduced for assessing muscle deformity (muscle deformity index, MDI) and subcutaneous fat variation (SFVI). The effects of MDI, SFVI and age on the difference of MAMA between the anthropometric and CT methods were investigated. RESULTS: MDIs were positively correlated with age in males (r=0.47, P<0.05) and females (r=0.66, P<0.001). SFVI was positively correlated with age only in females (r=0.54, P<0.01). Even in these patients, the relative MAMA estimated by anthropometry was significantly associated with that measured by CT (r=0.85, P<0.0001 in males and r=0.90, P<0.0001 in females). A Bland-Altman plot indicated that the difference between both methods was relatively small, although increased adiposity might be a source of overestimation for anthropometric MAMA measurement. CONCLUSIONS: MAMA estimated by anthropometry was a reliable indicator of muscle mass in patients with muscle atrophy and varying thickness of subcutaneous fat in lean patients.

Localization of matrix metalloproteinases (MMPs-2, 8, 9 and 20) in normal and carious dentine.

BACKGROUND: Dentine matrix metalloproteinases (MMPs) may participate in the destruction of dentine following demineralization by bacterial acids. This study investigated the localization of MMPs in carious dentine. METHODS: Frozen sections of dentine caries were prepared without demineralization and immersed in monoclonal antibody against MMP-2, -8, -9 and -20. The sections were labelled by IgG conjugated with gold colloidal particles, and observed under FE-SEM. Labelling indexes (number of gold particles/mum(2)) of outer and inner carious dentine, respectively, with and without bacterial infection, were compared with that of normal dentine. RESULTS: MMP-2 was distributed in both carious and normal dentine; the level of MMP-2 showed no significant difference among the outer caries, inner caries, and normal dentine. The labelling indexes of MMP-8 and MMP-9 both significantly decreased at the inner carious dentine compared with the level of normal dentine, but intensified again at the outer caries region. The labelling index of MMP-20 was the highest at normal dentine. CONCLUSIONS: The localization of MMPs was visibly detected using immunogold labelling. The localization of MMP-2 showed no significant difference among the three regions, while MMP-8 and MMP-9 showed significant reduction at the inner caries layer, and MMP-20 reduced toward the outer caries.

Protective effects of ebselen, a seleno-organic antioxidant on neurodegeneration induced by hypoxia and reperfusion in stroke-prone spontaneously hypertensive rat.

Cerebral ischemia followed by oxygen reperfusion induced apoptosis in hippocampal neurons in the stroke-prone spontaneously hypertensive rat (SHRSP) but not in Wistar Kyoto rats (WKY). We investigated whether 2-phenyl-1,2-benzisoselenazol-3(2H)-one, also called PZ51 (ebselen), useful for treating ischemic damage or antihypertension in the brain, can protect against ischemic neuronal damage in SHRSP. In this study, we compared the effects of ebselen, carvedilol, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) as well as vitamin E, added to cultures of neurons after reoxygenation (20% O(2)) following hypoxia (1% O(2)). SHRSP neurons died rapidly during reoxygenation following hypoxia but were rescued in large measure by 10 muM ebselen (neuronal death; 2.7+/-1.4%). In order of neuroprotective potency, the agents ranked as follows: ebselen>carvedilol>MCI-186>vitamin E. In vivo, strong neuroprotection by ebselen was observed in the hippocampal CA1 region of SHRSP (32.9+/-9.5 apoptotic neuron/1000 neurons, 30 mg/kg/day). Ebselen prevented apoptosis as confirmed by morphological observations in vivo. Its effect was associated with the expression of Bcl-2 and Bax. These findings suggest that ebselen has a marked inhibitory effect on neuronal damage during stroke. Ebselen may be effective in the prevention and/or treatment of neurodegenerative diseases associated with excessive apoptosis in patients with stroke.

High expression of GADD-45alpha and VEGF induced tumor recurrence via upregulation of IL-2 after photodynamic therapy using NPe6.

