Detailed characterisation of the Opitz syndrome protein MIDl, its homologues and molecular interactions

Opitz G/BBB syndrome (OS) is a genetically heterogeneous congenital disorder with both X-linked and autosomal forms. Although there are established diagnostic criteria, the phenotypic variability among patients can be considerable. Most commonly, OS patients present with craniofacial, laryngotracheoesophageal, cardiac, and genital anomalies. OS has been linked to Xp22.3 and 22q 11.2; and additional patients with unique chromosomal anomalies have allowed the tentative assignment of three other autosomal loci at 14ql 1.2, 13q32.3ter and 5p12-13. Yet despite differing genetic bases, the clinical presentation in either f01m is essentially indistinguishable, despite marked variability in phenotypic presentation even among related family members. Our laboratory has previously shown that approximately 50% of patients with a diagnosis of OS harbour mutations in the X-linked MIDI gene. MIDI encodes a 667 amino acid RING finger, B-box, coiled-coil (RBCC) microtubule binding protein that is expressed widely throughout embryonic development and congruent with tissues affected in OS. A highly related protein, MID2, is the closest homologue to MID 1 and has been found to similarly associate with the microtubule network either as homomultimers, or as heteromultimers with MID 1. In this thesis, several approaches were undertaken in order to better understand the sub-cellular and molecular aspects of OS. Firstly, a search for MIDlinteracting proteins was undertaken, and secondly an analysis of homologous proteins was completed with a view to providing insight based on the function of similar proteins. To identify protein partners of MID 1 that might be components of the large MID complexes, a yeast two-hybrid screen was performed. Using this approach, Alpha 4, a regulatory subunit of PP2-type phosphatases was identified as a strong interactor of MID 1 and additionally through separate testing, MID2. Cell localisation studies showed that both MID 1 and MID2 tethered Alpha 4 to the microtubules. Co-expression of domain-deleted and mutant forms of MID 1 with Alpha 4 localised the interaction domain of MID 1 to the B-boxes, which was confirmed by yeast two-hybrid assays. In addition to MID 1 and MID2 interacting with Alpha 4, it was also found that MID 1 and MID2 interacted with oneanother through their coiled-coil domains. This further implicated MID2 as a potential modifier of the OS phenotype. Therefore, in order to better understand the basic functional aspects of MID 1 and MID2, a detailed search for other homologues was canied out. The RBCC tripartite (TRIM) motif skewed protein BLAST searches for N-terminal matches for MID 1/MID2 homologues, so combinations of N-terminal and C-te1minal domains were also used to identify structurally related proteins. BLAST and PSIBLAST searches identified the homologous proteins, TRIM9 and TRIM36 as well as partial transcripts of two new proteins TRIFIC and TNL (IRIM.2.-like). Subsequently, full sequences were determined for both of these genes. This group of six related RBCC proteins were designated as the RBCC C-I subfamily. To further identify sequence identities within the C-1 subfamily and the rest of the RBCC protein superfamily, a relatively novel technique using HMMER was employed. This showed that the most highly scoring region across the C-I subfamily was not a known domain but a region between the coiled-coil and Fibronectin type-III domains. This new region, called the COS box, was shown to be restricted to a select group of RBCC and non-RBCC proteins, all which associate with microtubules. Mutation of paiiicular amino acids in the COS box completely abolished microtubule association, and addition of a MIDI C-terminal fragment containing the COS box to a non-microtubule associating RBCC protein directed the hybrid protein to microtubules. These findings completely change the previous understanding that the coiled-coil or C-terminus of MID 1 and other RBCC proteins were responsible for microtubule association. The work presented in this thesis shows that normal association of MID 1 to microtubules is dependent on the COS box. In addition, the B-box domain is necessary for tethering Alpha 4, a protein phosphatase 2A regulatory protein, to the microtubules. The homology of MID 1 and MID2, which extends functionally to homo and heterodimerisation, microtubule association, and interaction with Alpha 4, has led to the suggestion of potential heterogeneity m Opitz syndrome. In this light, Alpha 4 was subsequently shown to be mutated m a phenotypically similar syndrome, further supporting the importance of the molecular interaction between these proteins. Potential molecular pathways in which these proteins act are discussed, as are the effects of disruption of these pathways, and the impact such changes may have on the development of key tissues affected in the diseases.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

Energetische Bewertung der Bereitstellung ausgewählter lokaler Lebensmittel am Beispiel dreier Fleischarten

