ADAMTS5 is required for normal trabeculated bone development in the mandibular condyle.

OBJECTIVE: Determine the role of the extracellular matrix protease ADAMTS5 in development of the trabeculated bone of the mandibular condyle. METHODS: The mandibular condyles of wild type and mice deficient in the protease ADAMTS5 were examined for histopathology with Safranin O staining. Microcomputed tomography was performed to analyze the developing bone of the mandibular condyle. RNAscope and immunohistochemistry were utilized to investigate cell type and extracellular matrix expression. RESULTS: Mice deficient in Adamts5, (Adamts5tm1Dgen/J) exhibit an increase in trabecular separation (n = 37 wild type; n = 27: P < 0.0001) and reduction of trabecular thickness P = 0.0116 and bone volume fraction P = 0.0869 in the mandibular condylar head compared to wild type littermates. The altered bone parameters were more pronounced in male Adamts5-/- mice compared to female Adamts5-/- mice (TbSp; P = 0.03). Adamts5 was co-expressed with versican and Gli1 in mesenchymal, stem-like cells in the transition zone where the trabeculated bone is adjacent to mature hypertrophic chondrocytes. Loss of Adamts5 caused a reduction of Bglap expressing osteoblasts throughout mandibular condylar development and in young adult mice. The protease Mmp13, that is involved in mineralization and is expressed by hypertrophic chondrocytes and osteoblasts, was reduced in the mandibular condyle of Adamts5 deficient mice. CONCLUSION: This is the first report of a novel and critical role for Adamts5 in bone formation within the mandibular condyle of the temporomandibular joint. These data indicate Adamts5 may be required in the transdifferentiation of hypertrophic chondrocytes to osteoblasts during trabecular bone formation in development of the mandibular condyle.

The Zonal Architecture of the Mandibular Condyle Requires ADAMTS5.

Temporomandibular joint (TMJ) osteoarthritis (TMJOA) disrupts extracellular matrix (ECM) homeostasis, leading to cartilage degradation. Upregulated a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 leads to cleavage of its substrate aggrecan (Acan) and is considered a hallmark of TMJOA. However, most research on ADAMTS5-Acan turnover has focused on hyaline cartilage, not fibrocartilage, which comprises the TMJ. The mandibular condylar cartilage (MCC) of the TMJ is organized in zones, and chondrocytes are arranged in axial rows, yet the molecular mechanisms required to generate the MCC zonal architecture have not been elucidated. Here, we test the hypothesis that ADAMTS5 is required for development of the TMJ MCC. Adamts5+/+ and Adamts5-/- murine TMJs were harvested at postnatal day 7 (P7), P21, 2 mo, and 6 mo of age; histomorphometrics indicated increased ECM. Immunohistochemistry and Western blots demonstrated the expanded ECM correlated with increased Acan localization in Adamts5-/- compared to Adamts5+/+. Cell volume was also decreased in the MCC of Adamts5-/- due to both a reduction in cell size and less mature hypertrophic chondrocytes. Analysis of chondrogenic maturation markers by quantitative real-time polymerase chain reaction indicated Col2a1, Col10a1, and Sox9 were significantly reduced in Adamts5-/- MCC compared to that of Adamts5+/+. The older (6 mo) Adamts5-/- MCC exhibited changes in chondrogenic cell arrangements, including clustering and chondrogenic atrophy, that correlated with early stages of TMJOA using modified Mankin scoring. These data indicate a potentially novel and critical role of ADAMTS5 for maturation of hypertrophic chondrocytes and establishment of the zonal architecture that, when disrupted, may lead to early onset of TMJOA.

Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Krüppel-like factor: identification of a new multigene family.

We have identified and characterized the gene for a novel zinc finger transcription factor which we have termed lung Krüppel-like factor (LKLF). LKLF was isolated through the use of the zinc finger domain of erythroid Krüppel-like factor (ELKF) as a hybridization probe and is closely related to this erythroid cell-specific gene. LKLF is expressed in a limited number of tissues, with the predominant expression seen in the lungs and spleen. The gene is developmentally controlled, with expression noted in the 7-day embryo followed by a down-regulation at 11 days and subsequent reactivation. A high degree of similarity is noted in the zinc finger regions of LKLF and EKLF. Beyond this domain, the sequences diverge significantly, although the putative transactivation domains for both LKLF and EKLF are proline-rich regions. In the DNA-binding domain, the three zinc finger motifs are so closely conserved that the predicted DNA contact sites are identical, suggesting that both proteins may bind to the same core sequence. This was further suggested by transactivation assays in which mouse fibroblasts were transiently transfected with a human beta-globin reporter gene in the absence and presence of an LKLF cDNA construct. Expression of the LKLF gene activates this human beta-globin promoter containing the CACCC sequence previously shown to be a binding site for EKLF. Mutation of this potential binding site results in a significant reduction in the reporter gene expression. LKLF and EKLF can thus be grouped as members of a unique family of transcription factors which have discrete patterns of expression in different tissues and which appear to recognize the same DNA-binding site.

Proteomic Alterations Associated with Biomechanical Dysfunction are Early Processes in the Emilin1 Deficient Mouse Model of Aortic Valve Disease.