NPe6 is a novel second-generation photosensitizer used for photodynamic therapy (PDT). PDT using NPe6 and diode laser (664 nm) induces cell death, inflammatory reactions, immunological responses and damage to the microvasculature. In this study, we evaluated the influence of the immunological responses and of enhanced angiogenesis on the anti-tumor effect of NPe6-PDT using cytokine-overexpressing Lewis lung carcinoma (LLC), LLC-IL-2 cells both in vitro and in vivo. We showed by DNA microarray analysis in vitro that IL-2 and GADD-45alpha (growth arrest and DNA damage 45 alpha) mRNA expressions were induced by 3 h after NPe6-PDT applied at a dose killing 90% of the cells (LD90). IL-2-overexpressing cells (LLC/IL-2 cells) were resistant to the loss of clonogenicity as compared to the parental LLC cells in vitro. Furthermore, in female C57BL/6 mice, NPe6-PDT produced a cure rate of 66.7% in LLC tumors, whereas the cure rate was only 16.6% in LLC/IL-2 tumors, and overexpression of IL-2 caused failure of NPe6-PDT, with tumor recurrence, in vivo. These results suggest that IL-2 expression may play an unfavorable role in attenuation of the antitumor effect of NPe6-PDT. It has been reported that the expression of vascular endothelial growth factor (VEGF), in particular, may cause tumor recurrence after PDT and exert unfavorable effect in relation to attenuate the anti-tumor activity of PDT. Results of immunohistochemical analysis of LLC/IL-2 tumors have revealed that the expressions of GADD-45alpha and VEGF are induced in these tumors after PDT, and in particular, 12 h after PDT, the expression levels were much higher as compared with those in the LLC tumors. The results of our studies using in vitro and in vivo models suggest that the cell death caused by PDT was inhibited by induction of GADD-45alpha expression and that tumor recurrence was promoted by the enhancement of VEGF expression mediated by IL-2 upregulation. Therefore, it is speculated that the use of an IL-2 inhibitor may improve the efficacy of NPe6-PDT.

Effects of dentin characteristics on interfacial nanoleakage.

Water emanating from dentinal tubules during air-drying and light-curing of adhesives leads to entrapment of droplets at the resin-dentin interface and contributes to nanoleakage. This study tested the null hypothesis that characteristics of substrate dentin and type of adhesive used for bonding would not affect the occurrence of nanoleakage. Three self-etch adhesives were used to bond to 4 types of dentin with different characteristics in 12 groups. After silver challenge, nanoleakage percentage was measured within the hybrid layer of each sample. The deep dentin cut perpendicular to tubules always showed a significantly higher nanoleakage percentage compared with that of the other 3 types of dentin. The percentages of nanoleakage within the hybrid layers were not statistically different among adhesives. However, when bonding to deep perpendicular dentin, both all-in-one adhesives revealed more distinct nanoleakage within the adhesive layer compared with that achieved with Clearfil SE Bond, a two-step self-etch adhesive. The results did not support the null hypothesis.

A possible relationship between the anti-cancer potency of photodynamic therapy using the novel photosensitizer ATX-s10-Na(II) and expression of the vascular endothelial growth factor in vivo.

ATX-s10-Na(II) is a novel second-generation photo-sensitizer for photodynamic therapy (PDT). PDT using ATX-s10 and diode laser (670 nm) induces an apoptotic response, inflammatory reaction, immune reaction and damage to the microvasculature. In particular, the vascular shut-down effect plays an important role in the anti-tumor activity of ATX-s10-PDT. It has been reported that PDT induces hypoxia and expression of the vascular endothelial growth factor (VEGF) via the hypoxia-inducible factor 1 (HIF1)-alpha pathway. We hypothesized that the expression of VEGF may cause tumor recurrence after PDT and exert unfavorable effect against the anti-tumor activity of ATX-s10-PDT. In this study, we showed by DNA microarray analysis in vitro that VEGF mRNA expression was induced 3 h after laser irradiation in ATX-s10-PDT. We compared the anti-tumor activity of ATX-s10-PDT against lung cancer cell lines SBC-3 and SBC-3/VEGF, the latter overexpressing VEGF; there was no significant difference in the sensitivity to the PDT between the two cell lines as assessed by clonogenic assay. Furthermore, no statistically significant difference in the anti-tumor effect of PDT, as measured by tumor cures, was found between SBC-3 and SBC-3/VEGF tumors in female Balb/c-nu/nu nude mice in vivo. In conclusion, ATX-s10-PDT may prevent tumor recurrence despite induction of VEGF and promotion of tumor angiogenesis, which are known to enhance tumor proliferation and survival.

