The fur seal — a model lactation phenotype to explore molecular factors involved in the initiation of apoptosis at involution

Mammary gland involution requires co-ordination of milk production, immune responses, apoptosis and remodeling. Initiation and progression of each of these components involves integral control by the mammary gland. Although cell-based culture models and genetically manipulated animals have shed light on these processes, the factors controlling each step in the involution cascade are still poorly understood. The fur seal displays a unique lactation phenotype. During the lactation cycle the mammary gland downregulates milk production and initiates an immune response but fails to initiate the apoptotic phase of involution, allowing the female fur seal to undertake long foraging trips of up to 28 days between suckling bouts. Upon return to shore the female continues feeding her pup following resumption of lactation and milk production. Expression profiling of genes involved in this lactation cycle provides valuable tools for investigation of the factors responsible for the initiation of apoptosis at involution.

Characterizing the molecular phenotype of an Atp7a(T985I) conditional knock in mouse model for X-linked distal hereditary motor neuropathy (dHMNX)

ATP7A is a P-type ATPase essential for cellular copper (Cu) transport and homeostasis. Loss-of-function ATP7A mutations causing systemic Cu deficiency are associated with severe Menkes disease or its milder allelic variant, occipital horn syndrome. We previously identified two rare ATP7A missense mutations (P1386S and T994I) leading to a non-fatal form of motor neuron disorder, X-linked distal hereditary motor neuropathy (dHMNX), without overt signs of systemic Cu deficiency. Recent investigations using a tissue specific Atp7a knock out model have demonstrated that Cu plays an essential role in motor neuron maintenance and function, however the underlying pathogenic mechanisms of ATP7A mutations causing axonal degeneration remain unknown. We have generated an Atp7a conditional knock in mouse model of dHMNX expressing Atp7a(T985I), the orthologue of the human ATP7A(T994I) identified in dHMNX patients. Although a degenerative motor phenotype is not observed, the knock in Atp7a(T985I/Y) mice show altered Cu levels within the peripheral and central nervous systems, an increased diameter of the muscle fibres and altered myogenin and myostatin gene expression. Atp7a(T985I/Y) mice have reduced Atp7a protein levels and recapitulate the defective trafficking and altered post-translational regulatory mechanisms observed in the human ATP7A(T994I) patient fibroblasts. Our model provides a unique opportunity to characterise the molecular phenotype of dHMNX and the time course of cellular events leading to the process of axonal degeneration in this disease.

Molecular architecture of Aβ fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aβ plaque formation

OBJECTIVES: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

PMC42, a novel model for the differentiated human breast

Cultured human breast carcinoma cell lines are important models for investigating the pathogenesis of breast cancer. Their use, however, is limited because of loss of expression of breast-specific markers and the development of a dedifferentiated phenotype after continuous culture. PMC42 is a unique human breast carcinoma line, previously shown to express secretory and myoepithelial markers. We have induced PMC42 cells to form hollow organoids in culture, similar to in vivo breast structures, using a combination of hormones including estrogen, progesterone, dexamethasone, insulin, and prolactin in combination with a permeable extracellular matrix. The organoids comprised polarized cells located around a central lumen. Expression of β-casein was demonstrated in cells within organoids using reverse transcriptase-polymerase chain reaction, Western blot analysis, and confocal immunofluorescence. In this in vitro system, milk-specific gene expression was induced through hormone and matrix interactions which may be similar to those operating in vivo. PMC42 is a novel model for investigations into the molecular mechanisms of carcinogenesis and differentiation in the human breast.

The zebrafish as a model system for human disease

The zebrafish (Danio rerio) has been widely utilised for the study of developmental biology, which has lead to the evolution of sophisticated cellular and molecular approaches. More recently, the rapid progress of various zebrafish genomic infrastructure initiatives is facilitating the development of zebrafish models of human disease. This review aims to describe several representative examples of how the zebrafish can be successfully used to identify novel genes and assign gene function, providing invaluable clues to human pathophysiology.

Information integration in molecular bioscience

Integrating information in the molecular biosciences involves more than the cross-referencing of sequences or structures. Experimental protocols, results of computational analyses, annotations and links to relevant literature form integral parts of this information, and impart meaning to sequence or structure. In this review, we examine some existing approaches to integrating information in the molecular biosciences. We consider not only technical issues concerning the integration of heterogeneous data sources and the corresponding semantic implications, but also the integration of analytical results. Within the broad range of strategies for integration of data and information, we distinguish between platforms and developments. We discuss two current platforms and six current developments, and identify what we believe to be their strengths and limitations. We identify key unsolved problems in integrating information in the molecular biosciences, and discuss possible strategies for addressing them including semantic integration using ontologies, XML as a data model, and graphical user interfaces as integrative environments.

