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.

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.

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.

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.

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.

Stimulation of SV40 DNA replication and transcription by Alu family sequence.

The sequence motif GGAGGC (Alu core) is present in the Alu family repeats, where it is required for RNA polymerase III promoter function. This motif is also found in the SV40 origin (ori) of replication. Here, an oligonucleotide containing the Alu sequence was inserted into pSV2CAT, a plasmid composed of the SV40 enhancer/promoter/ori linked to the bacterial chloramphenicol acetyltransferase gene (CAT), to see the effect of the Alu sequence on SV40 DNA replication and transcription. Results of transfection experiments in human HeLa cells showed that the Alu sequence stimulated sequence-specifically replication and transcription in the SV40 system. Stimulation effects on DNA replication were observed when the Alu sequence was placed upstream of enhancer/promoter/ori in either orientation, while effects on transcription were detected only when it was inserted in the normal orientation. These effects correlate with sequence-specific binding of two proteins (40 kDa and 120 kDa) to this motif. In fact, binding was abolished by a mutation in the cognate sequence that disrupted stimulation of replication and transcription. Both proteins bind duplex DNA, while the 40 kDa one also binds the minus strand with high affinity.

Purification and Properties of Flavin- and Molybdenum-Containing Aldehyde Oxidase from Coleoptiles of Maize.

Aldehyde oxidase (AO; EC 1.2.3.1) that could oxidize indole-3-acetaldehyde into indole-3-acetic acid was purified approximately 2000-fold from coleoptiles of 3-d-old maize (Zea mays L.) seedlings. The apparent molecular mass of the native enzyme was about 300 kD as estimated by gel-filtration column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of 150-kD subunits. It contained flavin adenine dinucleotide, iron, and molybdenum as prosthetic groups and had absorption peaks in the visible region (300-600 nm). To our knowledge, this is the first demonstration of the presence of flavin adenine dinucleotide and metals in plant AO. Other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde also served as good substrates, but N-methylnicotinamide, a good substrate for animal AO, was not oxidized. 2-Mercaptoethanol, p-chloromercu-ribenzoate, and iodoacetate partially inhibited the activity, but well-known inhibitors of animal AO, such as menadione and estradiol, caused no reduction in activity. These results indicate that, although maize AO is similar to animal enzymes in molecular mass and cofactor components, it differs in substrate specificity and susceptibility to inhibitors. Immunoblotting analysis with mouse polyclonal antibodies raised against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptiles. The possible role of this enzyme is discussed in relation to phytohormone biosynthesis in plants.

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.

[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).

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.

Analysis of hemangiopericytic meningiomas by immunohistochemistry, electron microscopy and cell culture.

Intracranial hemangiopericytic meningiomas (HM) from seven patients were examined by immunostaining, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and cell culture. Positive staining for Factor VIII-related antigen was restricted to capillary endothelial cells. There was no reaction with anti-glial fibrillary acidic protein (GFAP) serum or anti-S-100 protein serum. In these neoplasms TEM displayed extracellular basement membrane-like material (BMLM), cytoplasmic intermediate filaments (IF) associated with a dense body, dilated rough-surfaced endoplasmic reticulum containing BMLM, a small area of interdigitation of cell membranes, and a unique intercellular punctate or linear density. By SEM these tumors had intercellular shell-like or reticular structures and irregularly branched capillaries which were compressed by ellipsoidal- or carrot-shaped tumor cells. Short-term monolayer culture showed rapid and vigorous growth of tumor cells, and the formation of pseudolumens but not of whorls. The TEM of cultured cells also showed cytoplasmic IF associated with the dense body. By SEM the cultured cells were flat and had a discoid nucleus with conspicuous nucleolar hillocks. Our results show that HM are mainly poorly specialized mesenchyme-related tumors of the meninges; some possess a potential for aggressive growth and some for differentiation into smooth muscle cells. Further study is needed to determine their histogenesis.

Platelet Ca2+ is not increased in stroke-prone spontaneously hypertensive rats: comparative study with spontaneously hypertensive rats.