Breast cancer resistance protein (BCRP) affected acquired resistance to gefitinib in a "never-smoked" female patient with advanced non-small cell lung cancer.

Development of acquired resistance to gefitinib after an initial good response is common. Recently, it was reported that this acquired resistance is related to a secondary mutation associated with a substitution of threonine by methionine at codon 790 (T790M) of the epidermal growth factor receptor (EGFR) gene. In this report, we present a "never smoking" woman with advanced lung cancer who showed acquired resistance to gefitinib, and analysis of autopsy samples revealed no evidence of EGFR mutations in either exons 18-21 or codon 790, and positive immunostaining for breast cancer resistance protein (BCRP). We describe, for the first time, a case in which expression of BCRP was associated with acquired resistance to gefitinib, independent of EGFR mutations.

Effect of dentin depth on hybridization quality using different bonding tactics in vivo.

OBJECTIVES: Incomplete resin infiltration and polymerization of adhesive contributed to nanoleakage formation. This study tested the null hypothesis that adoption of different bonding tactics and dentine depth will not affect hybridization quality in vivo. METHODS: Class V cavities were prepared on the labial/buccal surface of monkey teeth. They were bonded by Single Bond (a two-step total-etch adhesive), Clearfil SE Bond (a two-step self-etch adhesive), or Clearfil S(3) Bond (an all-in-one self-etch adhesive). Combined nanoleakage analysis and quantitative immunolabeling evaluation were carried out in the hybrid layer formed in both cervical superficial and deep dentine. RESULTS: Single Bond showed reticular and spotted nanoleakage while Clearfil SE Bond and Clearfil S(3) Bond presented only a spotted one. While Single Bond showed increased concentration of labeling of type I collagen within the deep part of the hybrid layer, two self-etch adhesives-Clearfil SE Bond and Clearfil S(3) Bond revealed a homogeneous labeling pattern, even if the latter presented a significantly increased labeling index in deep dentine. CONCLUSIONS: Different bonding tactics showed different nanoleakage patterns and immunolabeling index, and was influenced by dentine depth at different levels in vivo.

A time-dependent effect of PDGF-BB on adhesion and growth of cultured fibroblasts to root surfaces.

OBJECTIVE: Platelet-derived growth factor (PDGF-BB) is suggested to be a potent stimulator and a strong mitogenic agent for human periodontal ligament cells (PDL). This study aimed at assessing the effectiveness of PDGF-BB application on periodontally diseased root surfaces through attachment and growth of fibroblast cells. MATERIALS AND METHODS: Fifteen periodontally involved and five healthy teeth were selected, prepared from proximal surfaces and distributed into four groups (10 specimens per group): I: healthy; II: untreated diseased; III: scaling and root planning (SRP); and IV: SRP and PDGF-BB. Each group had three subdivisions (three specimens per group) which were incubated at three different time periods. The remaining specimen for each group was used to examine surface topography. Fibroblasts were pooled on root specimens and incubated. Results were evaluated by using scanning electron microscopy. Repeated cell counting was done within a representative standard area. RESULTS: The best results regarding PDL cell shape and density were obtained at day 3 in all experimental groups, except the diseased group. Although SRP samples showed slightly higher results in numbers of attached fibroblasts than diseased samples, they demonstrated a similar negative effect denoting incompatible root surfaces for fibroblast attachment. SRP plus PDGF-BB and healthy samples showed a comparable positive effect, suggesting a good root surface biocompatibility. Inter-group differences showed no significant differences on day 1, but statistically significant differences were found on both day 3 and day 7 incubation periods favoring groups I and IV over groups II and III. CONCLUSIONS: Platelet-derived growth factor showed a positive effect on adhesion and growth of cultured fibroblasts to periodontally diseased surfaces. Thus, PDGF-BB may have a promising role in clinical periodontics.

