The function of platelet-derived growth factor in the differentiation of mouse tongue striated muscle.

OBJECTIVE: To determine the function of platelet-derived growth factor (PDGF) in the final differentiation phase of tongue striated muscle cells. MATERIALS AND METHODS: We analyzed the expressions of PDGF-A, -B, platelet-derived growth factor receptor (PDGFR)-α, and PDGFR-ß in mouse tongues between embryonic days (E) 11 and 15. Furthermore, we examined the effects of human recombinant PDGF-AB and the peptide antagonist for PDGFRs using an organ culture system of mouse embryonic tongue. Mouse tongues at E12 were cultured in BGJb medium containing human recombinant PDGF-AB for 4 days or the peptide antagonist for PDGF receptors for 8 days. RESULTS: PDGF-A, -B, PDGFR-α, and -ß were expressed in the differentiating muscle cells between E11 and 15. The human recombinant PDGF-AB induced increases in the mRNA expressions of myogenin and muscle creatine kinase (MCK) and the number of fast myosin heavy chain (fMHC)-positive cells, markers for the differentiation of muscle cells. On the other hand, the peptide antagonist for PDGFRs induced suppressions in the mRNA expressions of myogenin and MCK, and the number of fMHC-positive cells. Both the PDGF-AB and the antagonist failed to affect the expressions of cell proliferation markers. CONCLUSION: These results suggest that PDGF functions as a positive regulator in the final differentiation phase of tongue muscle cells in mouse embryos.

Establishment of indicator for screening of child abuse and neglect in primary school-age children.

AIM: In Japan, elementary schools are committed to early discovery of child abuse and neglect. Under Japanese law, dentists are required to be involved in child welfare and early detection of child abuse. However, the extent to which dental practitioners cooperate for prevention of child abuse with schools remains limited to date. Therefore, we undertook a community-based project that aimed to develop screening indicators to identify potentially abused children based on their oral health condition and behavioural characteristics in education settings. We have already reported on the relationship between oral health condition and child abuse. The present study established an indicator that can facilitate identification and prevention of child abuse/neglect. METHODS: Study design: Cross-sectional study. Questionnaires were given to teachers at an elementary school to ascertain behavioural characteristics observed in children who experienced abuse. CONCLUSION: We developed a check sheet for proper assessment, which requires as little effort as possible, and an index for screening children in need based on teaching staff's observation of students' daily behaviour in school settings. Highly selected items are advantageous as they lead to a decrease in non-response or responses, which can help in improving the accuracy of the response to each question.

Ab initio study on the hydrogen desorption from MH-NH3 (M = Li, Na, K) hydrogen storage systems.

The hydrogen storage system LiH + NH(3) ↔ LiNH(2) + H(2) is one of the most promising hydrogen storage systems, where the reaction yield can be increased by replacing Li in LiH with other alkali metals (Na or K) in order of Li < Na < K. In this paper, we have studied the alkali metal M (M = Li, Na, K) dependence of the reactivity of MH with NH(3) by calculating the potential barrier of the H(2) desorption process from the reaction of an M(2)H(2) cluster with an NH(3) molecule based on the ab initio structure optimization method. We have shown that the height of the potential barrier becomes lower in order of Li, Na, and K, where the difference of the potential barrier in Li and Na is relatively smaller than that in Na and K, and this tendency is consistent with the recent experimental results. We have also shown that the H-H distance of the H(2) dimer at the transition state takes larger distance and the change of the potential energy around the transition state becomes softer in order of Li, Na, and K. There are almost no M dependence in the charge of the H atom in NH(3) before the reaction, while that of the H atom in M(2)H(2) takes larger negative value in order of Li, Na, and K. We have also performed molecular dynamics simulations on the M(2)H(2)-NH(3) system and succeeded to reproduce the H(2) desorption from the reaction of Na(2)H(2) with NH(3).

Fed and fasted states on heart rate variability, hemodynamic heart rate and blood pressure in adults submitted to moderate aerobic exercise.

