Non-syndromic oligodontia with a novel mutation of PAX9.

Agenesis of the permanent teeth is a congenital anomaly that is frequently seen in humans. Oligodontia is a severe type of tooth agenesis involving 6 or more congenitally missing teeth, excluding the third molars. Previous studies have indicated that mutations in the homeobox gene MSX1, paired domain transcription factor PAX9, and EDA are associated with non-syndromic oligodontia. This study reports a Japanese family (eight of 14 family members affected) with non-syndromic oligodontia who preferentially lacked molar teeth. In this family, a novel frameshift mutation (321_322insG) was identified in the paired domain of PAX9. The frameshift mutation caused altered amino acids in the paired domain and premature termination of translation by 26 amino acids. When transfected into COS-7 cells, the mRNA expression of 321_322insG PAX9 was comparable with that of wild-type PAX9. However, the mRNA of 321_322insG PAX9 was more unstable than that of wild-type PAX9. This mRNA instability caused a marked decrease in protein production, as evaluated by Western blot analysis and immunostaining. These findings suggest that the 321_322insG mutation causes insufficient function of PAX9 protein and haploinsufficiency as a genetic model of familial non-syndromic oligodontia with a PAX9 mutation.

Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia.

INTRODUCTION: Cleidocranial dysplasia (CCD, MIM#119600), for which the responsible gene is RUNX2, is a genetic disorder characterized by hypoplasia or aplasia of the clavicles, patent fontanelles, and a short stature. Supernumerary teeth and delayed eruption and impaction of permanent teeth are frequently associated with CCD. Our previous study reported wide intrafamilial variation in supernumerary tooth formation associated with a mutation in the RUNT-domain of RUNX2, suggesting a low correlation between the genotype and supernumerary tooth formation. To further clarify this point, a more precise evaluation was performed. DESIGN: Gene mutational analysis of nine Japanese individuals with CCD was performed. Dental and skeletal characteristics were examined based on patient examinations and radiographs. RESULTS: Four different gene mutations, including one novel mutation in RUNX2 gene (NM_001024630), were identified. Among them, four individuals had the R225Q mutation, three siblings had the P224S mutation, and the other two individuals had different frame-shift mutations. Wide variations in supernumerary tooth formation were observed in individuals with identical gene mutations, and discordance was seen between monozygotic twins. Asymmetric supernumerary tooth formation was noted in five out of the nine individuals. CONCLUSION: Individuals with identical gene mutations showed a wide variation in the supernumerary tooth formation. Not only the genotype but also environmental factors and a complex system including epigenetics and copy number variation might regulate supernumerary tooth formation in CCD.

A Mongolian patient with hypohidrotic ectodermal dysplasia with a novel P121S variant in EDARADD.

INTRODUCTION: Hypohidrotic ectodermal dysplasia is a genetic disorder characterized by diminished or a lack of sweating, congenital missing teeth, and sparse or absent hair. Three genes, EDA, EDAR, and EDARADD, all related to tumor necrosis factor signaling, have been reported as responsible genes for this disorder. Among them, the largest numbers of mutations have been identified in EDA, and only two mutations identified in EDARADD. MATERIALS AND METHODS: DNA analysis of EDA, EDAR, and EDARADD was performed on a Mongolian patient by polymerase chain reaction-direct sequencing. RESULTS: The 5-year-old Mongolian individual had no erupted deciduous or permanent teeth. A panoramic radiograph showed only one tooth in the right mandible. His hair and eyebrows were sparse, but he did not have a short stature. He showed diminished sweating. The nails of his fingers and toes were normal. Based on these conditions, he was diagnosed with hypohidrotic ectodermal dysplasia. There was no gene mutation of EDA or EDAR. A novel heterozygous variant (P121S; c.361C>T) was identified in the death domain of EDARADD (NM_080738). No other member of his family was affected, and this variant was not identified in his parents or maternal grandparents. CONCLUSION: This study reports an individual affected with hypohidrotic ectodermal dysplasia with a novel heterozygous P121S variant in the death domain of EDARADD.

In vivo application of amelogenin suppresses root resorption.

