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Biomedical subjects

R L Margolis

Publications and source records attributed to R L Margolis.

At least 73 records · Page 4Linked to original sources
PubMed1998

Suppression of tubulin tyrosine ligase during tumor growth.

The C terminus of the tubulin alpha-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL-). TTL- cells contain a unique tubulin isotype (delta2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL- cells and TTL- cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with delta2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.

3T3 Cells↗
PubMed1997

2-Aminopurine induces spindle cell morphology in MM14 myoblasts in the absence of differentiation signals.

MM14 murine myoblast cells remain in an undifferentiated and proliferative state if they are maintained in the continuous presence of basic fibroblast growth factor (FGF-2) and serum factors, but terminally differentiate into myocytes and myotubes if deprived of FGF-2 during G1 of the cell cycle. We find that 2-aminopurine (2-AP) induces a reversible, rapid, and profound alteration in the morphology of proliferating MM14 cells to a polarized shape which is similar to that observed during normally induced differentiation. This change requires neither a differentiation signal nor de novo protein synthesis. In contrast, we do not observe a morphological change in response to 2-AP in nonmyogenic cell lines. The morphological alteration of MM14 cells in response to 2-AP requires reorganization of the microtubule and actin cytoskeletons, in common with changes during normal differentiation. Additionally, we show that cytoskeletal rearrangements that occur during both normal differentiation and in response to 2-AP require the activity of the small GTPase Rho. Differentiation defective MM14 cells (DD-1 cells), which lack MyoD, also undergo a profound morphological alteration in the presence of 2-AP. These results indicate that morphological changes consistent with myoblast differentiation are regulated by a pathway independent of MyoD transcriptional control.

2-Aminopurine↗
PubMed1997

Chromosomes with two intact axial cores are induced by G2 checkpoint override: evidence that DNA decatenation is not required to template the chromosome structure.

Here we report that DNA decatenation is not a physical requirement for the formation of mammalian chromosomes containing a two-armed chromosome scaffold. 2-aminopurine override of G2 arrest imposed by VM-26 or ICRF-193, which inhibit topoisomerase II (topo II)-dependent DNA decatenation, results in the activation of p34cdc2 kinase and entry into mitosis. After override of a VM-26-dependent checkpoint, morphologically normal compact chromosomes form with paired axial cores containing topo II and ScII. Despite its capacity to form chromosomes of normal appearance, the chromatin remains covalently complexed with topo II at continuous levels during G2 arrest with VM-26. Override of an ICRF-193 block, which inhibits topo II-dependent decatenation at an earlier step than VM-26, also generates chromosomes with two distinct, but elongated, parallel arms containing topo II and ScII. These data demonstrate that DNA decatenation is required to pass a G2 checkpoint, but not to restructure chromatin for chromosome formation. We propose that the chromosome core structure is templated during interphase, before DNA decatenation, and that condensation of the two-armed chromosome scaffold can therefore occur independently of the formation of two intact and separate DNA helices.

2-Aminopurine↗
PubMed1997

Southern analysis for detection of CAG repeat expansions associated with dentatorubral pallidoluysian atrophy.

Dentatorubral pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by expansion of an unstable, tandemly repeated trinucleotide sequence, (CAG)n, in a novel gene on human chromosome 12p12-pter. Molecular diagnosis of DRPLA uses the polymerase chain reaction (PCR) to amplify and characterize the number of CAG repeats carried by individuals. The PCR analysis is fairly straightforward when two alleles are identified. However, when only a single allele is observed, it is difficult to know whether the sample is homozygous or whether there was failure to amplify the second allele. We describe a Southern analysis for detection of the DRPLA CAG repeat, providing an independent method for the assessment of expanded alleles.

Atrophy↗
PubMed1997

cDNAs with long CAG trinucleotide repeats from human brain.

Twelve diseases, most with neuropsychiatric features, arise from trinucleotide repeat expansion mutations. Expansion mutations may also cause a number of other disorders, including several additional forms of spinocerebellar ataxia, bipolar affective disorder, schizophrenia, and autism. To obtain candiate genes for these disorders, cDNA libraries from adult and fetal human brain were screened at high stringency for clones containing CAG repeats. Nineteen cDNAs were isolated and mapped to chromosomes 1, 2, 4, 6, 7, 8, 9, 12, 16, 19, 20, and X. The clones contain between 4 and 17 consecutive CAG, CTG, TCG, or GCA triplets. Clone H44 encodes 40 consecutive glutamines, more than any other entry in the nonredundant GenBank protein database and well within the range that causes neuronal degeneration in several of the glutamine expansion diseases. Eight cDNAs encode 15 or more consecutive glutamine residues, suggesting that the gene products may function as transcription factors, with a potential role in the regulation of neurodevelopment or neuroplasticity. In particular, the conceptual translation of clone CTG3a contains 18 consecutive glutamines and is 45% identical to the C-terminal 306 residues of the mouse numb gene product. These genes are therefore candidates for diseases featuring anticipation, neurodegeneration, or abnormalities of neurodevelopment.

