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J G Sutcliffe

Publications and source records attributed to J G Sutcliffe.

At least 19 recordsLinked to original sources
PubMed1992

Localization of the protein kinase C phosphorylation/calmodulin-binding substrate RC3 in dendritic spines of neostriatal neurons.

The rodent protein RC3 is expressed mainly by forebrain neurons during postnatal development and maturity. RC3 and its bovine homolog neurogranin/B-50 immunoreactive C-kinase substrate (BICKS) contain overlapping sites for protein kinase C phosphorylation and calmodulin binding that resemble those of the presynaptic 43-kDa growth-associated protein (GAP-43). However, morphological evidence suggests that RC3 has a postsynaptic localization. To test this hypothesis, we used two polyclonal antisera against synthetic peptides corresponding to nonoverlapping sequences within RC3 and compared cellular distributions of their binding in neostriatum of adult rats by immunohistochemistry, Golgi impregnation/gold toning, and correlative light/electron microscopy. Somatic and punctate patterns of RC3 immunoreactivity were observed. Somatic RC3 was found in cyto- and nucleoplasmic compartments of all neuronal phenotypes (medium spiny, medium aspiny, and large aspiny cells). Punctate RC3 was found mostly in dendritic spines. In contrast to the 43-kDa growth-associated protein, RC3 was seen infrequently in axons. We conclude that RC3 accumulates postsynaptically in dendritic spines of neostriatal neurons. We propose that RC3 acts as a "third messenger" substrate of protein kinase C-mediated molecular cascades during synaptic development and remodeling.

Amino Acid Sequence↗
PubMed1991

Pituitary hyperplasia and gigantism in mice caused by a cholera toxin transgene.

Cyclic AMP is thought to act as an intracellular second messenger, mediating the physiological response of many cell types to extracellular signals. In the pituitary, growth hormone (GH)-producing cells (somatotrophs) proliferate and produce GH in response to hypothalamic GH-releasing factor, which binds a receptor that stimulates Gs protein activation of adenylyl cyclase. We have now determined whether somatotroph proliferation and GH production are stimulated by cAMP alone, or require concurrent, non-Gs-mediated induction of other regulatory molecules by designing a transgene to induce chronic supraphysiological concentrations of cAMP in somatotrophs. The rat GH promoter was used to express an intracellular form of cholera toxin, a non-cytotoxic and irreversible activator of Gs. Introduction of this transgene into mice caused gigantism, elevated serum GH levels, somatotroph proliferation and pituitary hyperplasia. These results support the direct triggering of these events by cAMP, and illustrate the utility of cholera toxin transgenes as a tool for physiological engineering.

Amino Acid Sequence↗
PubMed1991

The human retinal degeneration slow (RDS) gene: chromosome assignment and structure of the mRNA.

Retinal degeneration slow (rds) is a mouse neurological mutation that is characterized phenotypically by abnormal development of rod and cone photoreceptors followed by their slow degeneration. This phenotype resembles the pathologic abnormalities seen in retinitis pigmentosa. The mouse rds gene has recently been cloned. Here we present the sequence of a full-length cDNA clone of the human RDS mRNA. We show that in human retina there are two RDS transcripts of 3.0 and 5.5 kb. By analysis of DNA from a panel of human X hamster somatic cell hybrids, and by direct in situ hybridization, we show that the RDS gene is located on the proximal short arm of human chromosome 6. Finally, we present information on the frequency of several observed restriction fragment length polymorphisms using the RDS cDNA. This information is of potential value for testing linkage of the RDS gene to the disease phenotype in families with retinitis pigmentosa.

Amino Acid Sequence↗
PubMed1991

The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated glycoprotein.

Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.

Amino Acid Sequence↗
PubMed1991

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.

Amino Acid Isomerases↗
PubMed1991

Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.

Animals↗
PubMed1990

Genetic ablation of a mouse gene expressed specifically in brain.

The 1B1075 gene was initially identified from a cDNA clone of a rat brain messenger RNA expressed in particular subsets of CNS neurons and pituitary cells. Although the protein encoded by this gene is of unknown function, its sequence suggests that it may be related to secretogranin proteins, which are found in association with secretory granules in a variety of peptidergic endocrine and neuronal cells. Here we show that the mouse 1B1075 gene is located between the dilute (d) and short ear (se) genes on chromosome 9. Many different deletion mutations have previously been isolated in the genetic region that includes these genes. By producing mice carrying two deletions that overlap at the 1B1075 locus, the gene for this brain-specific message can be completely eliminated from otherwise viable animals. The animals missing the 1B1075 gene provide an important new tool for determining the function of this gene in the brain. In addition, these results provide a new molecular entry point for detailed characterization of other genes in the d-se region.

Animals↗
PubMed1990

Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein.

A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.

Amino Acid Sequence↗
PubMed1990

Patterns of cerebral cortex mRNA expression.