In der vorliegenden Studie erfolgt eine energetische Bewertung der Bereitstellung dreier Fleischarten als Beispiel für lokale Lebensmittel. Anwendung findet eine modifizierte Form zur Berechnung des Kumulierten Energieaufwandes (KEA). Zu diesem Zweck erfolgt die Untersuchung lokaler Bereitstellungsprozesse für Schweinefleisch, Rindfleisch und Lammfleisch sowie ungarischer Bereitstellungs-prozesse für Schweinefleisch (in Ungarn regional bereitgestellt) mittels standardisierter Fragebögen hinsichtlich der Produktions- und Vermarktungsabläufe. Darüber hinaus findet eine vergleichende Bewertung einer Prozesskette für argentinisches Rindfleisch statt. Anhand der Module Transport, Mast-Elektroenergie, Mast- s.E. (sonstige Energie), Schlachtung/Zerlegung sowie Distribution findet die Ergebnisdarstellung mittels Allokation auf die funktionelle Einheit von 1 kg Fleisch ohne Knochen statt. Nach Auswertung aller energierelevanten Daten ergibt sich die Feststellung, dass die Umsätze an Endenergie und Primärenergie sowie die CO2-Emissionen mit steigender Betriebsgröße abnehmen. Die Verbrauchereinschätzung, dass lokal/regional erzeugte Lebensmittel geringere Umweltbelastungen -hinsichtlich Energieumsatz und CO2-Emission- verursachen als global erzeugte Lebensmittel, kann ebenso wie in bisher durchgeführten Untersuchungen zu Energiebilanzierungen von Prozessketten der Lebensmittel-bereitstellung (Fruchtsaft, Lammfleisch, Wein, Äpfel) nicht aufrechterhalten werden. Vielmehr verifizieren die Ergebnisse der hier durchgeführten qualitativen Analysen von Fallbeispielen die von SCHLICH 2004 geprägte TheorieThis work presents an energy evaluation of the supply chains of pork, beef and lamb. The execution of the analyses is oriented at a modified form of the CED (cumulated energy demand). For this purpose the ways of production and marketing of pork (Germany-Hessen and Hungary), beef (Germany-Hessen) an lamb (Germany-Hessen) from local supply chains are investigated. Data collection is carried out using standardized questionnaires. In addition to the mentioned analyses an evaluation of a global supply chain for beef from argentina is carried out as to compare local and global supply chains. Each supply chain will be subdivided to the modules transport, breeding-electrical energy, breeding-other energy, slaughtering/dissembling and distribution. The evaluation is achieved by allocation the energy turnover and carbon dioxyde emissions to the functional unit of 1 kg meat without bones. It is found that the energy turnover and CO2-emissions show a declining devolution related to an increasing size of businesses. Private consumer estimation expecting a lower energy turnover and environmental impact associated to meat of local origin can not be confirmed. In the same way former case studies on different food –juices, lamb, apples and wine- do, the work at hand demonstrates that the specific ecological impact does not depend on transport distance but rather on business size. It supports the theory o

MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

BACKGROUND: Patients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies. Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in ~50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder. A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules. RESULTS: To identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens. Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins. Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region. In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules. CONCLUSIONS: Our findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype. Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia) syndrome. Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome

Altered ureteric branching morphogenesis and nephron endowment in offspring of diabetic and insulin-treated pregnancy

<div><p>There is strong evidence from human and animal models that exposure to maternal hyperglycemia during <i>in utero</i> development can detrimentally affect fetal kidney development. Notwithstanding this knowledge, the precise effects of diabetic pregnancy on the key processes of kidney development are unclear due to a paucity of studies and limitations in previously used methodologies. The purpose of the present study was to elucidate the effects of hyperglycemia on ureteric branching morphogenesis and nephrogenesis using unbiased techniques. Diabetes was induced in pregnant C57Bl/6J mice using multiple doses of streptozotocin (STZ) on embryonic days (E) 6.5-8.5. Branching morphogenesis was quantified <i>ex vivo</i> using Optical Projection Tomography, and nephrons were counted using unbiased stereology. Maternal hyperglycemia was recognised from E12.5. At E14.5, offspring of diabetic mice demonstrated fetal growth restriction and a marked deficit in ureteric tip number (control 283.7±23.3 vs. STZ 153.2±24.6, mean±SEM, <i>p</i>&lt;0.01) and ureteric tree length (control 33.1±2.6 mm vs. STZ 17.6±2.7 mm, <i>p</i> = 0.001) vs. controls. At E18.5, fetal growth restriction was still present in offspring of STZ dams and a deficit in nephron endowment was observed (control 1246.2±64.9 vs. STZ 822.4±74.0, <i>p&lt;</i>0.001). Kidney malformations in the form of duplex ureter and hydroureter were a common observation (26%) in embryos of diabetic pregnancy compared with controls (0%). Maternal insulin treatment from E13.5 normalised maternal glycaemia but did not normalise fetal weight nor prevent the nephron deficit. The detrimental effect of hyperglycemia on ureteric branching morphogenesis and, in turn, nephron endowment in the growth-restricted fetus highlights the importance of glycemic control in early gestation and during the initial stages of renal development.</p> </div

Hox10 Genes Function in Kidney Development in the Differentiation and Integration of the Cortical Stroma