BACKGROUND: Heart rate variability (HRV) has proven to be a powerful non-invasive tool to investigate cardiac autonomic control and, seems to be influenced by nutritional status and exercise practice. However, the acute effects of fed or fasting states on HRV and blood pressure (BP) during low-to-moderate intensity aerobic exercise are currently unknown. Therefore, we investigated the baseline values and behavior of HRV, BP, and heart rate (HR) before and after low-to-moderate intensity aerobic exercise in fed and fasted states in healthy adults. METHODS: 12 healthy individuals with mean age (SD) 59.0 (9.1) years performed two tests on a treadmill at 80% of the mean velocity of the 6-min walking test separated by 48 h: 12 h fasted (FST) or 1 h fed (FED). HRV, BP and HR were analyzed at rest, posttest, and at the third, fifth, and seventh minutes of recovery. RESULTS: HRV and HR presented no significant alterations between nutritional conditions. HR at baseline was not different between nutritional conditions. Diastolic blood pressure was increased during the fasted baseline state. CONCLUSIONS: The results of the current study provide that 12 h overnight fasting does not seem to be enough to affect significant changes in the autonomic modulation in healthy adults submitted to low-to-moderate intensity aerobic exercise.

Tacrolimus and methotrexate for the prophylaxis of graft-versus-host disease after unrelated donor cord blood transplantation for adult patients with hematologic malignancies.

Eighteen patients with hematologic malignancies underwent cord blood transplantation (CBT) from unrelated donors after being conditioned with myeloablative or reduced-intensity regimens, and received tacrolimus and methotrexate (15 mg/m(2) on day 1, 10 mg/m(2) on days 3 and 6) as graft-versus-host disease (GVHD) prophylaxis. The median number of nucleated cells in infused cord blood was 2.66 x 10(7)/kg (range 1.90 to 4.15 x 10(7)/kg). Engraftment was achieved in 16 of 18 patients. The median time to absolute neutrophil count >0.5 x 10(9)/L was 21.5 days (range 17 to 32), and the median time to platelet count >2.0 x 10(9)/L was 36 days (range 26 to 57). Of the 16 evaluable patients, five and eight had grades I and II acute GVHD, respectively, and none had grades III/IV acute GVHD. The cumulative incidence of grade II acute GVHD was 44.4%. Chronic GVHD occurred in 7 of 15 evaluable patients: limited type in three patients, extensive type in four patients. Of the 18 patients, 14 were alive and disease-free between 173 and 1514 days after CBT (median 746 days). The probability of disease-free survival at 2 years was 79.1%. These results, although in a retrospective study, suggested that tacrolimus and short-term methotrexate effectively prevented the occurrence of severe acute GVHD after unrelated CBT, and may contribute to a high survival rate.

Visit-to-visit blood pressure variability, average BP level and carotid arterial stiffness in the elderly: a prospective study.

In a cross-sectional study, visit-to-visit blood pressure (BP) variability was shown to be associated with artery remodelling. Here, we investigated the impact of visit-to-visit BP variability and average BP on the carotid artery remodelling progression in high-risk elderly according to different classes of antihypertension medication use/non-use. BP measurements and carotid ultrasound were performed in the common carotid artery in 164 subjects (mean age 79.7 years at baseline, 74.7% females) with one or more cardiovascular risk factors. Based on 12 visits (1 × /month for 1 year), we calculated visit-to-visit BP variability expressed as the standard deviation (s.d.), coefficient of variation (CV), maximum BP, minimum BP and delta (maximum-minimum) BP. We measured mean intima-media thickness (IMT) as well as stiffness parameter ß were measured at baseline and at the mean 4.2-year follow-up. In a multiple regression analysis, the maximum, minimum, s.d. and average of systolic BP (SBP) were significantly associated with a change in ß-values between the baseline and follow-up after adjustment for age, smoking, lower high-density lipoprotein level, baseline ß-value and follow-up period. There were no significant associations between the visit-to-visit BP variability measures and the change in mean IMT. Significant associations of maximum, minimum, s.d. and average SBP were found with increased ß-values in the subjects without calcium channel blocker (CCB) use and in the subjects using renin-angiotensin system inhibitors (RASIs). Thus, exaggerated visit-to-visit SBP variability and a high average SBP level were significant predictors of progression in carotid arterial stiffness in high-risk elderly without CCBs use and in those using a RASI.

Signaling via the anti-CD30 mAb SGN-30 sensitizes Hodgkin's disease cells to conventional chemotherapeutics.