Investigation of plasmodiophora brassicae (clubroot disease) in vegetable brassica using arabidopsis thaliana as a model system

Clubroot, caused by Plasmodiophora brassicae, is the most devastating soil-borne disease of vegetable brassicas. It occurs all over the world and is responsible for crop losses of up to 10% every year. In Australia, the disease is being managed effectively with chemicals and cultural practices, but ideally control can be improved in the long term by the introduction of resistant cultivars. The life cycle ofP. brassicae and mode of action of plant resistance has not been fully elucidated because of the technical difficulties of working with an obligate, soil-borne plant pathogen. However, Arabidopsis thaliana, which is a host ofP. brassicae, has great potential as a model system for studying the life cycle, the infection process and development of resistance. We have developed a sand-liquid-culture system for growing Arabidopsis that allows easy observation of all life stages and, most importantly, the primary plasmodial stages within the root hair. The method was first optimised for observations of the lifecycle of the pathogen in a susceptible Arabidopsis ecotype (Col-3) where all stages of the lifecycle have now been observed and characterised. Further screening of Arabidopsis ecotypes for disease resistance has utilised one of the most virulent Australian pathotypes of brassica (ECD number 16/19/31). To date, Arabidopsis ecotype Ta-0 has shown a level of tolerance to the disease even though the roots get infected. It has been reported earlier that resistance toP. brassicae in Arabidopsis is due to one or a small number of genes. To examine changes in gene expression during the early, critical stages of infection, RNA was extracted from the susceptible and resistant ecotypes at two time points, 4 days and 17 days after inoculation. Microarray analysis will be used to investigate genome wide changes in gene expression during infection but also to identify candidate genes that may confer resistance to Australian isolates of the pathogen.

Molecular analysis of the human copper uptake protein hCTR1

This study examined the role of the high-affinity copper uptake protein hCTR1 in cellular copper homeostasis and found hCTR1 was internalized in response to raised copper levels. The work from this thesis supports a model in which the regulation of hCTR1 is partially or wholly dependent upon internal copper levels.

Ion clustering in molecular dynamics simulations of sodium iodide solutions

Model systems of sodium iodide dissolved in dimethyl ether or 1,2-dimethoxyethane (glyme) were studied in order to investigate the structural and dynamic properties of ionic solutions in small and polymeric ethers. Full molecular dynamics simulations were performed at a range of different salt concentrations. An algorithm was designed which assigns ions to clusters and then calculates all the terms which contribute to ionic conductivity. In dilute solutions, free ions are the most common ionic species, followed by ion pairs. As the concentration increases, pairs become the most common species, with significant concentrations of clusters with 3 through 6 ions. Changing the solvent from dimethyl ether to glyme significantly decreases the ion clustering due to the chelate effect in which the two oxygens on a solvent stabilize an associated cation. The conductivity in stable systems is shown to be primarily the result of the movement of free ions and the relative movement of ions within neutral pairs. The Nernst-Einstein relation, commonly used in the discussion of polymer electrolytes, is shown to be inadequate to quantitatively describe conductivity in the model systems.

Defence mechanisms of a resistant monocot model to phytophthora cinnamomi

The plant pathogen Phytophthora cinnamomi causes devastating disease in natural and agricultural systems worldwide. While some plants can survive, little is known about the underlying mechanisms of resistance. This research used histochemical and genome-wide analysis to identify key cellular and molecular defence mechanisms within the resistant plant Zea mays.

An inclusion complex of β-Cyclodextrin-L-Phenylalanine : 1H NMR and molecular docking studies

An inclusion host-guest complex between β-cyclodextrin (β-CD) and L-phenylalanine (LPhe) was investigated using ¹H nuclear magnetic resonance spectroscopy and molecular docking techniques. ¹H chemical shift changes of β-CD were used to calculate the stability constant (K_stb) of the complex. On the basis of the Hildebrand-Benesi method, the K_stb of the 1:1 complex in D₂O solution at 300 K, pD 7.6 was of 25.5 M^-1, implying a fast intermolecular exchange rate process. Interestingly, docking simulation indicates the toroidal space can be occupied by L-Phe with two favorable arrangements. For the predicted model with the higher probability score, the L-Phe aromatic ring is facing to the secondary hydroxyl groups of β-CD. Results from NMR and docking simulation are in good agreement with the x-ray structures of β-CD/L-phenylalanine derivatives.

