A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

The mandibular condylar cartilage (MCC) is an essential component of the temporomandibular joint, which orchestrates the vertical growth of the mandibular ramus through endochondral ossification with distinctive modes of cell differentiation. Parathyroid hormone-related protein (PTHrP) is a master regulator of chondrogenesis; in the long bone epiphyseal growth plate, PTHrP expressed by resting zone chondrocytes promotes chondrocyte proliferation in the adjacent layer. However, how PTHrP regulates chondrogenesis in the MCC remains largely unclear. In this study, we used a Pthrp-mCherry knock-in reporter strain to map the localization of PTHrP+ cells in the MCC and define the function of PTHrP in the growing mandibular condyle. In the postnatal MCC of PthrpmCherry/+ mice, PTHrP-mCherry was specifically expressed by cells in the superficial layer immediately adjacent to RUNX2-expressing cells in the polymorphic layer. PTHrP ligands diffused across the polymorphic and chondrocyte layers where its cognate receptor PTH1R was abundantly expressed. We further analyzed the mandibular condyle of PthrpmCherry/mCherry mice lacking functional PTHrP protein (PTHrP-KO). At embryonic day (E) 18.5, the condylar process and MCC were significantly truncated in the PTHrP-KO mandible, which was associated with a significant reduction in cell proliferation across the polymorphic layer and a loss of SOX9+ cells in the chondrocyte layers. The PTHrP-KO MCC showed a transient increase in the number of Col10a1+ hypertrophic chondrocytes at E15.5, followed by a significant loss of these cells at E18.5, indicating that superficial layer-derived PTHrP prevents premature chondrocyte exhaustion in the MCC. The expression of Runx2, but not Sp7, was significantly reduced in the polymorphic layer of the PTHrP-KO MCC. Therefore, PTHrP released from cells in the superficial layer directly acts on cells in the polymorphic layer to promote proliferation of chondrocyte precursor cells and prevent their premature differentiation by maintaining Runx2 expression, revealing a unique PTHrP gradient-directed mechanism that regulates MCC chondrogenesis.

Mesenchymal Progenitor Regulation of Tooth Eruption: A View from PTHrP.

Tooth eruption is a unique biological process by which highly mineralized tissues emerge into the outer world, and it occurs concomitantly with tooth root formation. These 2 processes have been considered independent phenomena; however, recent studies support the theory that they are indeed intertwined. Dental mesenchymal progenitor cells in the dental follicle lie at the heart of the coupling of these 2 processes, providing a source for diverse mesenchymal cells that support formation of the highly functional tooth root and the periodontal attachment apparatus, while facilitating formation of osteoclasts. These cells are regulated by autocrine signaling by parathyroid hormone-related protein (PTHrP) and its parathyroid hormone/PTHrP receptor PPR. This PTHrP-PPR signaling appears to crosstalk with other signaling pathways and regulates proper cell fates of mesenchymal progenitor cell populations. Disruption of this autocrine PTHrP-PPR signaling in these cells leads to defective formation of the periodontal attachment apparatus, tooth root malformation, and failure of tooth eruption in molars, which essentially recapitulate primary failure of eruption in humans, a rare genetic disorder exclusively affecting tooth eruption. Diversity and distinct functionality of these mesenchymal progenitor cell populations that regulate tooth eruption and tooth root formation are beginning to be unraveled.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors.

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Diverse contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages.

OBJECTIVES: Craniofacial skeletal development requires deliberate coordination of two distinct mechanisms of endochondral and intramembranous ossification. Col2a1-expressing cells encompass growth-associated skeletal progenitors in endochondral bones of the limb. The objective of this study was to determine the contribution of Col2a1-expressing cells to the craniofacial skeletal cell lineages. We hypothesize that Col2a1-expressing progenitors significantly contribute to various modes of ossification associated with the craniofacial development. METHODS: Cellular fates of Col2a1-expressing cells were studied based on a cre-loxP system using a Col2a1-cre transgene and an R26R-tdTomato reporter allele. We analysed three distinct locations of the craniofacial skeletal complex representing unique ossification mechanisms: the cranial base, the calvaria and the mandibular condyle. RESULTS: Col2a1-cre consistently marked a majority of skeletal cells in the cranial base. Interestingly, Col2a1-cre also marked a large number of osteoblasts and suture mesenchymal cells in the calvaria, in addition to chondrocytes in the underlying transient cartilage. In the mandibular condyle, Col2a1-cre marked chondrocytes and osteoblasts only during the growth phase. CONCLUSIONS: Col2a1 is expressed by progenitors of the skeletal lineage in canonical endochondral bone formation occurring in the cranial base. In contrast, other ossification mechanisms of the craniofacial complex utilize Col2a1-expressing cells in a different manner, whereby Col2a1 may be expressed in more differentiated or transient cell types of the skeletal lineage.

