Casitas b-lineage lymphoma-deficient mice are pretected against high-fat diet-induuced obesity and insulin resistance

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity involved in regulating the degradation of receptor tyrosine kinases. We have recently reported that c-Cbl^–/– mice exhibit a lean phenotype and enhanced peripheral insulin action likely due to elevated energy expenditure. In the study reported here, we examined the effect of a high-fat diet on energy homeostasis and glucose metabolism in these animals. When c-Cbl^–/– mice were fed a high-fat diet for 4 weeks, they maintained hyperphagia, higher whole-body oxygen consumption (27%), and greater activity (threefold) compared with wild-type animals fed the same diet. In addition, the activity of several enzymes involved in mitochondrial fat oxidation and the phosphorylation of acetyl CoA carboxylase was significantly increased in muscle of high-fat–fed c-Cbl–deficient mice, indicating a greater capacity for fat oxidation in these animals. As a result of these differences, fat-fed c-Cbl^–/– mice were 30% leaner than wild-type animals and were protected against high-fat diet–induced insulin resistance. These studies are consistent with a role for c-Cbl in regulating nutrient partitioning in skeletal muscle and emphasize the potential of c-Cbl as a therapeutic target in the treatment of obesity and type 2 diabetes.

Mucosal vascular addressin cell adhesion molecule-1 is expressed outside the endothelial lineage on fibroblasts and melanoma cells

Mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) is predominantly expressed on high endothelial venules in inflamed tissues where it assists with leucocyte extravasation. Here we report that MAdCAM-1 has the potential to be more widely expressed outside the endothelial cell lineage than previously appreciated. Thus, MAdCAM-1 RNA transcripts and cell-surface protein were expressed by NIH 3T3 fibroblasts following activation with tumour necrosis factor-alpha (TNF-alpha), and by freshly isolated and cultured primary mouse splenic and tail fibroblasts in the absence of TNF-alpha stimulation. They were constitutively expressed by B16F10 melanoma cells, and expression was enhanced by cell activation with TNF-alpha. Mucosal vascular addressin cell adhesion molecule-1 was expressed on the apical surface of isolated cells, but became predominantly localized to cell junctions in confluent cell monolayers, suggesting it may play a role in the homotypic aggregation of cells. Tumour necrosis factor-alpha enhanced the expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter in NIH 3T3 and B16F10 cells. A DNA fragment extending from nt -1727 to -673 was sufficient to confer cell-type selective expression. Mucosal vascular addressin cell adhesion molecule-1 expressed by NIH 3T3 cells was biologically active, as it supported the adhesion of TK-1 T cells in an alpha4beta7-dependent fashion. The expression of MAdCAM-1 by fibroblasts, and melanomas suggests MAdCAM-1 may play a role in regulating host responses in the periphery, leucocyte transmigration across nonendothelial boundaries, or the homotypic interactions of some malignant melanomas.

Epithelial-stromal interactions in mammary lineage development and carcinogenesis

This study determined that the microenvironment influences mammary cellular development and that this microenvironment differs between normal and malignant tissue. Cancer-associated fibroblasts, loss of an extracellular protein and presence of a growth factor were demonstrated to influence cancer cell migration, presenting a basis for the understanding of breast cancer metastasis.

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of β-casein occurred in the monotreme lineage, as opposed to more ancient duplications of α-casein in the eutherian lineage, while marsupials possess only single copies of α- and β-caseins. Despite this variability, the close proximity of the main α- and β-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

Early Connections : reflections on the canonical lineage of Southeast Asian temples

Temples were constructed across Southeast Asia following the spread of Brahmanic/Hindu culture between the fifth to eight centuries CE. Epigraphic evidence, architectural and stylistic similarities between temples in the region are strongly indicative of historic cross cultural links between the traditions. This paper presents the findings of a research project that pieces together fragments of evidence from early temple sites in Southeast Asia to establish the linkages between the Southeast Asian temple building traditions. The focus of the paper is on tracing the canonical connections between these traditions through an examination of temple sites in Cambodia and Java respectively. The legacy of this ancient diasporic movement remains celebrated today in the admiration of Southeast Asian monuments such as Angkor Wat and Prambanan. However this architecture evolved over time through a process of long experimentation with philosophies, world-views, and methods. . In order to permit a deeper examination of canonical connections, the authors use methods such as photogrammetry, digital and physical models to reconstruct the architectural forms. A detailed analysis of the canonical geometry and compositional form of these temples is undertaken with reference to Indic texts and temples. Comparing the relationships between cosmology, geometry and physical form in this earlier sites with both Indian and developed Southeast Asian models, it is intended that its generative role within Southeast Asian architectural historiography can be clarified and more fully celebrated.