A pool of 163 clones, isolated by screening 60,000 members of a Macaca fascicularis cerebral cortex cDNA library with a cortex-minus-cerebellum subtracted probe prepared by the phenol enhancement method, was analyzed by Northern blot hybridization studies. One hundred fifty-three of these clones corresponded to 22 RNAs whose abundance was at least 2-fold higher in cerebral cortex poly(A)+ RNA samples than in samples of cerebellar poly(A)+ RNA. Seven of these RNAs, represented by 131 clones, were undetectable in cerebellum. Only 10 of the 163 clones proved to be false positives. The abundance of several of these cortex-enriched RNAs was altered in Alzheimer's disease brains. Several RNAs that were present in cerebral cortex but undetectable in cerebellum were generally enriched in telencephalon, although none was restricted to the cortex. One of the cortex enriched RNAs, whose nucleotide sequence is presented, encoded monkey preprocholecystokinin. Overall, this study provides insights into the powers and limitations of subtractive hybridization and into the patterns of gene expression in the central nervous system.

Alzheimer Disease↗
PubMed1990

Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control.

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Animals↗
PubMed1990

1B1075: a brain- and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles.

The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.

Aging↗
PubMed1989

Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds).

Mutant mice homozygous for 'retinal degeneration slow' (rds/rds) are characterized phenotypically by abnormal development of photoreceptor outer segments in the retina, followed by gradual degeneration of photoreceptors. This process of degeneration is complete by one year, with preservation of all other retinal cells. The biochemical defect that leads to the mutant phenotype is not known. Our strategy for cloning the rds gene was based upon three previously reported observations. First, the rds locus maps to chromosome 17. Second, experimental rds/rds----+/+ and rds/+----+/+ tetra-parental mice manifest patchy photoreceptor changes in the retina, suggesting that the wild-type rds locus is expressed within cells of the photoreceptor lineage. Finally, the process of degeneration is specific to photoreceptors. On the basis of these observations, we predicted that the rds mRNA is encoded by a gene on chromosome 17 and is normally expressed exclusively within photoreceptors in the retina. We here present evidence that this is the case.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence and cellular distribution of rat chromogranin B (secretogranin I) mRNA in the neuroendocrine system.

The mRNA of rat secretory-vesicle protein chromogranin B is abundant in brain, adrenal medulla, and anterior pituitary. The primary translation product predicted from the cDNA sequence of this 2,337-nucleotide transcript corresponds to a hydrophilic 655-residue protein preceded by a signal peptide. Both termini of the mature 75-kD protein show extensive similarity to other chromogranins; the more variable internal region is characterized by glutamic acid clusters and numerous pairs of basic residues. In rodent brain, mRNA accumulation starts around embryonic days 13-14 and peaks by postnatal day 20. In situ hybridization in brain sections shows that the mRNA is enriched in the hippocampal formation, the endocrine hypothalamus, the olfactory system, and in anatomically distinct structures in the pons-medulla.

Amino Acid Sequence↗
PubMed1989

Myelin-associated glycoprotein (MAG) and rat brain-specific 1B236 protein: mapping of epitopes and demonstration of immunological identity.

The myelin-associated glycoprotein (MAG) and the brain 1B236 protein are 100-kDa glycoproteins containing 30% carbohydrate that exist in two developmentally regulated forms and are specific to the nervous system. Recent cDNA cloning experiments in several laboratories using primarily immunological means of identification have determined the complete primary sequence of a rat brain glycoprotein that seems to correspond to both MAG and 1B236, suggesting that these proteins are identical. However, MAG was previously considered to be an oligodendrocyte/myelin specific component in the CNS at all ages, whereas 1B236 was thought to be primarily a neuronal component in adult rats but synthesized by oligodendrocytes at the time of active myelination. The composite term 1B236/MAG was proposed to describe the molecule identified by the cDNAs. In order to explore further the relationship between MAG and 1B236, as well as their developmentally regulated forms, experiments were carried out on rat samples utilizing synthetic peptides corresponding to sequences throughout the 1B236 molecule, antisera raised to synthetic peptides in the C-terminus of 1B236 that distinguish between the two developmentally regulated forms, and well-characterized polyclonal and monoclonal antibodies raised to purified MAG. Epitope mapping demonstrated that reactive sites were distributed throughout the extracellular and intracellular domains of 1B236/MAG. Only antibodies reacting with the smaller of the two forms of 1B236/MAG detected the glycoprotein in the peripheral nervous system. Both anti-MAG and anti-1B236 antibodies revealed a drastic reduction of the level of 1B236/MAG in 25-day-old myelin-deficient rats and in adult quaking mice, and both types of antibodies revealed a slight shift of 1B236/MAG toward higher apparent Mr in quaking mice as had previously been reported for MAG. The results indicate that MAG and 1B236 are almost certainly identical since they cannot be distinguished immunologically by the reagents available and that quantitatively most of the glycoprotein is associated with oligodendrocytes and myelin rather than neurons at all ages.

Animals↗