The fate of Osterix-expressing mesenchymal cells in dental root formation and maintenance.

OBJECTIVES: Osterix (Osx)-expressing mesenchymal cells are progenitors for tooth root forming cells. The aim of this study was to reveal the fates of Osx-expressing cells during and after root formation using a lineage tracing experiment. MATERIAL AND METHODS: To reveal the fates of Osx-expressing dental mesenchymal progenitors, we took advantage of tamoxifen-inducible Cre reporter system. Osx-creER; R26R-tdTomato mice received tamoxifen (0.1 mg/body) at postnatal day 3 (P3). In this system, Osx-expressing at P3 (Osx-P3) cells undergo recombination, and they and their descendants continue to express Tomato red fluorescence protein permanently. Mandibles were dissected at serial time points ranging from P4 to P116 to investigate how Osx-P3 cells participated in root formation. Tomato+ cells on frozen sections were imaged under fluorescence microscopy. RESULTS: Osx-P3 cells and their descendants differentiated into all kinds of cells that contributed to the root and periodontal tissues, such as odontoblasts, cementoblasts, alveolar bone osteoblasts and periodontal ligament (PDL) cells during root formation. Even after root formation was completed, they persisted in dental pulp and PDL to provide progenitor cells for odontoblasts and cementoblasts. CONCLUSION: Osx-expressing cells play important roles in the entire processes of tooth root formation; their progeny continue to contribute to maintenance of tooth root even after root formation is complete.

A Supramolecular Substance, [2] Rotaxane, Induces Apoptosis in Human Molt-3 Acute Lymphoblastic Leukemia Cells.

The antitumor effects of a supramolecular substance, the [2] rotaxane (TRO-A0001), and its molecular mechanisms were investigated. TRO-A0001 suppressed the proliferation of cultured human Molt-3 acute lymphoblastic leukemia cells for 12-72 h in a dose-dependent manner. Based on flow cytometry, TRO-A0001 clearly induced apoptosis after 24 h. The mitochondrial membrane potential disappeared after treatment with 1.0 µM of TRO-A0001. Expression of the cleaved forms of capase-9 and caspase-3 was significantly increased in cells exposed to TRO-A0001, whereas the expression of XIAP, a type of inhibitor of apoptosis family, was decreased. These results suggest that [2] rotaxane TRO-A0001 may be a highly promising new antitumor medicine.

Longitudinal monitoring of CYP3A activity in patients receiving 3 cycles of itraconazole pulse therapy for onychomycosis.

WHAT IS KNOWN AND OBJECTIVE: Itraconazole, a CYP3A inhibitor, is used for the treatment for onychomycosis with a three-cycle pulse therapy over 3 months, but its effects on in vivo CYP3A activity during the entire course remain unknown. METHODS: Urinary 6ß-hydroxycortisol/cortisol ratios were determined in 19 patients with onychomycosis, before therapy, during three cycles of itraconazole pulse therapy (200 mg twice daily for a week in each monthly cycle) and at 3 month after completion of therapy. RESULTS AND DISCUSSION: The mean 6ß-hydroxycortisol/cortisol ratio was reduced by 68% from baseline (P < 0·05) after the 1st pulse dosing, but the inhibitory effect appeared to be resolved before the next pulse dosing and at 3 months post-treatment. The magnitude of inhibition appeared in proportion to the baseline CYP3A activity. WHAT IS NEW AND CONCLUSION: The inhibitory effect of itraconazole pulse therapy on the in vivo CYP3A activity appears clinically relevant at the end of each cycle, but the inhibition resolves, on average, within 3 weeks.

Reduction in superoxide dismutase expression in the epithelial mucosa of eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Eosinophils generate large amounts of oxidant species. The eosinophil-dominant type of chronic rhinosinusitis (CRS) with nasal polyps is related to more extensive disease and a decreased likelihood of surgical success. Superoxide dismutase (SOD) is the first-line and only antioxidant enzyme that converts superoxide to hydrogen peroxide. METHODS: The patients with CRS with nasal polyps were divided into eosinophilic and noneosinophilic groups. The expression of three isoforms of SOD, intracellular copper-zinc SOD (CuZnSOD), mitochondrial manganese SOD (MnSOD) and extracellular SOD (ECSOD), were examined by enzyme activity assay, immunohistochemistry and quantitative real-time RT-PCR sampled by laser capture microdissection. RESULTS: SOD activity in the eosinophilic and noneosinophilic groups was significantly reduced compared to that of the control groups. Immunostaining of both CuZnSOD and MnSOD in the eosinophilic group was significantly decreased compared with that in the noneosinophilic and control groups. CuZnSOD mRNA of the eosinophilic group was significantly decreased compared with that of the control group, whereas MnSOD mRNA in the eosinophilic group was significantly decreased compared with that in the noneosinophilic and control groups. Neither immunoreactivity nor mRNA of ECSOD was different among the three groups. The degree of epithelial damage and disease severity were inversely correlated with CuZnSOD and MnSOD immunoreactivity. CONCLUSIONS: The reduction in SOD activity and the downregulation of the SOD message are suggested to be related to eosinophil recruitment and epithelial damage of CRS with nasal polyps.