Amelogenin is recognized as an enamel protein associated with enamel formation. Besides this well-known function, remarkable root resorption has been seen in amelogenin-null mutant mice. Moreover, in vitro culture studies showed that amelogenin suppressed osteoclast differentiation. These studies raised the hypothesis that amelogenin can inhibit root resorption by reducing odontoclast number. To examine this hypothesis, we applied porcine amelogenins in a rat root resorption model, in which maxillary first molars were replanted after being air-dried. Compared with untreated and carrier-treated tooth roots, the application dramatically reduced the odontoclast number on root surfaces and inhibited cementum and root dentin resorption. Amelogenin significantly reduced the number of human odontoclastic cells in culture. It also inhibited RANKL expression in mouse bone marrow cell cultures. All these findings support our hypothesis that amelogenin application suppresses root resorption by inhibiting odontoclast number, and suggest that this is mediated by the regulation of RANKL expression.

Social stress alters expression of large conductance calcium-activated potassium channel subunits in mouse adrenal medulla and pituitary glands.

Large conductance calcium-activated potassium (BK) channels are very prominently expressed in adrenal chromaffin and many anterior pituitary cells, where they shape intrinsic excitability complexly. Stress- and sex-steroids regulate alternative splicing of Slo-alpha, the pore-forming subunit of BK channels, and chronic behavioural stress has been shown to alter Slo splicing in tree shrew adrenals. In the present study, we focus on mice, measuring the effects of chronic behavioural stress on total mRNA expression of the Slo-alpha gene, two key BK channel beta subunit genes (beta2 and beta4), and the 'STREX' splice variant of Slo-alpha. As a chronic stressor, males of the relatively aggressive SJL strain were housed with a different unfamiliar SJL male every 24 h for 19 days. This 'social-instability' paradigm stressed all individuals, as demonstrated by reduced weight gain and elevated corticosterone levels. Five quantitative reverse transcriptase-polymerase chain assays were performed in parallel, including beta-actin, each calibrated against a dilution series of its corresponding cDNA template. Stress-related changes in BK expression were larger in mice tested at 6 weeks than 9 weeks. In younger animals, Slo-alpha mRNA levels were elevated 44% and 116% in the adrenal medulla and pituitary, respectively, compared to individually-housed controls. beta2 and beta4 mRNAs were elevated 162% and 194% in the pituitary, but slightly reduced in the adrenals of stressed animals. In the pituitary, dominance scores of stressed animals correlated negatively with alpha and beta subunit expression, with more subordinate individuals exhibiting levels that were three- to four-fold higher than controls or dominant individuals. STREX variant representation was lower in the subordinate subset. Thus, the combination of subunits responding to stress differs markedly between adrenal and pituitary glands. These data suggest that early stress will differentially affect neuroendocrine cell excitability, and call for detailed analysis of functional consequences.

Comprehensive gene expression analysis in human periodontal ligaments of the mandibular third molars performing vertical movement and the maxillary second premolars with occlusal contact.

OBJECTIVES: The periodontal ligament (PDL) is thought to be an important tissue in vertical movement during tooth eruption, but the precise molecular mechanism is not known. Thereto, comprehensive gene expression was analyzed in human PDL of mandibular third molars performing vertical movement and maxillary second premolars with occlusal contact. DESIGN: The expression profile of 9,243 genes in the PDL of one subject was compared between vertically moving third molars and second premolars with occlusal contact by DNA microarray. RESULTS: The expression of 27 genes showed more than a 10-fold difference between third molars and second premolars. The expression of CALB1 (encoding calbindin 1), CYP26A1 (encoding cytochrome P450, family 26, subfamily A, polypeptide 1), SPOCK3 (encoding testican-3), CCK (encoding cholecystokinin) and SCRG1 (encoding scrapie responsive protein 1) was more than 30-fold higher in PDLs of the third molars than the second premolars. CALB1 is reported to increase at the pressure side of PDL during experimental orthodontic tooth movement in rats. Interestingly, in this study, CALB1 expression showed the largest difference. In contrast, CRCT1 (encoding cysteine-rich C-terminal 1), SPRP3 (encoding small proline-rich protein 3), IL8 (encoding interleukin 8) and MMP12 (encoding matrix metalloproteinase 12) showed more than 100-fold higher expression in PDLs of the second premolars than the third molars. CONCLUSION: The present comprehensive gene expression in PDLs provides new insights into the molecular mechanism during the vertical tooth movement.

Diversity of supernumerary tooth formation in siblings with cleidocranial dysplasia having identical mutation in RUNX2 : possible involvement of non-genetic or epigenetic regulation.

