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J A Hammarback

Publications and source records attributed to J A Hammarback.

At least 19 recordsLinked to original sources
PubMed2006

Diurnal gene expression patterns of T-type calcium channels and their modulation by ethanol.

The transient (T-type) calcium channel participates in the generation of normal brain rhythms as well as abnormal rhythms associated with a range of neurological disorders. There are three different isoforms of T-type channels and all are particularly enriched in the thalamus, which is involved in generating many of these rhythms. We report a novel means of T-type channel regulation in the thalamus that involves diurnal regulation of gene expression. Using real time polymerase chain reaction we detected a diurnal pattern of gene expression for all T-type channel transcripts. The peak of gene expression for the CaV3.1 transcript occurred close to the transition from active to inactive (sleep) states, while expression for both CaV3.2 and CaV3.3 peaked near the transition of inactive to active phase. We assessed the effect of chronic consumption of ethanol on these gene expression patterns by examining thalamic tissues of ethanol-consuming cohorts that were housed with the controls, but which received ethanol in the form of a liquid diet. Ethanol consumption resulted in a significant shift of peak gene expression of approximately 5 h for CaV3.2 toward the normally active phase of the mice, as well as increasing the overall gene expression levels by approximately 1.7-fold. Peak gene expression was significantly increased for both CaV3.2 and CaV3.3. Measurements of CaV3.3 protein expression reflected increases in gene expression due to ethanol. Our results illustrate a novel regulatory mechanism for T-type calcium channels that is consistent with their important role in generating thalamocortical sleep rhythms, and suggests that alterations in the pattern of gene expression of these channels could contribute to the disruption of normal sleep by ethanol.

Analysis of Variance↗
PubMed2001

Expression of microtubule-associated protein 2 in benign and malignant melanocytes: implications for differentiation and progression of cutaneous melanoma.

Cutaneous melanocytic neoplasms are known to acquire variable characteristics of neural crest differentiation. Melanocytic nevus cells in the dermis and desmoplastic melanomas often display characteristics of nerve sheath differentiation. The extent and nature of neuronal differentiation characteristics displayed by primary and metastatic melanoma cells are not well understood. Here, we describe induction of a juvenile isoform of microtubule-associated protein 2 (MAP-2c) in cultured metastatic melanoma cells by the differentiation inducer hexamethylene bisacetamide. Up-regulation of this MAP-2 isoform, a marker for immature neurons, is accompanied by extended dendritic morphology and down-regulation of tyrosinase-related protein 1 (TYRP1/gp75), a melanocyte differentiation marker. In a panel of cell lines that represent melanoma tumor progression, MAP-2c mRNA and the corresponding approximately 70-kd protein could be detected predominantly in primary melanomas. Immunohistochemical analysis of 61 benign and malignant melanocytic lesions showed abundant expression of MAP-2 protein in melanocytic nevi and in the in situ and invasive components of primary melanoma, but only focal heterogeneous expression in a few metastatic melanomas. In contrast, MAP-2-positive dermal nevus cells and the invasive cells of primary melanomas were TYRP1-negative. This reciprocal staining pattern in vivo is similar to the in vitro observation that induction of the neuronal marker MAP-2 in metastatic melanoma cells is accompanied by selective extinction of the melanocytic marker TYRP1. Our data show that neoplastic melanocytes, particularly at early stages, retain the plasticity to express the neuron-specific marker MAP-2. These observations are consistent with the premise that both benign and malignant melanocytes in the dermis can express markers of neuronal differentiation.

Acetamides↗
PubMed2000

Regulation of microtubule-associated protein 1B (MAP1B) subunit composition.

The MAP1B and MAP1A genes each produce an mRNA that encodes a polyprotein. When cleaved, each polyprotein yields a single heavy chain and a single light chain, which become noncovalently associated. In previous work, it was found that the MAP1B light chains and heavy chains exist in a 2:1 ratio. Through use of quantitative immunoblot techniques, this finding was further examined in the developing rat brain. MAP1B heavy chain (HC) and light chain (LC1), as well as the light chain of MAP1A (LC2), were prepared in purified form for use as standards and/or immunogens for generation of antibodies for immunoblotting. Brain homogenates and microtubule-enriched fractions from developing rats were assayed for MAP1B HC and LC1 content. Results indicated that postnatal rat brain homogenates contain LC1 in a 6:1 to 8:1 molar ratio to the MAP1B HC. Purified microtubules also contain LC1 in excess of MAP1B HC, but at a ratio of 2:1. We propose that most of the excess LC1 in homogenates is either insoluble or not bound to microtubules. The findings raise the possibility of a function for the "excess" LC1 that does not require association with MAP1 HC and/or microtubules. Given a synthetic mechanism that produces MAP1B HC and LC1 in a 1:1 ratio at both transcription and translation steps, we propose that the "excess" LC1 in brain homogenates is a consequence of LC1 having a greater half-life than the MAP1B HC. Consistently with this hypothesis, a major pool of MAP1B HC is rapidly degraded after blocking protein synthesis with cycloheximide, whereas LC1 levels remain constant even after 24 hr of cycloheximide treatment.