Organogenesis requires the differentiation and integration of distinct populations of cells to form a functional organ. In the kidney, reciprocal interactions between the ureter and the nephrogenic mesenchyme are required for organ formation. Additionally, the differentiation and integration of stromal cells are also necessary for the proper development of this organ. Much remains to be understood regarding the origin of cortical stromal cells and the pathways involved in their formation and function. By generating triple mutants in the Hox10 paralogous group genes, we demonstrate that Hox10 genes play a critical role in the developing kidney. Careful examination of control kidneys show that Foxd1-expressing stromal precursor cells are first observed in a cap-like pattern anterior to the metanephric mesenchyme and these cells subsequently integrate posteriorly into the kidney periphery as development proceeds. While the initial cap-like pattern of Foxd1-expressing cortical stromal cells is unaffected in Hox10 mutants, these cells fail to become properly integrated into the kidney, and do not differentiate to form the kidney capsule. Consistent with loss of cortical stromal cell function, Hox10 mutant kidneys display reduced and aberrant ureter branching, decreased nephrogenesis. These data therefore provide critical novel insights into the cellular and genetic mechanisms governing cortical cell development during kidney organogenesis. These results, combined with previous evidence demonstrating that Hox11 genes are necessary for patterning the metanephric mesenchyme, support a model whereby distinct populations in the nephrogenic cord are regulated by unique Hox codes, and that differential Hox function along the AP axis of the nephrogenic cord is critical for the differentiation and integration of these cell types during kidney organogenesis

Heterozygous Mutations of FREM1 Are Associated with an Increased Risk of Isolated Metopic Craniosynostosis in Humans and Mice

The premature fusion of the paired frontal bones results in metopic craniosynostosis (MC) and gives rise to the clinical phenotype of trigonocephaly. Deletions of chromosome 9p22.3 are well described as a cause of MC with variably penetrant midface hypoplasia. In order to identify the gene responsible for the trigonocephaly component of the 9p22.3 syndrome, a cohort of 109 patients were assessed by high-resolution arrays and MLPA for copy number variations (CNVs) involving 9p22. Five CNVs involving FREM1, all of which were de novo variants, were identified by array-based analyses. The remaining 104 patients with MC were then subjected to targeted FREM1 gene re-sequencing, which identified 3 further mutant alleles, one of which was de novo. Consistent with a pathogenic role, mouse Frem1 mRNA and protein expression was demonstrated in the metopic suture as well as in the pericranium and dura mater. Micro-computed tomography based analyses of the mouse posterior frontal (PF) suture, the human metopic suture equivalent, revealed advanced fusion in all mice homozygous for either of two different Frem1 mutant alleles, while heterozygotes exhibited variably penetrant PF suture anomalies. Gene dosage-related penetrance of midfacial hypoplasia was also evident in the Frem1 mutants. These data suggest that CNVs and mutations involving FREM1 can be identified in a significant percentage of people with MC with or without midface hypoplasia. Furthermore, we present Frem1 mutant mice as the first bona fide mouse model of human metopic craniosynostosis and a new model for midfacial hypoplasia

PPARα Deficiency in Inflammatory Cells Suppresses Tumor Growth

Inflammation in the tumor bed can either promote or inhibit tumor growth. Peroxisome proliferator-activated receptor (PPAR)α is a central transcriptional suppressor of inflammation, and may therefore modulate tumor growth. Here we show that PPARα deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of the endogenous angiogenesis inhibitor thrombospondin-1 and prevents tumor growth. Bone marrow transplantation and granulocyte depletion show that PPARα expressing granulocytes are necessary for tumor growth. Neutralization of thrombospondin-1 restores tumor growth in PPARα-deficient mice. These findings suggest that the absence of PPARα activity renders inflammatory infiltrates tumor suppressive and, thus, may provide a target for inhibiting tumor growth by modulating stromal processes, such as angiogenesis

The effect of high-altitude on human skeletal muscle energetics: 31P-MRS results from the caudwell xtreme everest expedition

Many disease states are associated with regional or systemic hypoxia. The study of healthy individuals exposed to high-altitude hypoxia offers a way to explore hypoxic adaptation without the confounding effects of disease and therapeutic interventions. Using 31P magnetic resonance spectroscopy and imaging, we investigated skeletal muscle energetics and morphology after exposure to hypobaric hypoxia in seven altitude-naïve subjects (trekkers) and seven experienced climbers. The trekkers ascended to 5300 m while the climbers ascended above 7950 m. Before the study, climbers had better mitochondrial function (evidenced by shorter phosphocreatine recovery halftime) than trekkers: 16±1 vs. 22±2 s (mean ± SE, p<0.01). Climbers had higher resting [Pi] than trekkers before the expedition and resting [Pi] was raised across both groups on their return (PRE: 2.6±0.2 vs. POST: 3.0±0.2 mM, p<0.05). There was significant muscle atrophy post-CXE (PRE: 4.7±0.2 vs. POST: 4.5±0.2 cm2, p<0.05), yet exercising metabolites were unchanged. These results suggest that, in response to high altitude hypoxia, skeletal muscle function is maintained in humans, despite significant atrophy