SGN-30, a monoclonal antibody with activity against CD30+ malignancies, is currently in phase II clinical evaluation for treatment of Hodgkin's disease (HD) and anaplastic large cell lymphoma. The mechanisms underlying SGN-30's antitumor activity were investigated using cDNA array of L540 cells. SGN-30 treatment activated NF-kappaB and modulation of several messages including the growth regulator p21WAF1/CIP1 (p21) and cellular adhesion marker ICAM-1. p21 protein levels increased coincident with growth arrest and Annexin V/PI staining in treated HD cells. To determine if SGN-30-induced growth arrest would sensitize tumor cells to chemotherapeutics used against HD, L540cy and L428 cells were exposed to SGN-30 in combination with a panel of cytotoxic agents and resultant interactions quantified by the Combination Effects Method. Interactions between SGN-30 and all cytotoxic agents examined were additive or better. These in vitro data translated to increased efficacy of SGN-30 and bleomycin against L540cy tumor xenografts. In addition to direct cell killing, SGN-30 affects growth arrest and drug sensitization through growth regulating and proapoptotic machinery. Importantly, these data suggest that SGN-30 can enhance the efficacy of standard chemotherapies used to treat patients with CD30+ malignancies.

The expressions of insulin-like growth factors, their receptors, and binding proteins are related to the mechanism regulating masseter muscle mass in the rat.

OBJECTIVE: The mechanism regulating skeletal muscle mass is unclear. The purpose of the present study was to investigate the extent to which insulin-like growth factors (IGFs), their receptors (IGFRs), and binding proteins (IGFBPs) are involved in the regulation of skeletal muscle mass. DESIGN: We measured the mRNA expression levels for IGFs, IGFRs, and IGFBPs in the rat masseter muscle hypertrophied by oral administration of clenbuterol for 3 weeks and determined the correlations between the weight of masseter muscle and the mRNA expression levels. RESULTS: The mRNA expression levels for IGF-I and II, IGFR1 and 2, and IGFBP4 and 6 showed clenbuterol-induced elevations and positive correlations with the weight of masseter muscle. That for IGFBP3 only exhibited a clenbuterol-induced decrease and a strong negative correlation with the weight of masseter muscle. The mRNA expression levels for IGFBP2 and 5 showed no significant changes between the control and clenbuterol groups, and no significant correlations. IGFBP1 mRNA was not detectable. CONCLUSION: These results suggest that IGF-I, II, IGFR1 and 2, and IGFBP3, 4 and 6 are related to the mechanism regulating masseter muscle mass in the rat.

Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family.

A new human gene encoding a receptor-type tyrosine kinase was isolated by a weak cross-hybridization with v-ros oncogene. A cDNA of about 7.7 kb carried a 4.2 kb open reading frame, and the predicted amino acid sequence of 1338 residues contained extracellular, transmembrane and tyrosine kinase domains. Although its extracellular domain is approximately 220 amino acids longer than those of the products of the fms family, i.e. c-fms, c-kit and platelet-derived growth factor receptor genes, the overall structure including cysteine motifs in its extracellular domain and a long peptide insertion in its tyrosine kinase domain indicates that this new gene is closely related to the fms family. Consequently, the gene was designated as flt (fms-like tyrosine kinase) gene. The expression of the flt gene was strongly suppressed in most of the tumor cell lines examined so far, whereas this mRNA was expressed in a variety of normal tissues of adult rat.

A new communication system between hepatocytes and sinusoidal endothelial cells in liver through vascular endothelial growth factor and Flt tyrosine kinase receptor family (Flt-1 and KDR/Flk-1).

Hepatocyte Growth Factor (HGF)/Scatter Factor secreted from sinusoidal endothelial cells and Kupffer cells in liver activates the c-Met tyrosine kinase receptor expressed on hepatocytes. Here we report yet another possible communication system through a different ligand and tyrosine kinase receptor in an opposite direction. We isolated and determined the primary structure of the entire coding region of rat flt-1 (fms-like tyrosine kinase), a receptor for Vascular Endothelial Growth Factor (VEGF). Using rat flt-1 cDNA as a probe we found that the flt-1 mRNA was expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. The transcripts of another VEGF receptor KDR/Flk-1 structurally related to Flt-1 was also expressed specifically in sinusoidal endothelial cells. On the other hand, VEGF mRNA was expressed weakly in hepatocytes, but not in the nonparenchymal cell fraction. Furthermore, in an in vitro culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells. These results suggest that hepatocytes regulate the proliferation and survival of the sinusoidal endothelial cells in liver in a paracrine manner. Therefore two reciprocal communication systems, VEGF-Flt receptor family and HGF-Met receptor, may exist in hepatic tissue.