Animals↗
PubMed2000

Microtubule-associated protein 1 subunit expression in primary cultures of rat brain.

Microtubule-associated protein 1A (MAP1A) and MAP1B are developmentally regulated proteins linked to axon formation. They each consist of a unique heavy chain and three common light chains. We used immunofluorescence microscopy to qualitatively assess the variability in MAP1 subunit expression between individual cells. The ratio of light chain 1 to MAP1 heavy chain varies greatly between cells with some cells expressing MAP1A heavy chain in the apparent absence of light chain 1. The results imply the existence of MAP1 molecules that differ in light chain composition. Transfection experiments indicate that the light chains differ in microtubule binding activity and subcellular targeting activity. This further suggests that the regulation of MAP1 light chain content can control MAP1 function.

Animals↗
PubMed1996

Gene localization and developmental expression of light chain 3: a common subunit of microtubule-associated protein 1A(MAP1A) and MAP1B.

Microtubule-associated proteins 1A (MAP1A) and MAP1B are abundant neuronal MAPs thought to be involved in neurite formation and stabilization. The relative levels of MAP1A and MAP1B change dramatically during development, with MAP1B expression highest in forming neurons, and MAP1A expression highest in mature neurons. We examined the expression of light chain 3 (LC3), a subunit of both MAP1A and MAP1B, to see if its expression paralleled that of either heavy chain. Anti-LC3 immunohistochemistry reveals that LC3 in rat brain is restricted to neurons that are expressing either the MAP1A or MAP1B heavy chain. Although LC3 is expressed exclusively in cells expressing heavy chains, developmental changes in the total amount of LC3 protein are not proportional to changes in the amount of either the MAP1A or MAP1B heavy chain. LC3 protein expression measured by quantitiative immunoblotting is twice as high in postnatal brain as in embryonic and adult brain. The localization of the LC3 gene to human chromosome 20cen-q13 demonstrates that LC3 is the only MAP1 subunit that is not linked to the heavy chain genes. Because LC3 is a component of both the MAP1A and MAP1B microtubule-binding domains, the heavy-chain independent regulation of LC3 expression might modify MAP1 microtubule-binding activity during development.

Aging↗
PubMed1996

Neuronal abnormalities in microtubule-associated protein 1B mutant mice.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.

Animals↗
PubMed1994

Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B.

Light chain 3 (LC3) is a subunit of the neuronal microtubule-associated proteins (MAPs), MAP1A and MAP1B. Recent findings show that the cDNAs for MAP1A and MAP1B encode polyproteins that contain the MAP1A or MAP1B heavy chain and a LC2 or LC1 subunit, respectively. We have sequenced a cDNA encoding rat LC3, and its sequence is not found in the MAP1A/LC2 or MAP1B/LC1 polyprotein cDNAs. The deduced amino acid sequence of LC3 is highly conserved between rat and mouse. Rat LC3 is a 16.4-kDa protein with a predicted pI of 9.2. It is encoded on a 1.7-kilobase mRNA. Our anti-LC3 antiserum shows that LC3 is abundant only in neurons and that the majority of LC3 in brain co-purifies with microtubules. Purified recombinant rat LC3 retains the ability to associate with microtubules assembled in the presence of brain MAPs and with microtubules assembled from purified tubulin. We propose that LC3 functions primarily as a MAP1A and MAP1B subunit and that its expression could regulate the microtubule binding activity of MAP1A and MAP1B.

Amino Acid Sequence↗
PubMed1994

Genetic and physical mapping of human recoverin: a gene expressed in retinal photoreceptors.

