Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

Synthesis and evaluation of alkenyl indazoles as selective Aurora kinase inhibitors

A series of alkenyl indazoles were synthesized and evaluated in Aurora kinase enzyme assays. Several promising leads were optimized for selectivity towards Aurora B. Excellent binding affinity and good selectivity were achieved with optimized compounds in isolated Aurora subfamily assays.     

Bacterial transferase MraY inhibitors: Synthesis and biological evaluation

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds.     

Specificity of the tumor necrosis factor-induced protein 6-mediated heavy chain transfer from inter-alpha-trypsin inhibitor to hyaluronan: implications for the assembly of the cumulus extracellular matrix

The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins.     

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.Download video file.^{(130M, mp4)}     

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.     

Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies.

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.     

Depression and adipose polyunsaturated fatty acids in an adolescent group

The purpose of the present study was to investigate the relation between adipose tissue polyunsaturated fatty acids, an index of long-term or habitual fatty acid dietary intake and depression. The sample consisted of 90 adolescents from the island of Crete. There were 54 girls and 36 boys, aged 13-18. The mean age was 15.2 years. Subjects were examined by the Preventive Medicine and Nutrition Clinic of the University of Crete. Depression was assessed through the use of the Beck Depression Inventory (BDI) and the Center for Epidemiologic Studies Depression Scale (CES-D). Unlike other studies, there were no significant relations between adipose tissue n-3 or n-6 polyunsaturated fatty acids and depression. BDI correlated positively with adipose tissue C20:3n-6/C18:3n-6 ratio, while CES-D correlated positively with adipose tissue (C20:3n-6+C22:5n-3)/(C18:3n-6+C20:5n-3) ratio. Depressed subjects (BDI>16, CES-D>16) had significantly elevated adipose tissue C20:3n-6/C18:3n-6 and (C20:3n-6+C22:5n-3)/(C18:3n-6+C20:5n-3) ratios, than non-depressed subjects. The observed positive relation between depression and the particular fatty acid ratios, in the present study, appears to indicate increasing activity of elongases, the enzymes responsible for elongating polyunsaturated fatty acids into their longer-chain derivatives, with increasing depression. This is the first literature report of a possible relation between elongases and depression. The observed relation may stem from a possible over-expression of the HELO1 (ELOVL5) gene, the gene encoding a protein responsible for elongating long-chain polyunsaturated fatty acids, in the adipose tissue of depressed adolescents.     

Asymmetric chlorine-bridged zig-zag Cu(II) chains linked together in 2D sheets by strong Watson-Crick type intermolecular double hydrogen bonds of bis(pyrimidin-2-yl)amine ligands: synthesis, structure and antiferromagnetism

A novel polymeric, asymmetric chloro-bridged copper chain with general formula [μ-Cl-CuCl(dipm]_n (in which dipm = bis(pyrimidin-2-yl)amine) has been synthesized and characterised by X-ray crystallography and infrared spectroscopy. The chains are organized in 2D sheets by intermolecular double H bonds between pairs of dipm molecules. In addition, EPR and magnetic measurements have been performed, and these have been related to the dinuclear structural details.The geometry around the copper(II) ion is distorted square pyramidal with the basal plane formed by the two nitrogen atoms of the dipm ligand and two chloride atoms, one of which is bridging. The Cu-N distances are 2.0342(15) and 2.0125(15) Å and Cu-Cl distances are 2.2899(6) and 2.2658(6) Å. The apical position of Cu is occupied by a chloride atom of a neighbouring unit atom at a distance of 2.6520(6) Å, resulting in the polynuclear array in which one chloride anion and the copper ion forms a zig-zag chain. The magnetic interaction between the Cu-Cu atoms is weak antiferromagnetic with a singlet-triplet separation (J) of −3.2(1) cm⁻¹.     

Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with 1–10 mM binding affinity (K_D) were iteratively optimized to give leads with 200 nM biochemical activity and low μM cellular activity in a Replicon assay.