X-ray crystal structure determination and molecular dynamics simulation of prophospholipase A2 inhibited by amide-type substrate analogues.

X-ray crystal structures of bovine pancreas prophospholipase A2 (proPLA2) inhibited by two amide-type inhibitors, [(R)-2-dodecanoyl-amino-1-hexanolphosphocholine (DAHPc) and (R)-2-dodecanoylamino-1-hexanolphosphoglycol (DAHPg)], were determined to R = 0.208 and 0.215 using reflections with up to 2.1 A resolution, respectively. Both complex crystals lacked defined electron densities for the prosequence of the N-terminal and for a loop region consisting of residues 65-70, retaining the disordered feature observed in free proPLA2 despite stabilization due to complex formation. The polar and nonpolar moieties of the amide-type inhibitors were located in the calcium-binding pocket and in the N-terminal alpha-helical hydrophobic region of the enzyme, respectively. As for the amide group of the inhibitor, which is lacking in the true substrate, a strong hydrogen bond was formed between the NH of the inhibitor and the unprotonated N(delta1) atom of His-48, resulting in the tight binding of the inhibitor to proPLA2, as well as to PLA2. The 20-30 times more potent inhibitory activity of DAHPg than DAHPc toward PLA2 could be explained by hydrogen bond formation between the glycol OH of DAHPg and the carbonyl O of Asp-49. The seven residues of the N-terminal prosequence of proPLA2, though disordered, block the access of a water molecule to Ala-1 of PLA2 or change the hydrogen-bonding property of Ala-1 alpha-amino group, resulting in breakage of the water-mediated hydrogen-bond network which is commonly formed in PLA2. The results of molecular dynamics (MD) calculation in an aqueous solution at 300 K indicate that this, rather than the close contact between the prosequence and the residues 65-70 loop region, is the main reason why the latter region becomes flexible in proPLA2, compared with in PLA2.

Inhibition of internal entry site (IRES)-mediated translation by a small yeast RNA: a novel strategy to block hepatitis C virus protein synthesis.

The observation that poliovirus mRNA is not translated in the yeast Saccharomyces cerevisiae has led to the discovery of a small RNA (60 nt, called IRNA, inhibitor RNA) which was later shown to specifically inhibit internal ribosome entry site (IRES)-mediated translation of naturally uncapped mRNAs. Translation of cellular capped mRNAs was not significantly inhibited by IRNA. IRNA also specifically inhibited hepatitis C virus (HCV) IRES-mediated translation in vitro and in vivo. A hepatoma cell line constitutively expressing IRNA was refractory to infection by a chimeric poliovirus (PV/HCV) in which PV IRES is replaced by HCV-IRES. In contrast, a PV/EMCV chimeric virus containing the EMCV IRES was not significantly inhibited in the IRNA-hepatoma cell line compared to the control hepatoma cells. UV-crosslinking studies showed that the IRNA binds a number of cellular proteins that appear to be important for IRES-mediated translation. Interaction of these proteins with the viral IRES elements is believed to be important in recruiting ribosomes to the 5( UTR of viral RNAs. The binding of the purified La autoantigen to the HCV IRES element was efficiently and specifically competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

A simple method of HLA-DRB typing using enzymatically amplified DNA and immobilized probes on microtiter plate.

We have developed a simple and economical method for HLA-DNA typing, called microtiter plate hybridization (PCR-MPH), which could replace standard PCR-SSO. This method is similar to that of an ELISA. Briefly, the PCR products labeled at the 5' termini with biotin were hybridized with probes immobilized on a microtiter well, and the bound PCR products were detected by streptavidin-conjugated enzymes followed by color development. A system for HLA-DRB1 "generic" typing (e.g., DR1, DR2), using microtiter wells coated with 12 different SSOs has been established. The HLA-DRB types classified using this method agreed well with those obtained by conventional serologic typing. The advantages of this microtiter plate-hybridization method for routine HLA-DNA typing are a short assay time, easy processing of large numbers of samples, and the potential for automation.