PURPOSE: Recoverin is a calcium-binding protein that may be involved in phototransduction in mammalian retinal photoreceptors, and is considered to be a candidate gene for retinitis pigmentosa. This study was undertaken to develop the recoverin locus into a polymorphic marker for future linkage studies on retinitis pigmentosa families. METHODS: A human genomic cosmid clone was isolated and used to map the recoverin gene to a human chromosome through hybridization to a panel of somatic hybrid cell line DNAs, and to human metaphase chromosomes by fluorescence in situ hybridization. A dinucleotide repeat polymorphism located within the coding region of the recoverin gene was identified, and used to genetically map the recoverin gene relative to index markers. In addition, three restriction fragment length polymorphisms revealed by the cosmid clone were identified and characterized. RESULTS: Hybridization to the somatic hybrid cell line DNAs localized the recoverin gene to chromosome 17. Recoverin was further localized to 17p12-p13 by fluorescence in situ hybridization. The dinucleotide repeat polymorphism and restriction fragment length polymorphisms at the recoverin locus have a cumulative polymorphic information content = 0.71. CONCLUSIONS: These polymorphic markers and additional closely linked markers will be useful for linkage analysis of families with retinitis pigmentosa.

Base Sequence↗
PubMed1993

Molecular cloning and nucleotide sequence of a cDNA encoding recoverin from human retina.

In the process of photoreceptor signal transduction, light initiates an enzymatic cascade that leads to hydrolysis of cyclic GMP (cGMP) and closure of cGMP-gated sodium-calcium channels resulting in photoreceptor hyperpolarization. Recoverin is a calcium-binding protein that is thought to reverse the effects of light on cGMP levels by activating guanylate cyclase. Guanylate cyclase produces cGMP to overcome the cGMP-hydrolysing effect of phosphodiesterase, and reopens the sodium-calcium channels in photoreceptor outer segments. We have cloned and sequenced a cDNA encoding recoverin in human retina. The human nucleotide sequence is 88% identical to the bovine sequence, and contains a 600-base pair (bp) open reading frame encoding 200 amino acids. In situ hybridization of cultured Y79 human retinoblastoma cells with a radioactive recoverin cDNA probe showed intense, specific labeling of the cytoplasm, indicating the presence of mRNA encoding recoverin. Direct sequencing of a Y79 retinoblastoma cDNA polymerase chain reaction (PCR) product confirmed the presence of recoverin in this human cell line.

Amino Acid Sequence↗
PubMed1993

Expression of recoverin mRNA in the human retina: localization by in situ hybridization.

The localization of mRNA encoding recoverin in the human retina was examined by in situ hybridization. The riboprobe utilized in this study was transcribed from a cDNA clone containing the complete coding region of human recoverin. The riboprobe hybridized extensively with the photoreceptor inner segments, and the outer nuclear layer. Specific hybridization was also seen in a subpopulation of cells in the inner nuclear layer, and occasionally in cells of the ganglion cell layer (GCL). Recoverin-like immunoreactivity was observed in all photoreceptors, and in a subpopulation of bipolar cells. Very robust immunolabeling was observed in a small population of cells in the GCL. This study suggests that recoverin, or a recoverin-like protein, is produced by at least three different cell types in the human retina: photoreceptors, bipolars and rare cells in the ganglion cell layer.

Adult↗
PubMed1993

Tenascin expression in the mouse: in situ localization and induction in vitro by bFGF.

The glycoprotein tenascin is found in the extracellular matrix in regions of cell motility, cell proliferation, and tissue modelling. We have used novel tenascin cDNA probes to localize tenascin transcripts in the developing mouse and to study the regulation of tenascin expression by growth factors in vitro. At postnatal day 1 tenascin mRNAs are abundant in regions of bone and cartilage formation, as well as in the ependymal layer of the central nervous system. Previous studies have demonstrated that transforming growth factor-beta type 1 (TGF-beta 1) can induce tenascin expression in vitro. As TGF-beta 1 is absent or scarce in the developing brain, it is likely that other growth factors, alone or in addition to TGF-beta 1, may regulate tenascin expression during development. Therefore, we have compared the effects of TGF-beta 1 and a growth factor that is found in both developing connective tissue and the central nervous system, basic fibroblast growth factor (bFGF), on tenascin expression in a mouse embryo fibroblast cell line (Swiss 3T3 cells). Immuno-slot blot analysis of Swiss 3T3 cell-conditioned culture medium demonstrates that bFGF is a more potent inducer of tenascin expression than TGF-beta 1. Furthermore, bFGF and TGF-beta 1 have an additive effect on levels of tenascin, but not fibronectin, in the conditioned medium. Western blots revealed that different forms of tenascin are induced by bFGF and TGF-beta 1: the tenascin induced by the former has a molecular mass of approximately 250 kDa, the latter induces an approximately 200 kDa form of tenascin. The induction of large tenascin by bFGF was confirmed by northern blot analysis, which revealed increased levels of an 8 kb tenascin transcript after 24 h by as little as 4 ng/ml of bFGF in serum-free medium. Thus bFGF, alone or in combination with TGF-beta 1, is a potential regulator of tenascin expression in vitro. bFGF may alter not only the relative abundance of tenascin and fibronectin in the extracellular matrix, but also the splice variant of tenascin expressed by a given cell type.