Engineering molecular self-assembled fibrillar networks by ultrasound

The architecture of self-organized three-dimensionally interconnected nanocrystal fibrillar networks has been achieved by ultrasound from a solution consisting of separate spherulites. The ultrasound stimulated structural transformation is correlated to the striking ultrasonic effects on turning nongelled solutions or weak gels into strong gels instantly, with enhancement of the storage modulus up to 3 magnitudes and up to 4 times more gelling capability. The basic principle involved in the ultrasound-induced structural transformation is established on the basis of the nucleation-and-growth model of a fiber network formation, and the mechanism of seeding multiplication, aggregation suppressing, and fiber distribution and growth promotion is proposed. This novel technique enables us to produce self-supporting gel functional materials possessing significantly modified macroscopic properties, from materials previously thus far considered to be “useless”, without the use of chemical stimuli. Moreover, it provides a general strategy for the engineering of self-organized fiber network architectures, and we are consequently able to achieve the supramolecular functional materials with controllable macroscopic properties.

A neuro-immune model of myalgic encephalomyelitis/chronic fatigue syndrome

This paper proposes a neuro-immune model for Myalgic Encephalomyelitis/Chronic fatigue syndrome (ME/CFS). A wide range of immunological and neurological abnormalities have been reported in people suffering from ME/CFS. They include abnormalities in proinflammatory cytokines, raised production of nuclear factor-κB, mitochondrial dysfunctions, autoimmune responses, autonomic disturbances and brain pathology. Raised levels of oxidative and nitrosative stress (O&NS), together with reduced levels of antioxidants are indicative of an immuno-inflammatory pathology. A number of different pathogens have been reported either as triggering or maintaining factors. Our model proposes that initial infection and immune activation caused by a number of possible pathogens leads to a state of chronic peripheral immune activation driven by activated O&NS pathways that lead to progressive damage of self epitopes even when the initial infection has been cleared. Subsequent activation of autoreactive T cells conspiring with O&NS pathways cause further damage and provoke chronic activation of immuno-inflammatory pathways. The subsequent upregulation of proinflammatory compounds may activate microglia via the vagus nerve. Elevated proinflammatory cytokines together with raised O&NS conspire to produce mitochondrial damage. The subsequent ATP deficit together with inflammation and O&NS are responsible for the landmark symptoms of ME/CFS, including post-exertional malaise. Raised levels of O&NS subsequently cause progressive elevation of autoimmune activity facilitated by molecular mimicry, bystander activation or epitope spreading. These processes provoke central nervous system (CNS) activation in an attempt to restore immune homeostatsis. This model proposes that the antagonistic activities of the CNS response to peripheral inflammation, O&NS and chronic immune activation are responsible for the remitting-relapsing nature of ME/CFS. Leads for future research are suggested based on this neuro-immune model.

The resurrection plant Sporobolus stapfianus: An unlikely model for engineering enhanced plant biomass?

The resurrection grass Sporobolus stapfianus Gandoger can rapidly recover from extended periods of time in the desiccated state (water potential equilibrated to 2% relative humidity) (Gaff and Ellis, Bothalia 11:305–308 1974; Gaff and Loveys, Transactions of the Malaysian Society of Plant Physiology 3:286–287 1993). Physiological studies have been conducted in S. stapfianus to investigate the responses utilised by these desiccation-tolerant plants to cope with severe water-deficit. In a number of instances, more recent gene expression analyses in S. stapfianus have shed light on the molecular and cellular mechanisms mediating these responses. S. stapfianus is a versatile research tool for investigating desiccation-tolerance in vegetative grass tissue, with several useful characteristics for differentiating desiccation-tolerance adaptive genes from the many dehydration-responsive genes present in plants. A number of genes orthologous to those isolated from dehydrating S. stapfianus have been successfully used to enhance drought and salt tolerance in model plants as well as important crop species. In addition to the ability to desiccate and rehydrate successfully, the survival of resurrection plants in regions experiencing short sporadic rainfall events may depend substantially on the ability to tightly down-regulate cell division and cell wall loosening activities with decreasing water availability and then grow rapidly after rainfall while water is plentiful. Hence, an analysis of gene transcripts present in the desiccated tissue of resurrection plants may reveal important growth-related genes. Recent findings support the proposition that, as well as being a versatile model for devising strategies for protecting plants from water-loss, resurrection plants may be a very useful tool for pinpointing genes to target for enhancing growth rate and biomass production.