Low-dose histone deacetylase inhibitor treatment leads to tumor growth arrest and multi-lineage differentiation of malignant rhabdoid tumors

PURPOSE: Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1 SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT. EXPERIMENTAL DESIGN: Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with low-dose HDACi can lead to sustained cell growth arrest and differentiation. RESULTS: Sustained low-dose panobinostat (LBH589) treatment led to changes in cellular morphology associated with a marked increase in the induction of neural, renal, and osteoblast differentiation pathways. Genome-wide transcriptional profiling highlighted differential gene expression supporting multilineage differentiation. Using mouse xenograft models, sustained low-dose LBH589 treatment caused tumor growth arrest associated with tumor calcification detectable by X-ray imaging. Histological analysis of LBH589-treated tumors revealed significant regions of ossification, confirmed by Alizarin Red staining. Immunohistochemical analysis showed increased TUJ1 and PAX2 staining suggestive of neuronal and renal differentiation, respectively. CONCLUSIONS: Low-dose HDACi treatment can terminally differentiate MRT tumor cells and reduce their ability to self-renew. The use of low-dose HDACi as a novel therapeutic approach warrants further investigation.

Comparative phylogeography of alpine invertebrates indicates deep lineage diversification and historical refugia in the Australian Alps

Aim: Comparative phylogeographic analyses of alpine biota from the Northern Hemisphere have linked patterns of genetic diversification to glacial expansion and contraction events in the Pliocene and Pleistocene. Furthermore, the extent of diversification across species groups appears to be associated with vagility. In this study we test whether these patterns apply to a geologically stable system from eastern Australia with comparatively shallow elevational gradients and minimal influence from historical glacial activity. Location: The Australian Alps, Victoria, eastern Australia. Methods: We considered phylogeographic patterns across five alpine invertebrate species based on mitochondrial and nuclear DNA sequence data. Bayesian inference methods were used to estimate species phylogenies and divergence times among lineages. GIS tools were used to map interpopulation genetic divergence and intrapopulation genetic diversity estimates and to visualize spatial patterns across species, providing insights into patterns of endemism and demographic history. Results: Phylogeographic patterns and the timing of lineage diversification were consistent across taxonomic groups. Mountain summits harbour highly differentiated haplogroups, including summits connected by high-elevational plateaus, pointing to diversifications being maintained since the early to mid-Pleistocene. These findings are consistent with previous studies of alpine mammals and reptiles, demonstrating a high degree of endemism in this region, regardless of species vagility. Main conclusions: The fine spatial scales at which deep genetic differentiation among alpine communities was observed in this study are unprecedented. This suggests that glacial periods have had less of an impact on species distributions and genetic diversity than they have in alpine systems in the Northern Hemisphere. Historical gene flow among sky-island populations has been limited despite connecting snowlines during glacial periods, suggesting that factors other than snow cover have influenced patterns of gene flow in this region. These findings emphasize the unique phylogeographic history affecting Victorian alpine biodiversity, and the importance of conserving biodiversity from multiple mountain summits in this region of high endemism.

