Dana M. García

The α-Actinin Gene Family: A Revised Classification

The sequencing of a genome is the first stage of its complete characterization. Subsequent work seeks to utilize available sequence data to gain a better understanding of the genes which are found within a genome. Gene families comprise large portions of the genomes of higher vertebrates, and the available genomic data allow for a reappraisal of gene family evolution. This reappraisal will clarify relatedness within and between gene families. One such family, the α-actinin gene family, is part of the spectrin superfamily. There are four known loci, which encode α-actinins 1, 2, 3, and 4. Of the eight domains in α-actinin, the actin-binding domain is the most highly conserved. Here we present evidence gained through phylogenetic analyses of the highly conserved actin-binding domain that α-actinin 2 was the first of the four α-actinins to arise by gene duplication, followed by the divergence of α-actinin 3 and then α-actinins 1 and 4. Resolution of the gene tree for this gene family has allowed us to reclassify several α-actinins which were previously given names inconsistent with the most widely accepted nomenclature for this gene family. This reclassification clarifies previous discrepancies in the public databases as well as in the literature, thus eliminating confusion caused by continued misclassification of members of the α-actinin gene family. In addition, the topology found for this gene family undermines the 2R hypothesis theory of two rounds of genome duplication early in vertebrate evolution.

Summer Scientific Research for Teachers: The Experience and its Effect

Doing science – conducting actual research side by side with researchers—is perhaps the best way to achieve scientific literacy (Duchovnay andJoyce, 2000)IntroductionScientific inquiry is fundamental to the science education reform movement.It is the dominant theme of the 1990s reform documents such as the NationalScience Education Standards [NSES] (National Research Council, [NRC], 1996a)and the Benchmarks for Science Literacy (Association for the Advancement ofScience [AAAS], 1993). The development of scientific inquiry skills is consideredto be the most effective way to create a society of scientifically literate citizens(NRC, 1996a). As stated in the NSES, “when engaging in inquiry, students...actively develop their understanding of science by combining scientific knowledgewith reasoning and thinking skills” (NRC, 1996a p. 2). Inquiry methods oftenaddress issues that are important to the community such as environmental andhuman health. It is believed that students will be better able to discuss issues andmake informed decisions about scientific issues if they have developed reasoningskills through inquiry methods.Teachers who have learned science facts from textbooks and lectures, butlack scientific research experiences, may have difficulty teaching science by inquiry(Hodson, 1993; Roth, McGinn, and Brown, 1997). Beisenherz & Dantonio (1991)stated that, “teachers cannot be lectured at, demonstrated to, and asked toThis material is based upon work supported in part by gifts and grants from the NationalScience Foundation (ESI #9731321), the Eisenhower Professional Development GrantsProgram (#98024) administered by the Texas Higher Education Coordinating Board,and the Southwest Bell Corporation Foundation. Any opinions, findings, andconclusions or recommendations expressed in this article are those of the author(s)and do not necessarily reflect the views of the funding agencies.

Activating transcription factor 3 (ATF3) expression in the neural retina and optic nerve of zebrafish during optic nerve regeneration

Fish, unlike mammals, can regenerate axons in the optic nerve following optic nerve injury. We hypothesized that using microarray analysis to compare gene expression in fish which had experienced optic nerve lesion to fish which had undergone a similar operation but without optic nerve injury would reveal genes specifically involved in responding to optic nerve injury (including repair), reducing detection of genes involved in the general stress and inflammatory responses. We discovered 120 genes were significantly (minimally two-fold with a P-value < or = 0.05) differentially expressed (up or down) at one or more time point. Among these was ATF3, a member of the cAMP-response element binding protein family. Work by others has indicated that elevated cAMP could be important in axon regeneration. We investigated ATF3 expression further by qRT-PCR, in situ hybridization and immunohistochemistry and found ATF3 expression is significantly upregulated in the ganglion cell layer of the retina, the nerve fiber layer and the optic nerve of the injured eye. The upregulation in retina is detectable by qRT-PCR by 24 h after injury, at which time ATF-3 mRNA levels are 8-fold higher than in retinas from sham-operated fish. We conclude ATF3 may be an important mediator of optic nerve regeneration-promoting gene expression in fish, a role which merits further investigation.

Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish.

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

BackgroundIn fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents.ResultsThe general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion.ConclusionsOur results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

BackgroundNuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there.ResultsUsing confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence.ConclusionWe therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

GFAP and nuclear lamins share an epitope recognized by monoclonal antibody J1-31

Monoclonal antibody J1-31 was raised against plaque materials taken from brains of patients who had suffered from multiple sclerosis (MS). Preliminary characterization of the antigen revealed it to be a protein of M(w) 68-70 kDa with both a cytoplasmic and nuclear localization. Here we report the results of isolation and peptide sequencing of the antigen from human brains, and immunocytochemical analysis of the antigen in F98 glioma cells. Purification and peptide sequencing indicate that the antibody recognizes a form of glial fibrillary acidic protein, possibly a phosphorylated variant. However, confocal immunocytochemistry and western analysis of F98 glioma cells raise the possibility that it also recognizes a phosphorylated epitope found in nuclear lamins. Analysis of the expression of the J1-31 epitope in F98 cells with respect to time in culture, cell density, and DNA synthesis showed a developmental relationship: cells that were engaged in rapid growth and DNA synthesis exhibited strong J1-31 staining in nuclei, whereas quiescent cells did not. We conclude that mAB J1-31 remains a useful antibody for studying multiple sclerosis, and is likely to prove useful in studies of the dynamics of nuclear lamins, particularly in models for wound-healing.

Intermediate filaments of zebrafish retinal and optic nerve astrocytes and Müller glia: differential distribution of cytokeratin and GFAP

BackgroundOptic nerve regeneration (ONR) following injury is a model for central nervous system regeneration. In zebrafish, ONR is rapid - neurites cross the lesion and enter the optic tectum within 7 days; in mammals regeneration does not take place unless astrocytic reactivity is suppressed. Glial fibrillary acidic protein (GFAP) is used as a marker for retinal and optic nerve astrocytes in both fish and mammals, even though it has long been known that astrocytes of optic nerves in many fish, including zebrafish, express cytokeratins and not GFAP. We used immunofluorescence to localize GFAP and cytokeratin in wild-type zebrafish and transgenic zebrafish expressing green fluorescent protein (GFP) under control of a GFAP promoter to determine the pattern of expression of intermediate filaments in retina and optic nerve.FindingsGFAP labeling and GFAP gene expression as indicated by GFP fluorescence was found only in the Müller glial cells of the retina. Within Müller cells, GFP fluorescence filled the entire cell while GFAP labelling was more restricted in distribution. No GFAP expression was observed in optic nerves. Cytokeratin labeling of astrocytes was observed throughout the optic nerve and less intensely in cells in the retinal inner plexiform layer. The retinal inner limiting membrane was strongly labeled by anti-cytokeratin.ConclusionsStudies of astrocyte function during ONR in zebrafish cannot solely rely on GFAP as an astrocyte marker or indicator of reactivity. Future studies of ONR in zebrafish should include evaluation of changes in cytokeratin expression and localization in the optic nerve.

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light‐adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype‐specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)‐induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol‐induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.

Phosphorylated neurofilaments and SNAP-25 in cultured SH-SY5Y neuroblastoma cells

Components of the extracellular matrix (ECM) of mammals have profound effects on the behavior and differentiation of many different cell types. Here, we report the results of biochemical and immunocytochemical investigations of the expression of SNAP-25 and phosphorylated neurofilament proteins (NFs) by cells grown on coverslips, cells cultured in EHS-ECM gels, and cells in situ in rat brain. SNAP-25 and phosphorylated NFs were detected by immunofluorescence in all these environments but were not detectable by Western analysis in extracts of cells grown on coverslips. The results support the interpretation that EHS-ECM induces differentiation of SH-SY5Y cells in culture and suggest this system as a model system for study of nerve tissue formation and repair.