We have reported that cytosolic Ca2+ concentration ([Ca2+]i) is increased in platelets from spontaneously hypertensive rats (SHR) in both basal and thrombin-stimulated conditions. To determine whether the correlation between blood pressure and cellular Ca2+ metabolism exists in stroke-prone SHR (SHRSP), we investigated Ca2+ handling using fura 2 and aggregation response in platelets of 12- to 13-week-old male SHRSP, SHR, and Wistar-Kyoto rats (WKY). Systolic pressure was highest in SHRSP and lowest in WKY (213 +/- 8, 172 +/- 7, and 135 +/- 5 mm Hg, respectively). Basal [Ca2+]i was significantly higher in SHR than WKY (45.9 +/- 4.5 versus 41.2 +/- 4.8 nmol/L, P<.05), and that in SHRSP (40.2 +/- 2.8 nmol/L) was similar to that in WKY. Thrombin (0.1 IU/mL)-stimulated [Ca2+]i rise was greater in SHR and smaller in SHRSP than in WKY in the presence of extracellular Ca2+ (530 +/- 50 and 408 +/- 52 versus 475 +/- 50 nmol/L, respectively; P<.05). The recovery rate from the peak [Ca2+]i response to thrombin was greatest in SHRSP and least in WKY. Ionomycin (5 micromol/L)-stimulated [Ca2+]i rise was similar in WKY, SHR, and SHRSP (731 +/- 97, 743 +/- 88, and 683 +/- 70 nmol/L, respectively). Thrombin-induced maximum platelet aggregation response was higher in SHR and lower in SHRSP than WKY (82 +/- 4 percent and 61 +/- 15 percent versus 73 +/- 6 percent, respectively; P<.05). In contrast to SHR, basal [Ca2+]i in SHRSP was similar to that in WKY, and thrombin-stimulated [Ca2+]i was attenuated. These result suggest that platelet Ca2+ handling differs between SHR substrains and that an increased [Ca2+]i is not obligatory in genetically hypertensive rats.

Effects of insulin on calcium metabolism and platelet aggregation.

The influence of insulin on platelets in vitro has not been exhaustively investigated. To clarify whether insulin affects Ca2+ metabolism in platelets directly or through alteration of other systems regulating intracellular Ca2+ homeostasis, we examined the effect of insulin both alone and in combination with prostaglandin E1 on platelet aggregation and Ca2+ metabolism. Incubation of rat platelets with insulin reduced thrombin-induced Ca2+ influx but did not change thrombin-evoked release of Ca2+ from internal stores or the size of internal Ca2+ stores. The interactive effects of insulin with prostaglandin E1 were only additive, and insulin did not augment the effects of prostaglandin E1 on platelet Ca2+ metabolism. In contrast, insulin did not inhibit thrombin-induced platelet aggregation but did augment inhibition of platelet aggregation by prostaglandin E1. Our results suggest that insulin inhibits platelet function by both prostaglandin E1-dependent and -independent mechanisms.

Intravenous administration of L-arginine inhibits angiotensin-converting enzyme in humans.

The iv administration of L-arginine, a precursor of endothelium-derived relaxing factor/nitric oxide, is known to decrease blood pressure in humans by its direct vasodilatory effects. The purpose of the present study was to determine whether L-arginine infusion modifies the renin-angiotensin (Ang)-aldosterone system as well as blood pressure and renal hemodynamics. L-Arginine and saline vehicle were iv administered to 10 healthy male subjects in random order on different days. L-Arginine infusion (500 mg/kg over 30 min) decreased mean blood pressure (from 81.2 +/- 2.7 to 74.0 +/- 2.5 mm Hg; P < 0.001) and renal vascular resistance (from 0.085 +/- 0.007 to 0.074 +/- 0.006 mm Hg/mL.min; P < 0.01) and increased heart rate (from 60.3 +/- 2.7 to 69.7 +/- 2.1 beats/min; P < 0.001) and renal plasma flow (from 616.6 +/- 37.8 to 701.0 +/- 49.2 mL/min; P < 0.05). L-Arginine reduced serum Ang-converting enzyme activity (from 10.4 +/- 0.6 to 8.9 +/- 0.5 nmol/mL.min; P < 0.05) and plasma Ang-II (from 19.3 +/- 3.3 to 12.7 +/- 2.8 pg/mL; P < 0.001), but had no effect on PRA or the glomerular filtration rate. The saline vehicle did not alter any of these parameters. The iv administration of L-arginine (endothelium-derived relaxing factor/nitric oxide) may reduce the plasma Ang-II concentration by inhibiting Ang-converting enzyme. The mechanism by which L-arginine infusion decreases blood pressure can be at least in part explained by inhibition of the renin-Ang system.