INTRODUCTION: Cleidocranial dysplasia (CCD, MIM #119600) is an autosomal-dominant disorder characterized by hypoplasia or aplasia of clavicles, patent fontanelles and short stature. The responsible gene has been identified as RUNX2. CCD is also accompanied by characteristic dental abnormalities, e.g. supernumerary teeth, delayed eruption and impaction of permanent teeth. Intrafamilial variations of skeletal abnormalities are reported but those of dental abnormalities are obscure. To clarify this point, a precise examination of the dental features of CCD siblings having identical mutation was performed. DESIGN: Gene mutational analysis of three Japanese CCD siblings and their father was performed. Skeletal and dental characteristics were examined by the inquiry and radiographs. RESULTS: Three siblings uniformly showed patent fontanelles and short stature. They and their father had a novel missense mutation in the RUNT-domain (P210S) of RUNX2. The siblings were completely discordant for the dental characteristics with the position and number of supernumerary teeth being completely different. The youngest, a 12-year-old boy, had six supernumerary teeth, which appeared symmetrically around the maxillary canines and mandibular premolars. The second, a 15-year-old girl, had four supernumerary teeth which appeared around the mandibular incisors. The oldest, a 17-year-old boy, had 11 supernumerary teeth, which were symmetrically around the mandibular lateral dentition and asymmetrically around the maxillary incisors and premolars. CONCLUSION: The present study suggests the involvement of non-genetic or epigenetic regulation in supernumerary tooth formation in CCD.

A case of amelogenesis imperfecta, cleft lip and palate and polycystic kidney disease.

OBJECTIVE: Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders characterized by developmental abnormalities of tooth enamel. The AI is also seen as part of multi-organ abnormalities, e.g. with cone-rod dystrophy, hypothalamo-hypophyseal insufficiency and renal failure. The present patient with AI and nephrocalcinosis exhibited a phenotype different from previous cases with renal failure. To highlight the characteristics of this rare case, extensive analysis that included histological, biochemical and genetic examinations was performed. PATIENT: The present Japanese male patient exhibited dentition with AI and bilateral cleft lip and palate. Ground sections of his extracted tooth showed that it was hypomaturation-type AI, unlike previous cases with nephrocalcinosis were hypoplastic-type. He showed nephrocalcinosis and hematuria at 15 years of age but these symptoms appeared to be secondary to polycystic kidney disease. He showed skeletal Class II pattern with a retrognathic profile and retroclined incisors of both arches. A dolicofacial appearance was seen with an enlarged gonial angle. Biochemical makers including serum alkaline phosphatase, parathyroid hormone, calcitonin, calcium, and phosphate, were all in the normal range. Sequence analysis of the genes encoding amelogenin and enamelin, which are known to be responsible for hypoplastic-type AI, did not reveal any mutations. Since mouse null mutant of homeobox transcription factor, Msx2, exhibits a phenotype resembling AI, the human homolog of this gene, MSX2, was sequenced. There was a missense mutation of T447C that resulted in the conversion of methionine to threonine at 129.

Accelerated bone formation and increased osteoblast number contribute to the abnormal tooth germ development in parathyroid hormone-related protein knockout mice.

Our previous study showed that tooth germs at late embryonic stage [later than embryonic day 17.5 (E17.5)] and neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are compressed or penetrated by the surrounding alveolar bone tissue. In vivo and in vitro studies have shown that the development of the tooth germ proper is not disturbed, but insufficient alveolar bone resorption, due to the decreased number and hypofunction of osteoclasts, is the main cause of this abnormality. In addition to the insufficient alveolar bone resorption, progressive bone formation toward tooth germs was observed in homozygous mice, suggesting that accelerated bone formation also contributes to this abnormality. To further investigate this, homozygous mice at E14.0 and E15.5, when alveolar bone is forming, were used for histochemical and bone histomorphometric analyses. In contrast to the late embryonic stage, the alveolar bone did not yet compress developing tooth germs in homozygous mice on E14.0, but a larger amount of bone tissue was seen compared to wild-type littermates. Histomorphometric analysis of bone at E14.0 revealed that the osteoblast numbers and surfaces in the mandibles and in the bone collar of femora of homozygous mice were significantly higher than those of wild-type mice. However, unlike our previous study showing the osteoclast surface on E18.5 in homozygous mice to be significantly lower than that of wild-type mice, this study at E14.0 showed no significant difference between the two genotypes. To evaluate the amount of calcification around tooth germs, 3D images of mandibles were reconstructed from the calcein-labeled sections of the wild-type and mutant mice. Labeling was performed at E14.0, and the mice were sacrificed 1 h after the calcein injection to minimize the effect of bone resorption. Comparison of the 3D images revealed that the labeled surface was larger around developing tooth germs in homozygous mouse than in wild-type mouse. On day E15.5, osteoblasts approached the enamel organ of homozygous mice but this was not observed in wild-type mice. In this study, we report a systemic increase in osteoblast number and accelerated bone formation in homozygous PTHrP-knockout mice, both of which contribute to the abnormal tooth development.

Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel's cartilage.

Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel's cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel's cartilage and that muscle tissues influence mandible morphology.

Orthodontic treatment of a case of Becker muscular dystrophy.

Becker muscular dystrophy, similar to Duchenne muscular dystrophy, is a X-chromosomal linked anomaly characterized by progressive muscle wasting and weakness. Duchenne-type is known to have severe openbite with a steep mandibular plane, but there are no studies that describe the occlusal and skeletal patterns of the Becker-type. Here, we report the orthodontic treatment of a Becker muscular dystrophy patient. In the correction of his severe skeletal open bite general anesthesia or orthognathic surgery was not an option. Multiloop edgewise archwires were employed for orthodontic treatment. After 3 years and 8 months the open bite was corrected. During the retention period contact between the anterior teeth was maintained 8 months after active treatment despite a marked relapse tendency.

Ubc9 interacts with chicken ovalbumin upstream promoter-transcription factor I and represses receptor-dependent transcription.

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.

Development of a novel mouse osteoclast culture system including cells of mandibular body and erupting teeth.

Osteoclasts are multinucleated cells with the specialized function of resorbing calcified tissues. These cells develop from hemopoietic cells of the monocyte-macrophage lineage with the support of osteoblasts/stromal cells. Tooth eruption is a vertical movement of teeth via creation of an eruption pathway in and through the alveolar bone. The precise cellular and molecular determinants of tooth eruption are not yet clear, and a cell culture system that can reproduce the activity of osteoclast formation during tooth eruption is expected to be a useful tool to clarify the mechanism of eruption pathway formation. To this end, mandibular bodies, including incisors and molars, were isolated from 9- to 11-day-old mice undergoing active tooth eruption. Primary cells were obtained from mandibular bodies by enzymatic digestion and cultured in alphaMEM containing 15% FBS without any cytokine or growth factor or hormone in the culture (AFT culture, for alveolar bone, dental follicle, and tooth). A progressive increase in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells was observed in AFT culture. The osteoclastic cells generated were immunopositive for cathepsin K and calcitonin receptor, and formed resorption pits when cultured on dentine slices. Parathyroid hormone-related protein (PTHrP), expressed by the enamel organ of tooth, is reported to be an essential factor in creation of the eruption pathway. To verify this point, cells were isolated from mandibular bodies from which all teeth and dental follicles had been removed and cultured similarly (A culture, for alveolar bone). Osteoclastic cells were not formed and PTHrP production was hardly detected in the medium of A culture, in contrast to the high level of PTHrP in AFT culture. Since our previous study demonstrated that neonatal homozygous PTHrP-knockout mice show impaired osteoclastogenesis around tooth germs, AFT culture was performed by using this sample to examine whether this culture system can reproduce the status of osteoclastogenesis observed in vivo. The result showed that none of the osteoclastic cells were generated from cells of homozygous mice. We here report a novel mouse osteoclast culture system that reproduces the activity of osteoclast formation around erupting teeth without addition of any cytokine or growth factor or hormone to the medium. Histological examination of various transgenic and mutant mice now offers valuable findings on studies of tooth eruption and the present culture system using these animals would be a powerful tool in clarifying the cellular and molecular mechanisms of eruption pathway formation.

Disturbed tooth development in parathyroid hormone-related protein (PTHrP)-gene knockout mice.

Parathyroid hormone-related protein (PTHrP) is involved in epithelial-mesenchymal cell interactions during development of various tissues and organs. Tooth germ development is a classical model for this interaction. In tooth germs, PTHrP is expressed in the enamel organ (epithelial component), whereas its major receptor, the type I PTH/PTHrP receptor is expressed in cells of the alveolar bone and dental follicle (mesenchymal components). To clarify the role of PTHrP during fetal tooth germ development, PTHrP gene-knockout mice were used for histochemical and ultrastructural analysis. In wild-type mice, osteoclastic cells were aligned predominantly in the inner aspects of the alveolar bone surrounding the developing tooth germs throughout the late embryonic (after embryonic, 17.5 days) and neonatal animals examined. In contrast, osteoblasts were predominant in corresponding areas of fetal homozygous PTHrP-gene knockout mice with only occasional osteoclasts. In such areas, cell-free surfaces showing cement line-like tartrate-resistant acid phosphatase (TRAP) reactions were frequently observed. In neonatal homozygous mice, bone spicules were often shown to penetrate and/or compress the enamel organ and caused partial destruction of the tooth germs. Osteoclasts were few in number in the inner aspects of the alveolar bone, and had poorly developed ruffled border. No morphological abnormality was noted in cells of the tooth germs proper. On bone surfaces away from developing tooth germs, functional osteoclasts with structural features similar to those in wild-type mice were observed in homozygous mice. These observations suggest that PTHrP is required to maintain an appropriate spatiotemporal arrangement of bone cells and osteoclast function, which are necessary for the normal development of tooth germ and alveolar bone encasing the tooth germ. The observation also demonstrates that PTHrP deficiency affects the structure and function of osteoclasts exclusively those located in the vicinity of the growing tooth germ.