Male death resulting from hybridization between subspecies of the gypsy moth, Lymantria dispar.

We explored the origin of all-female broods resulting from male death in a Hokkaido population of Lymantria dispar through genetic crosses based on the earlier experiments done by Goldschmidt and by testing for the presence of endosymbionts that are known to cause male killing in some insect species. The mitochondrial DNA haplotypes of the all-female broods in Hokkaido were different from those of normal Hokkaido females and were the same as those widely distributed in Asia, including Tokyo (TK). Goldschmidt obtained all-female broods through backcrossing, that is, F1 females obtained by a cross between TK females (L. dispar japonica) and Hokkaido males (L. dispar praeterea) mated with Hokkaido males. He also obtained all-male broods by mating Hokkaido females with TK males. Goldschmidt inferred that female- and male-determining factors were weakest in the Hokkaido subspecies and stronger in the Honshu (TK) subspecies. According to his theory, the females of all-female broods mated with Honshu males should produce normal sex-ratio broods, whereas weaker Hokkaido sexes would be expected to disappear in F1 or F2 generations after crossing with the Honshu subspecies. We confirmed both of Goldschmidt's results: in the case of all-female broods mated with Honshu males, normal sex-ratio broods were produced, but we obtained only all-female broods in the Goldschmidt backcross and obtained an all-male brood in the F1 generation of a Hokkaido female crossed with a TK male. We found no endosymbionts in all-female broods by 4,'6-diamidino-2-phenylindole (DAPI) staining. Therefore, the all-female broods observed in L. dispar are caused by some incompatibilities between Honshu and Hokkaido subspecies.

Novel QTLs for photoperiodic flowering revealed by using reciprocal backcross inbred lines from crosses between japonica rice cultivars.

The rice japonica cultivars Nipponbare and Koshihikari differ in heading date and response of heading to photoperiod (photoperiod sensitivity). Using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analyses for heading date in a set of reciprocal backcross inbred lines (BILs) from crosses between Nipponbare and Koshihikari. Under natural-day conditions, transgressive segregation in days to heading (DTH) toward both early and late heading was observed in both BIL populations. QTL analyses revealed that two QTLs--on chromosomes 3 and 6--were involved in the difference in heading date between the parental cultivars. The Nipponbare allele at the QTLs on chromosomes 3 and 6 showed, respectively, increasing and decreasing effects on DTH in both BIL populations. The transgressive segregation observed in the BILs could be accounted for mainly by the complementary action of a set of alleles with opposing effects. Both QTLs were finely mapped as single Mendelian factors in secondary mapping populations (BC2F2 plants/BC2F3 lines). The QTL on chromosome 3 was mapped in the 1,140-kb interval between 94O03-4 (SSR) and OJ21G19-4 (SNP) and was designated Hd16. The QTL on chromosome 6 was mapped in the 328-kb interval between P548D347 (SSR) and 0007O20 (SSR) and was designated Hd17. Both Hd16 and Hd17 were involved in photoperiod sensitivity, as revealed by observation of the DTH of nearly isogenic lines of Nipponbare under short- and long-day conditions, suggesting that allelic differences in both Hd16 and Hd17 account for most of the difference in photoperiod sensitivity between the parental cultivars.

Determination of the antiproton-to-electron mass ratio by precision laser spectroscopy of pHe+.

A femtosecond optical frequency comb and continuous-wave pulse-amplified laser were used to measure 12 transition frequencies of antiprotonic helium to fractional precisions of (9-16)x10(-9). One of these is between two states having microsecond-scale lifetimes hitherto unaccessible to our precision laser spectroscopy method. Comparisons with three-body QED calculations yielded an antiproton-to-electron mass ratio of Mp/me=1836.152674(5).

[Non-Hodgkin malignant lymphoma of rib origin: report of a case].

A 69-year-old man with left chest and back pain was found to have an osteolytic mass (4.2 x 3.8 cm) in the left 8th rib by chest X-ray and computed tomography (CT) in August 2003. There were no abnormal findings in the abdomen, lung, mediastinum or bone except the left 8th rib. Although the spontaneous disappearance of pleural effusion and the elevated CRP suggested the possibility of myelitis, the malignancy of the rib could not be ruled out, and the surgery was performed in September 2003. The mass was resected en bloc together with the involved 8th rib. The histological diagnosis was primary non-Hodgkin lymphoma (diffuse, medium-sized to large B-cell lymphoma).