Aortic valve (AV) disease involves stiffening of the AV cusp with progression characterized by inflammation, fibrosis, and calcification. Here, we examine the relationship between biomechanical valve function and proteomic changes before and after the development of AV pathology in the Emilin1-/- mouse model of latent AV disease. Biomechanical studies were performed to quantify tissue stiffness at the macro (micropipette) and micro (atomic force microscopy (AFM)) levels. Micropipette studies showed that the Emilin1-/- AV annulus and cusp regions demonstrated increased stiffness only after the onset of AV disease. AFM studies showed that the Emilin1-/- cusp stiffens before the onset of AV disease and worsens with the onset of disease. Proteomes from AV cusps were investigated to identify protein functions, pathways, and interaction network alterations that occur with age- and genotype-related valve stiffening. Protein alterations due to Emilin1 deficiency, including changes in pathways and functions, preceded biomechanical aberrations, resulting in marked depletion of extracellular matrix (ECM) proteins interacting with TGFB1, including latent transforming growth factor beta 3 (LTBP3), fibulin 5 (FBLN5), and cartilage intermediate layer protein 1 (CILP1). This study identifies proteomic dysregulation is associated with biomechanical dysfunction as early pathogenic processes in the Emilin1-/- model of AV disease.

Revision in sequence of CAD aspartate transcarbamylase domain of Drosophila.

The Drosophila CAD gene, also known as rudimentary, encodes a protein that carries out the first three enzymatic steps of de novo pyrimidine biosynthesis. The sequence for this gene, as previously published, appears to contain several errors. The correction of six bases in a 250 bp stretch encoding the aspartate transcarbamylase domain leads to changes of frame in two areas of the predicted amino acid sequence, consisting of lengths of 30 and 15 amino acid residues, respectively. The revised sequence shows significantly improved positional identity with both Syrian hamster and Escherichia coli aspartate transcarbamylases.

[Experiences with the subfrontal approach to manage extensive fractures of the frontal skull base]. / Erfahrungen mit dem subfrontalen Zugang zur Rekonstruktion ausgedehnter Frakturen im Bereich der Frontobasis.

BACKGROUND: Surgical management of multiple traumatized patients with head and neck trauma is highly individualized and depends on a number of factors including etiology, intracranial pressure, concomitant injuries, patient age and the possibility of an interdisciplinary procedure. Severe head and neck trauma are often connected with fractures of the frontal skull base or nasoethmoido-orbital complex and CSF leakage. If there is suspicion of a CSF leakage surgical management to cover the dura-defect is essential. An intradural approach is necessary in case of concomitant intradural injuries while primary extradural access provides excellent exposure of the rhinobasis with low morbidity and good results. METHODS AND MATERIAL: We report about our surgical experiences of 55 patients with severe frontobasal trauma, who were operated between 1/1999 and 11/2003. The subfrontal approach according to Raveh we had chosen in 20 patients which were operated by an interdisciplinary team together with the neurosurgeons. With an average follow up of 36 month we report about early and late complications. RESULTS: 19/20 patients showed sufficient coverage of the CSF leakage, once a revision surgery was necessary. Finally this patient had also an unobjectionable coverage of the CSF leakage. We saw no major complication like meningitis or brain abscess, intracerebral haematoma or surgical injury of the orbital wall. The most important complication was an anosmia, which depending on the extension of the approach results in any patients. CONCLUSIONS: Our results show that the subfrontal approach is a reliable method to explore extensive frontal dural defects and to reconstruct fractures of the frontal skull base without additional trauma to the frontal lobe.

Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion.

On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins.

Expression and subcellular localization of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein in mouse and chicken over developmental time.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic-helix-loop-helix/Per- ARNT-Sim (bHLH/PAS) transcription factor that is involved in multiple signaling pathways. This study focuses on the tissue distribution and subcellular localization of ARNT during embryological development of the mouse and chicken. Two different species were chosen to determine the consistency of the ARNT staining pattern. Immunohistochemical techniques were used to stain sections of embryos over three developmental time points for each species. Mouse tissues evaluated from embryonic day 10.5, 12.5, and 15, exhibited predominant nuclear staining with little change in expression patterns over time. Chicken tissues evaluated from embryonic day 2, 4, and 10 also showed predominant nuclear staining within all cells and little change in expression over developmental time, as well as, low levels of cytoplasmic ARNT staining in some cells. Importantly, in all tissues, the level of ARNT staining within the nuclear compartment was greater than staining observed in the cytoplasm. Thus, the overall conclusions from these studies are that i) the predominant subcellular localization of ARNT protein is nuclear, and ii) that mouse and chicken appear to maintain ARNT protein expression in many cell types over developmental time. These data support vertebrate ARNT as a nuclear transcription factor and a model in which dimerization partners require nuclear localization for interaction.

Analysis of developmental switching in transgenic mice with 5' and 3' deletions in the human beta globin gene.

Our interest in the cis-acting elements that promote the up-regulation of the beta globin gene has led to a systematic deletion analysis of portions of the beta globin gene in the context of the HS2 and gamma globin gene using transgenic mice. In constructs that delete the 5' region to only 265 bp, high-level, erythroid-specific expression was observed. Further deletion to 122 bp, however, results in significantly reduced expression levels. A substitution of a minilocus control region for the single HS2 site was also produced, resulting in increased beta globin expression over that seen with the HS2 alone. These results are consistent with the presence of an enhancer-like element between -122 and -265. In addition, a construct in which the entire beta globin gene promoter was replaced by a thymidine kinase promoter was tested. Interestingly, no expression was detected in these transgenic mice. This may indicate the requirement for an erythroid-specific promoter to drive this gene. Finally, the 3' region of the beta globin gene was deleted in order to examine the effect of a previously defined 3' enhancer region. With deletion of this region, the expression of the human beta globin gene in transgenic mice is unchanged relative to the parental constructs.

The evolutionary history of the first three enzymes in pyrimidine biosynthesis.

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.