Molecular characterization of Hop mosaic virus: its serological and molecular relationships to Hop latent virus.

The 3'-terminal sequence of hop mosaic virus (HpMV) genomic RNA was determined. A cDNA of approximately 1.8 kbp was amplified from the HpMV genome by 3' RACE using a degenerate primer, which was designed to anneal to the overlapping region of open reading frames (ORFs) 2 and 3 of eight carlavirus genomes. The sequence contained three ORFs, encoding proteins of 7-, 34-, and 11-kDa, which corresponded to ORFs 4, 5, and 6 of the carlavirus genome, respectively. The amino acid sequence of ORF 5, encoding the coat protein (CP) of HpMV, shows the highest identity (67%) to that of Hop latent virus (HpLV). The HpMV CP N-terminal sequence differs from that of HpLV, but the central and C-terminal sequences of the CP of both viruses are similar. The sequence similarity possibly causes the cross-reaction of heterologous antibodies of HpMV and HpLV. Phylogenetic analyses based on the CP amino acid and 3' non-coding region sequences indicate close relationships among HpMV, HpLV, and Potato virus M. We report here the first molecular characterization of HpMV genomic RNA.

Structure and targeting of RyR1: implications from fusion of green fluorescent protein at the amino-terminal.

In skeletal muscle, an anterograde signal from the dihydropyridine receptor (DHPR) to the ryanodine receptor (RyR1) is required for excitation-contraction (EC) coupling and a retrograde signal from RyR1 to the DHPR regulates the magnitude of the calcium current carried by the DHPR. As a tool for studying biosynthesis and targeting, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the amino terminal of RyR1 and expressed it in dyspedic myotubes. The GFP-RyR1 was present in a restricted domain near the nucleus injected with cDNA and was fully functional, which places constraints on the location of the amino terminal in the folded structure of RyR1.

Seaweed prevents breast cancer?

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)-induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4 degrees C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemotherapeutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast cancer.

Effective fluconazole therapy for liver transplant recipients during continuous hemodiafiltration.

Fungal infections are still one of the main causes of death and complications after solid organ and bone marrow transplants. The authors evaluated the effect of continuous hemodiafiltration (CHDF) on the pharmacokinetics of fluconazole in liver transplant recipients. Six liver transplant patients (primary biliary cirrhosis, n = 2; fulminant hepatitis, n = 2; viral hepatitis, n = 2) were enrolled in this study. In one patient not receiving CHDF, the fluconazole levels increased with increasing dosages. In contrast, in patients undergoing CHDF, the dosage of fluconazole was increased from 100 mg/d to 200 mg/d, but fluconazole did not reach the targeted levels. It appears that the targeted trough level cannot be achieved by administration of fluconazole at a dosage of 100 to 200 mg/d during CHDF. A higher dosage (600-1000 mg/d) of fluconazole may be required to achieve the therapeutic drug level in patients undergoing CHDF. In patients undergoing CHDF, fluconazole was given at a dosage of 800 mg/d and reached the targeted levels. In addition, after CHDF, the dosage of fluconazole was decreased to 100 mg/d, and fluconazole reached the near-targeted trough level. These results demonstrate that CHDF removes fluconazole from the blood at an efficiently high rate, resulting in its ineffective blood level. To guarantee safe and effective fluconazole therapy, the trough levels should be monitored routinely during CHDF.

Biosynthesis of proteoglycan in bone and cartilage of parathyroid hormone-related protein knockout mice.

Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wildtype littermates were dissected into bony and cartilaginous regions and metabolically labeled with [35S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [35S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%-5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (Kd = 0.10-0.16) observed in the bony explants of both genotypes, a minor peak (Kd = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development.

Parathyroid hormone-related protein is required for normal intramembranous bone development.

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.