Prevalence of the factor V-Leiden mutation in four distinct American ethnic populations.

Resistance to activated protein C (APC) is the most common risk factor for venous thromboembolism, a major cause of morbidity and mortality with an incidence of about 1/1,000 per year. The Arg 506 to Gln mutation in exon 10 of the coagulation factor V gene (factor V-Leiden) has been found to be responsible for over 90% of the APC resistance cases and is an autosomal dominant trait. Initial studies have suggested that this mutation is restricted to individuals of European Caucasian extraction with an average allele frequency in European and American Caucasians of 4.4%, making it one of the most common monogenic disorders in the Caucasian population. A limited number of other ethnic populations have been tested and the mutation has been found only rarely. In our multiethnic survey of 602 individuals, Hispanic-Americans had the highest observed frequency of the factor V-Leiden mutant allele, 1.65%, while African-Americans had a somewhat lower frequency, 0.87%. No factor V-Leiden mutations were found in 191 Asian-Americans or 54 Native-Americans tested. These results indicate that the factor V-Leiden mutation segregates in populations with significant Caucasian admixture and is rare in genetically distant non-European groups. This ethnic stratification may be important in developing cost-effective selective screening programs to identify individuals at risk for thromboembolism and offer prophylactic therapy.

Detection of lymphomatous involvement of the lung by bronchoalveolar lavage. Application of immunophenotypic and gene rearrangement analysis.

We report three cases of pulmonary involvement of non-Hodgkin's lymphoma in which immunophenotypic or gene rearrangement analysis of bronchoalveolar lavage (BAL) cells demonstrated monoclonality of T- or B-cell lineage. The first patient had T-cell lymphoma and developed pulmonary lesions. Surface marker analysis of the BAL cells revealed that CD8-positive lymphoid cells were dominant and Southern blot analysis of T-cell receptor gene detected gene rearrangement demonstrating monoclonality of T-cell lineage. The second patient presented with diffuse micronodular shadows on chest radiograph. Marked B-lymphocytosis in BAL fluid prompted us to analyze their clonality. The third was a case in which recurrence of primary pulmonary lymphoma was suspected. In the second and third case, the finding of marked increase in the number of CD 19-positive B lymphocytes with a single class of light chains proved a monoclonal population in BAL cells. With the review of other cases in our study and the relevant literature, we conclude that the clonal analysis of BAL cells is helpful in establishing the diagnosis of pulmonary involvement of T- or B-cell lymphoma.

X chromosome methylation-based chimerism assay for sex-mismatched hematopoietic stem cell transplantation.

Analysis of hematopoietic chimerism is important for monitoring engraftment, graft failure, and disease recurrence. Although several techniques are now available, their sensitivity is unsatisfactory. In sex-mismatched stem cell transplantation (SCT) with a female donor, Y chromosome-specific sequences have proven the most sensitive marker. However, in the case of a male donor, no such reliable marker has been available to date. In this study, we report a novel method we developed to detect microchimerism in female recipients who receive SCT from male donors. The X-linked human androgen receptor gene (HUMARA) contains a highly polymorphic CAG trinucleotide repeat. Near this polymorphic site are methyl-sensitive HpaII restriction enzyme sites. After HpaII digestion, unmethylated male HUMARA sequences are completely digested, while methylated female ones remain intact among the male origin cells. This allows a highly efficient detection of a small number of female cells. Combined with the nested PCR technique, the X chromosome methylation-based chimerism assay could attain a 10(-4) level of sensitivity, which is 1000-fold higher than that of conventional assays. The applicability of the method was confirmed in two transplant cases. This highly sensitive method can also be applied to detect minimal residual disease or microchimerism in conditions other than hematopoietic SCT.

A comparative study of tropomyosin from vertebrate ventricles.