Demonstration of reversed flow in segmental branches of the portal vein with hand-held color Doppler ultrasonography after hematopoietic stem cell transplantation.

Hepatic veno-occlusive disease (VOD) is a severe complication of hematopoietic stem cell transplantation (SCT). When monitored with hand-held color Doppler ultrasonography during day -7 to +35 around SCT, reversed blood flow in the segmental branches of the portal vein was detected in nine of 56 patients who had undergone SCT. Three of nine patients had clinical evidence of VOD, but six patients did not fulfill the criteria for diagnosis of VOD initially. Two patients progressed to clinical VOD at a later date and the reversed portal flow disappeared with or without treatment for VOD in the other four patients. Monitoring for reversed portal flow with color Doppler ultrasonography may be a useful tool for the early diagnosis of VOD, and may improve prognosis by allowing early initiation of treatment.

[Bronchioloalveolar carcinoma with acute respiratory failure due to hemoptysis; report of a case].

A 69-year-old male was admitted to our hospital for hemoptysis and dyspnea. Because of his deteriorating respiratory distress, he was intubated and controlled by respirator for 3 days. He was diagnosed with adenocarcinoma of the lung by the sputum examination and chest computed tomography (CT) revealed an infiltration shadow in the peripheral superior ventral segment (S3) of the right upper lobe. He underwent right upper lobectomy with video-assisted thoracic surgery. Microscopic findings of the resected specimen measuring 10 x 10 x 7 cm revealed mucin-producing bronchioloalveolar carcinoma (BAC) with metastases in lymph nodes and the same lobe (S2b). We reported a rare case of BAC with hemoptysis.

CD28 and secretory immunoglobulin A-dependent activation of eosinophils: inhibition of mediator release by the anti-allergic drug, suplatast tosilate.

BACKGROUND: Eosinophils are major effector cells in allergic diseases. After their recruitment to sites of inflammation, they contribute to the pathophysiology of the disease by releasing granule proteins and cytokines. Suplatast tosilate (IPD-1151T), a new anti-allergic agent, has shown beneficial effect in the treatment of asthma, associated with reduced bronchoalveolar lavage eosinophil infiltration and eosinophilic cationic protein (ECP) release in serum and sputum. OBJECTIVE: We investigated whether suplatast tosilate could exert direct effects on human eosinophil activation. METHODS: Eosinophils from hypereosinophilic patients or normal donors were purified by Percoll gradient and the magnetic cell separation system. Chemotaxis was studied using the Boyden chamber technique using three chemoattractants, formyl-methionine-leucine-phenylalanine (fMLP), IL-5 and eotaxin. Oxidative metabolism was determined by a luminol-dependent chemiluminescence assay after activation with eotaxin or secretory IgA (sIgA). The release of ECP and eosinophil derived neurotoxin (EDN) was measured by radioimmunoassay and cytokine production was determined by ELISA following activation with sIgA or anti-CD28. RESULTS: The chemotactic response to fMLP, IL-5 and eotaxin was significantly inhibited by IPD-1151T. Suplatast tosilate was partially inhibiting the release of reactive oxygen species (ROS) induced by eotaxin and sIgA. Activation by sIgA and CD28 ligation resulted in the release of ECP and EDN, which was inhibited by IPD-1151T. Upon activation by anti-CD28, only IL-13 production was inhibited by IPD-1151T, whereas release of IL-2 and IFN-gamma was not affected. IL-10 release induced by sIgA was also inhibited by IPD-1151T. Additionally, the pro-inflammatory cytokine IL-6, which was secreted following anti-CD28 and sIgA stimulation, was strongly inhibited by IPD-1151T. CONCLUSION: Through inhibition of chemotaxis, IPD-1151T might limit the number of eosinophils at the inflammation site. Furthermore, it could reduce the pathological potential of eosinophils by inhibiting the release of ROS and cationic proteins, main inflammatory mediators produced by eosinophils. Moreover, the inhibition of immunoregulatory cytokines released by eosinophils could locally modify the immune response.

Young's syndrome associated with situs inversus totalis.

Primary ciliary dysfunction causes recurrent sino-pulmonary infections and shares the pathophysiology of the reproductive tract. A 34-year old man with bronchectasis and situs inversus totalis was investigated because of azoospermia. Endocrinological evaluations were all normal and scrotal exploration revealed the distended caput, but atrophic body to tail, of the epididymis. Light microscopy of testicular biopsy showed normal spermatogensis and motile sperms were easily obtained by testicular sperm extraction. Electron microscopy demonstrated normal sperm structure, whereas 65% of nasal cilias showed defect of central microtubules. This case, which bears a resemblance of Kartagener's syndrome (situs inversus totalis), is considered a unique variant of Young's syndrome.

The potential impact of drug transporters on nucleoside-analog-based antiviral chemotherapy.

Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported. Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs. This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter. The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance. We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.