Evolutionary history of a secondary terrestrial Australian diving beetle (Coleoptera, Dytiscidae) reveals a lineage of high morphological and ecological plasticity

The evolution of a secondary terrestrial lifestyle in diving beetles (Coleoptera: Dytiscidae) has never been analysed in a phylogenetic framework before. Here we study Terradessus caecus Watts, a terrestrial species of the subfamily Hydroporinae endemic to Australia. We infer its phylogenetic placement using Bayesian inference and maximum-likelihood methods based on a multilocus molecular dataset. We also investigate the divergence time estimates of this lineage using a Bayesian relaxed clock approach. Finally, we infer ancestral ecological preferences using a likelihood approach. We recover T. caecus nested in the genus Paroster Sharp with strong support. Therefore, we establish a synonymy for both species of Terradessus with Paroster: Paroster caecus (Watts) n.comb. and Paroster anophthalmus (Brancucci & Monteith) n.comb. Paroster is an endemic Australian genus that has a remarkable number of subterranean species in underground aquifers with highly derived morphologies. Our results highlight one of the most remarkable radiations of aquatic beetles with independent ecological pathways likely linked to palaeoclimatic disruptions in the Neogene. Paroster caecus (Watts) n.comb. originated from a mid-Miocene split following the onset of an aridification episode that has been ongoing to the present day. The deep changes in ecological communities in association with the drying-out of palaeodrainage systems might have pushed this lineage to colonize a new niche in terrestrial habitats.

Expression of Na+/H+ exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis

Sodium/proton exchangers (NHE) are transmembrane proteins that facilitate the exchange of a Na⁺ ion for a H⁺ ion across cellular membranes. The NHE are present in the gills of fishes and are believed to function in acid-base regulation by driving the extrusion of protons across the branchial epithelium in exchange for Na⁺ in the water. In this study, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the presence of a branchial NHE in the gills of the Atlantic hagfish, Myxine glutinosa. The subsequent partial cDNA sequence shares homology with other vertebrate and invertebrate NHE isoforms. In addition, using semi-quantitative, multiplex RT-PCR we demonstrate that mRNA expression of hagfish gill NHE is upregulated following an induced metabolic acidosis. Expression was increased to 4.4 times basal levels at 2-h post-infusion and had decreased to 1.6 times basal by 6 h. Expression had returned to basal levels by 24-h post-infusion. The inference from this study is that a gill NHE which is potentially important in acid-base regulation has been present in the vertebrate lineage since before the divergence of the hagfishes from the main vertebrate line.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.

Phylogenetic relationships of the globally distributed freshwater prawn genus Macrobrachium (Crustacea: Decapoda: Palaemonidae): biogeography, taxonomy, and the convergent evolution of abbreviated larval development

There has hitherto been little research into evolutionary and taxonomic relationships amongst species of the freshwater prawn genus Macrobrachium Bate across its global distribution. Previous work by the authors demonstrated that the endemic Australian species did not evolve from a single ancestral lineage. To examine whether other regional Macrobrachium faunas also reflect this pattern of multiple origins, the phylogeny of 30 Macrobrachium species from Asia, Central/South America and Australia was inferred from mitochondrial 16S rRNA sequences. Phylogenetic relationships demonstrate that, despite some evidence for regional diversification, Australia, Asia and South America clearly contain Macrobrachium species that do not share a common ancestry, suggesting that large-scale dispersal has been a major feature of the evolutionary history of the genus. The evolution of abbreviated larval development (ALD), associated with the transition from an estuarine into a purely freshwater lifecycle, was also mapped onto the phylogeny and was shown to be a relatively homoplasious trait and not taxonomically informative. Other taxonomic issues, as well as the evolutionary origins of Macrobrachium, are also discussed.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

The molecular systematics of Leiopotherapon unicolor (Günther, 1859): testing for cryptic speciation in Australia’s most widespread freshwater fish

Leiopotherapon unicolor is the most widespread freshwater fish species in Australia. A comprehensive allozyme and mitochondrial DNA 16S rRNA data set was assembled from 141 specimens of L. unicolor collected Australia-wide in order to test for cryptic speciation in this far-ranging species. Surprisingly, little genetic diversity was observed within L. unicolor and provided no evidence for the existence of cryptic species within this lineage. In contrast, a small sample set of L. aheneus used as the outgroup showed two highly divergent haplotypes strongly suggestive of cryptic speciation. L. unicolor has a number of ecological and life history attributes that may explain the lack of significant genetic divergence over substantial geographical distances. The occurrence of other widespread fish and crustacean species that also display only limited genetic diversity indicate that climate conditions more favourable to dispersal across central and northern Australia than is suggested by the extent of present-day aridity have occurred in the relatively recent geological past.