3T3 Cells↗
PubMed1992

Microtubule-associated proteins 1A and LC2. Two proteins encoded in one messenger RNA.

The deduced amino acid sequence for the filamentous microtubule-associated protein (MAP) 1A, thought to be involved in stabilizing the mature neuronal cytoskeleton, has been determined from a series of overlapping cDNA clones. Though previously described as biochemically and immunologically distinct from MAP1B, we now demonstrate that MAP1A is structurally related to MAP1B, a protein associated with neurite outgrowth and process plasticity. The two MAPs exhibit regional amino acid sequence similarities spanning their potential microtubule binding domains placing both into a new MAP family. The cDNA sequence encoding MAP1A was also found to encode one of its associated light chains (LC) called LC2. Both proteins are found on a single mRNA in the same open reading frame and are translated as a pre-MAP1A/LC2-protein. The topological relationship between MAP1A and LC2 coding sequences is, therefore, identical to that previously shown for MAP1B and LC1 (Hammarback, J. A., Obar, R. A., Hughes, S. M., and Vallee, R. B. (1991) Neuron 7, 129-139). Based on these and earlier results, we conclude that LC1 and LC2 are structurally related polypeptides generated from distinct MAP polyprotein precursors but free to exchange between the two MAPs.

Amino Acid Sequence↗
PubMed1991

Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued.

Cytoplasmic dynein is a microtubule-activated ATPase which produces force towards the minus ends of microtubules. It is thought to be responsible for retrograde axonal transport and other aspects of organelle motility and may have a role in the poleward movement of mitotic chromosomes. Cytoplasmic dynein is an oligomeric complex of two catalytic heavy chains and a number of accessory subunits. We now report the cloning and sequencing of a complementary DNA for one of these species, a cytoplasmic dynein-associated polypeptide of relative molecular mass 150,000 (Mr 150K). A full-length cDNA was found to contain an open reading frame of 4.0 kilobases, which is predicted to encode a polypeptide of Mr 145K. It has extensive homology with the product of the Drosophila gene Glued, which encodes a polypeptide of Mr 148K. The Glued mutation is dominant, with pleiotropic developmental defects in heterozygotes and an embryonic lethal phenotype in homozygotes. As dominant mutations may involve disruption of normal protein-protein interactions, the Glued mutation should provide insight into the mode of action of cytoplasmic dynein in vivo.

Amino Acid Sequence↗
PubMed1991

MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain.

Microtubule-associated protein 1B (MAP1B), an abundant developmentally regulated neuronal protein, is a stoichiometric complex of a heavy chain and two light chains (light chain 1 and light chain 3). We find that light chain 1 is encoded within the 3' end of a previously reported MAP1B heavy chain cDNA. Amino acid sequencing, epitope mapping, Northern blotting, and Southern blotting indicate that the light chain and heavy chain are encoded by the same mRNA within the same open reading frame. In addition, amino acid sequencing of a 120 kd microtubule-binding and light chain-binding fragment of the heavy chain reveals that light chain 1 binds near the heavy chain N-terminus. Together these data indicate that the heavy chain and light chain 1 are produced by proteolytic processing of a MAP1B polyprotein and form a complex microtubule-binding domain.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins.

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.

Amino Acid Sequence↗
PubMed1990

Antibody exchange immunochemistry.

Antibodies are found to transfer rapidly between antigen samples attached to separate solid supports. The half-times for this antibody exchange at 20 and 37 degrees C were 7 and 1 h, respectively. Antibody exchange was exhibited by all 17 polyclonal and monoclonal antibodies tested and was readily detected by enzyme-linked immunosorbent assay, immunoblotting, or immunocytochemical assays. Taking advantage of this phenomenon, antibodies can be affinity-purified using less than a picomole of antigen and small amounts of antibody.

Animals↗
PubMed1988

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity.

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.

Animals↗