Bone with a vascular flap induced from fat tissue with the use of rhBMP-2 in rats.

Here we report that successful bone formation with a vascular flap inside a cylindrical mold was induced from fat tissue with the use of recombinant human bone morphogenetic protein-2 in rats. Fat tissue connected to blood vessels was prepared to fit into the mold and implanted intramuscularly into the hind leg in Wistar rats. RhBMP-2 (20 micro g) was applied in a collagen sheet previously placed on the inside surface of the mold. Bone formation was confirmed radiologically and morphologically at 2, 4, and 8 weeks after the surgery. In the control group without rhBMP-2 or the group with ligation of the blood vessels before the implantation, bone formation was not observed. Our success in bone formation having a definite size, shape, and blood supply may lead to a therapeutic approach to effective bone reconstitution. The present study is the first report on bone induction from fat tissue by rhBMP-2 in vivo.

Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells.

Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca(2+) signaling pathways, however, are not well understood. We investigated Ca(2+) influx and release functions in HMSCs. In Ca(2+) imaging experiments, spontaneous Ca(2+) oscillations were observed in 36 of 50 HMSCs. The Ca(2+) oscillations were completely blocked by the application of 10 micro M cyclopiazonic acid (CPA) or 1 micro M thapsigargin (TG). A brief application of 1 micro M acetylcholine (ACh) induced a transient increase of [Ca(2+)](i) but the application of caffeine (10 mM) did not induce any Ca(2+) transient. When the stores were depleted with Ca(2+)-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca(2+) (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca(2+) currents (I(SOC)) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca(2+) currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca(2+) currents were recorded. Using RT-PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP(3)R) type I, II, and III and DHP receptors alpha1A and alpha1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca(2+) channels. These results suggest that in undifferentiated HMSCs, Ca(2+) release is mediated by InsP(3)Rs and Ca(2+) entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.

Compositional analysis of root cementum and dentin after Er:YAG laser irradiation compared with CO2 lased and intact roots using Fourier transformed infrared spectroscopy.

The present study examines the dental root after Er:YAG laser irradiation, compared with CO2 lased and non-treated surfaces, using Fourier Transformed Infrared (FTIR) spectroscopy. Freshly extracted human teeth were irradiated by Er:YAG laser at an energy output of 40 mJ/pulse, 10 Hz (0.4 watts), with or without water coolant, and by CO2 laser at an energy output of 0.5 watts in continuous wave mode without coolant. The surfaces were chalky and smooth after irradiation by Er:YAG laser with water coolant, were charred and irregular after irradiation by Er:YAG laser without water coolant, and were completely carbonized after CO2 laser irradiation. The FTIR profiles from samples of the surfaces that were irradiated by Er:YAG laser with water coolant were similar to those from non-treated samples, except for a slight decrease on the OH and amide bands, which are mainly related to organic components. This decrease was observed to be extreme after CO2 laser irradiation and moderate after Er:YAG laser irradiation without coolant. The formation of new bands showing toxic substances was observed to a large extent after CO2 laser irradiation and to a smaller extent after Er:YAG laser irradiation without water coolant. In contrast, no such bands were detected after Er:YAG laser irradiation with water coolant. The present results show that these laser treatments selectively ablated more organic components than inorganic components and that Er:YAG laser irradiation with water coolant did not cause major compositional changes or chemically deleterious changes in either root cementum or dentin.

Transient structures of wave patterns arising in the wave regeneration of subalpine coniferous forests.

