Characterization and expression of two matrix metalloproteinase genes during sea urchin development

Matrix metalloproteinases (MMPs) play an essential role in a variety of processes in development that require extracellular matrix remodeling and degradation. In this study, we characterize two MMPs from the sea urchin Strongylocentrotus purpuratus. These clones can both be identified as MMPs based on the presence of conserved domains such as the cysteine switch, zinc-binding, and hemopexin domains. In addition, both of these genes contain consensus furin cleavage sites and putative transmembrane domains, classifying them as membrane-type MMPs. We have named these clones SpMMP14 and SpMMP16 based on the vertebrate MMPs with which they share the greatest similarity. SpMMP14 is expressed in all cells from the egg to mesenchyme blastula stage embryo. Expression of this gene is strongest in the animal and vegetal poles early in gastrulation and in the animal pole only later in gastrulation. SpMMP16 is expressed at low levels in eggs. Expression of SpMMP16 becomes more pronounced in the vegetal pole region at the blastula and mesenchyme blastula stages and becomes confined to vegetal pole descendants, such as pigment cells, later in development. In the future, we hope to learn more about the possible functions of these genes in sea urchin development.     

Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice

Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.     

Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.     

Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish

The myostatin (MSTN)-null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or "double muscling" as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub-family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN-1 and -2. These analyses further indicated that all Salmonid genes described are actually MSTN-1 orthologs and that additional MSTN-2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)-1 and -2) revealed many differences, primarily within the latency-associated peptide regions, but also within the bioactive domains. The 2-kb promoter region of zfMSTN-2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN-1 promoter. In fact, zfMSTN-2 expression was limited to the early stages of somitogenesis, whereas zfMSTN-1 was expressed throughout embryogenesis. These data suggest that zfMSTN-2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.     

A proteomic analysis of murine bone marrow and its response to ionizing radiation

To characterize the mouse bone marrow tissue proteome and investigate the response to radiation damage we took bone marrow before and after 4-Gy gamma-irradiation from mouse strains (C57BL/6 and CBA/Ca) that differ in their short-term and long-term radiation responses and analyzed extracellular proteins by high-resolution 2-DE. Twenty proteins were identified from 71 protein spots in both C57BL/6 and CBA/Ca. We detected significant differences between control and irradiated bone marrow and between genotypes and identified many of the changed proteins by MS. In C57BL/6, 27 spots were significantly different between control and irradiated samples. In CBA/Ca, 18 spots showed significant changes following irradiation. Proteins such as serum albumin, apolipoprotein A-I, ferritin, haptoglobin (Hp) and alpha-1-antitrypsin were changed in irradiated bone marrow of both mouse strains, reflecting an ongoing acute-phase reaction. Several other proteins including serotransferrin, neutrophil collagenase, peroxiredoxin 2 and creatine kinase M chain were changed specifically in an individual mouse strain. The proteomic approach makes an important contribution to characterizing bone marrow proteome and investigating the tissue response of bone marrow to radiation, assists in identifying genotype-dependent responses and provides support for the importance of microenvironmental factors contributing to the overall response.     

Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers

Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.     

The neural substrates of memory suppression: a FMRI exploration of directed forgetting

The directed forgetting paradigm is frequently used to determine the ability to voluntarily suppress information. However, little is known about brain areas associated with information to forget. The present study used functional magnetic resonance imaging to determine brain activity during the encoding and retrieval phases of an item-method directed forgetting recognition task with neutral verbal material in order to apprehend all processing stages that information to forget and to remember undergoes. We hypothesized that regions supporting few selective processes, namely recollection and familiarity memory processes, working memory, inhibitory and selection processes should be differentially activated during the processing of to-be-remembered and to-be-forgotten items. Successful encoding and retrieval of items to remember engaged the entorhinal cortex, the hippocampus, the anterior medial prefrontal cortex, the left inferior parietal cortex, the posterior cingulate cortex and the precuneus; this set of regions is well known to support deep and associative encoding and retrieval processes in episodic memory. For items to forget, encoding was associated with higher activation in the right middle frontal and posterior parietal cortex, regions known to intervene in attentional control. Items to forget but nevertheless correctly recognized at retrieval yielded activation in the dorsomedial thalamus, associated with familiarity-based memory processes and in the posterior intraparietal sulcus and the anterior cingulate cortex, involved in attentional processes.     

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)～0.5 μM) or Mg(2+) (IC(50)～3 μM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.