A comparative study of vertebrate ventricle tropomyosin has been carried out from the viewpoint of molecular evolution. The ventricles containing one-component tropomyosin were generally known, and in this paper those containing two components were also found in 8 species among mammals, reptiles, amphibia, and fish, but not among birds. The two components were concluded to be authentic tropomyosin and not artifacts since they showed lower electrophoretic mobilities in the presence of urea, and they were precipitated at pH 4.5 and bound to F-actin. Studies on cysteine contents and cyanogen bromide cleavage peptide patterns revealed that the characteristics of the two tropomyosin components from pig, turtle, amphibia and carp ventricles varied increasingly in that order from typical alpha- and beta-characteristics as seen in rabbit skeletal muscle tropomyosin. The single component of chicken ventricle tropomyosin showed alpha component characteristics in its electrophoretic mobility and cysteine content, and beta component characteristics in cyanogen bromide cleavage peptide pattern. The two components of carp ventricle tropomyosin seemed to be the most primitive, having two cysteine residues per molecule and a cyanogen bromide cleavage peptide pattern different from those of the two components of rabbit skeletal muscle.

Analysis of the distribution of CAG repeats and X-chromosome inactivation status of HUMARA gene in healthy female subjects using improved fluorescence-based assay.

We investigated the polymorphic CAG-repeat distribution and the X-inactivation status of the human androgen receptor (HUMARA) gene in 58 female Japanese volunteers. Polymerase chain reaction amplification was performed using a fluorescent-dye-labeled primer under conditions specific for GC-rich targets, and fragments were analyzed. To estimate the length of these fragments, FAM-labeled (blue fluorescent) products were simultaneously compared with ROM-labeled size markers (red) that were created by sequencing various HUMARA fragments. The number of polymorphic CAG repeats of HUMARA in 116 alleles from 58 female subjects ranged from 15 to 28. Of the 58 volunteers, 51 (88.0%) were heterozygous. In 96% of the heterozygous female subjects, the allelic differences were no greater than 6 repeats. X-chromosome inactivation was calculated as the ratio of the area of the smaller peak to the sum of the areas of the smaller and larger peaks. The average ratio was 0.38 (range, 0.09-0.50). Preferential use of 1 allele, by more than 75% (ratio. <0.25). was observed in 5 volunteers (10.9%). The clonal nature of a patient with chronic myelogenous leukemia was easily identified. This method is sensitive enough to discriminate a difference of 1 triplet repeat.

Progress of cell proliferation in striated muscle tissues during development of the mouse tongue.

The developmental stages of and places for the proliferation of tongue muscle cells have not yet been determined. To determine the stages of and places for proliferation between embryonic day (E) 9 and birth, we analyzed the expression of cyclin D1 mRNA and the immunolocalization for proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive nuclei to total nuclei (PCNA-labeling index) was obtained in the anterior, middle, and posterior regions. Cyclin D1 mRNA was highly expressed between E11 and E13, but decreased thereafter until birth. The distribution of PCNA-positive cell nuclei was consistent with that of myogenic cells in the occipital somites at E9. The PCNA-labeling index was highest at E11, then decreased until birth without a significant difference among the 3 regions. These findings suggest that some tongue muscle progenitor cells begin proliferation in the occipital somites at E9, and that the proliferation in the whole tongue region occurred most actively between E11 and E13, then decreased until birth without regional differences.

Developmental changes in the nicotinic acetylcholine receptor in mouse tongue striated muscle.

There are no published studies on synaptogenesis focusing on the elimination of the superfluous nicotinic acetylcholine receptor (nAChR) outside the neuromuscular junction and the nAChR subunit switch from the embryonic-type (alpha2betagammadelta subunits) to the adult-type (alpha2betaepsilondelta subunits) in mouse tongues. To identify the time course of nAChR subunit elimination and switch, we analyzed the expression levels of alpha, epsilon, and gamma subunit mRNAs, and the immunolocalization of the delta subunit protein in the mouse tongue and corresponding hind limb. The analysis included the period from embryonic day (E) 11 to the newborn stage. The nAChR elimination and subunit switch began at E15 in the tongue and at E17 in the hind limb. They were nearly complete at birth in the tongue, but not in the hind limb. The early completion of synaptogenesis in the tongue at birth may be related to the early functional demands placed on the tongue, such as suckling and swallowing, immediately after birth.