In wave-regeneration phenomena observed in the subalpine coniferous forests, mainly consisting of Abies species, the blighted forests present various shapes in the course of development, spots at the initial stage turning into arches and finally into long whitish stripes. Because the wave-regeneration could not be followed in the field without long term studies, a simple model has been elaborated to simulate the various different dieback structures observed in the real forests. This model, based on cellular automata, is employed to analyze the power spectral density of canopy tree height fluctuations in the wave-regenerated forests. The results demonstrate that almost all the dieback structures observed in the field can be generated by this simple model, by varying the wind direction and its strength by some stochasticity. The power spectrum density presents various shapes in the course of development, white noise type at the initial stage turning into Lorentz type and finally into 1/f type power spectrum (spatial Fourier frequency).

Hepatocyte growth factor/scatter factor is implicated in the mode of stromal invasion of uterine squamous cervical cancer.

OBJECTIVE: The purpose of this study was to examine the relationship of hepatocyte growth factor/scatter factor (HGF/SF) to cell motility and invasion in uterine cervical cancer. METHODS: We examined the expression of HGF/SF and its receptor, c-met, in cervical cancer cell lines SKG-IIIa (squamous cell carcinoma) and Hela-S3 (adenocarcinoma) and in stromal cells of the cervical cancer tissue by reverse transcription-polymerase chain reaction. We studied the effect of HGF/SF on invasiveness of SKG-IIIa and Hela-S3 in an invasion model of the modified Boyden chamber method and by electron microscopy. SKG-IIIa cells were also seeded on the thick Matrigel-coated layer to evaluate the invasion patterns in three-dimensional directions. To investigate the mechanism of an inductive effect of HGF/SF on the invasiveness of SKG-IIIa, we examined the effect of HGF/SF on the expression of intercellular adhesion molecule E-cadherin, cell-substrate adhesion molecules CD44, alpha2beta1, and alpha6beta1, and intracellular skeleton fiber actin in SKG-IIIa in cell enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. RESULTS: HGF/SF messenger RNA (mRNA) was detected in stromal cells, and c-met mRNA was detected in SKG-IIIa and Hela-S3. Hela-S3 that initially showed weak intercellular contact freely invaded the Matrigel-coated multiporous membrane without the addition of HGF/SF. In contrast, SKG-IIIa that initially showed strong intercellular adhesion could invade the membrane after the addition of HGF/SF. The same results were represented by an addition of HECD-1, an anti-human E-cadherin antibody. In an experiment with cell culture in a thick Matrigel layer, control SKG-IIIa showed a mirror-ball-like invasion pattern, whereas HGF/SF-stimulated SKG-IIIa spread horizontally over the membrane and migrated through the membrane holes, presenting a tentacular invasion pattern. Migration of SKG-IIIa under the membrane was confirmed by scanning and transmission electron microscopy. The addition of HGF/SF in cell ELISA assay decreased the expression of E-cadherin and actin in SKG-IIIa, but it did not change the expression of CD44, alpha2beta1, and alpha6beta1. Immunofluorescence staining revealed that the expression of E-cadherin in cell membrane was disturbed by HGF/SF. CONCLUSIONS: Our data indicate that HGF/SF produced by stromal cells influences the mode of stromal invasion of squamous cervical cancer by selectively decreasing the expression of both E-cadherin and actin.

S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif-binding factor A.

S1 proteins A-D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross-linking. In hnRNPs, protein D2 occurred at nuclease-hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif-binding factor A (CBF-A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF-A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post-transcriptional processes in gene expression.

Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo.

When bone is lost due to injury and/or illness, the defects are generally filled with natural bone because artificial bone materials have problems of bioaffinity. However, natural bone also has supply and infection problems. If an artificial material has the same biological properties as bone, it can replace natural bone for grafting. We synthesized a hydroxyapaite (HAp) and collagen (Col) composite by a simultaneous titration coprecipitation method using Ca(OH)2, H3PO4 and porcine atelocollagen as starting materials. The composite obtained showed a self-organized nanostructure similar to bone assembled by the chemical interaction between HAp and Col. The consolidated composite by a cold isostatic pressure of 200 MPa indicated a quarter of the mechanical strength of bone. It also indicated the same biological properties as grafted bone: The material was resorbed by phagocytosis of osteoclast-like cells and conducted osteoblasts to form new bone in the surrounding area. This HAp/Col composite having similar nanostructure and composition can replace autologous bone grafts.