Adult↗
PubMed1997

The role of the PH domain and SH3 binding domains in dynamin function.

Dynamin, a 100 kD GTPase, is necessary for the normal development and function of mammalian neural tissue. In neurons, it is necessary for the biogenesis of synaptic vesicles, and in other cell types dynamin has a general and important role in clathrin mediated receptor endocytosis. Different isoforms function as molecular scissors either during the formation of coated vesicles from plasma membrane coated pits, or during the release of intracellular vesicles from donor membranes. The mechanism entails the formation of a horseshoe-shaped dynamin polymer at the neck of the budding vesicle, followed by neck scission through a GTP hydrolysis dependent activity. The primary sequence of dynamin contains several C-terminal SH3 binding proline motifs, a central pleckstrin homology (PH) domain, and an N-terminal GTPase domain. Each of these domains appears to play a distinct role in dynamin function. Dynamin is activated by stimulus coupled PKC phosphorylation in brain, possibly mediated through PKC interactions with the PH domain. Further, SH3 domain interactions with the C-terminal sequences and phophatidylinositol/G beta gamma interactions with the PH domain also increase dynamin GTPase activity. Each of these various regulatory mechanisms is important in dynamin function during vesicle budding, although the means by which these mechanisms integrate in the overall function of dynamin remains to be elucidated.

Amino Acid Sequence↗
PubMed1997

A novel, heritable, expanding CTG repeat in an intron of the SEF2-1 gene on chromosome 18q21.1.

There are currently 13 diseases known to be caused by unstable triplet repeat mutations; however, there are some instances (as with FRAXF and FRA16) when these mutations appear to be asymptomatic. In a search for polymorphic CTG repeats as candidate genes for bipolar disorder, we screened a genomic human chromosome 18-specific library and identified a 1.6 kb clone (7,6A) with a CTG24 repeat that maps to 18q21.1. The CTG repeat locus, termed CTG18.1, is located within an intron of human SEF2-1, a gene encoding a basic hellx-loop-hellx DNA binding protein involved in transcriptional regulation. The CTGn repeat is highly polymorphic and very enlarged alleles, consistent with expansions of up to CTG2100, were identified. PCR and Southern blot analysis in pedigrees ascertained for a Johns Hopkins University bipolar disorder linkage study and in CEPH reference pedigrees revealed a tripartite distribution of CTG18.1 alleles with stable alleles (CTG10-CTG37), moderately enlarged and unstable alleles (CTG53-CTG250), and very enlarged, unstable alleles (CTG800-CTG2100). Moderately enlarged alleles were not associated with an abnormal phenotype and have a combined enlarged allele frequency of 3% in the CEPH and bipolar populations. Very enlarged alleles, detectable only by Southern blot analysis of genomic digests, have thus far been found in only three individuals from our bipolar pedigrees, and to date, have not been found in any of the CEPH reference pedigrees. These enlarged alleles may arise, at least in part, via somatic mutation.

Alleles↗
PubMed1996

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Amino Acid Sequence↗
PubMed1996

Analysis of thirteen trinucleotide repeat loci as candidate genes for schizophrenia and bipolar affective disorder.

A group of diseases are due to abnormal expansions of trinucleotide repeats. These diseases all affect the nervous system. In addition, they manifest the phenomenon of anticipation, in which the disease tends to present at an earlier age or with greater severity in successive generations. Many additional genes with trinucleotide repeats are believed to be expressed in the human brain. As anticipation has been reported in schizophrenia and bipolar affective disorder, we have examined allele distributions of 13 trinucleotide repeat-containing genes, many novel and all expressed in the brain, in genomic DNA from schizophrenic (n = 20-97) and bipolar affective disorder patients (23-30) and controls (n = 43-146). No evidence was obtained to implicate expanded alleles in these 13 genes as causal factors in these diseases.

Base Sequence↗
PubMed1996

Cloning, expression, and properties of the microtubule-stabilizing protein STOP.

Nerve cells contain abundant subpopulations of cold-stable microtubules. We have previously isolated a calmodulin-regulated brain protein, STOP (stable tubule-only polypeptide), which reconstitutes microtubule cold stability when added to cold-labile microtubules in vitro. We have now cloned cDNA encoding STOP. We find that STOP is a 100.5-kDa protein with no homology to known proteins. The primary structure of STOP includes two distinct domains of repeated motifs. The central region of STOP contains 5 tandem repeats of 46 amino acids, 4 with 98% homology to the consensus sequence. The STOP C terminus contains 28 imperfect repeats of an 11-amino acid motif. STOP also contains a putative SH3-binding motif close to its N terminus. In vitro translated STOP binds to both microtubules and Ca2+-calmodulin. When STOP cDNA is expressed in cells that lack cold-stable microtubules, STOP associates with microtubules at 37 degrees C, and stabilizes microtubule networks, inducing cold stability, nocodazole resistance, and tubulin detyrosination on microtubules in transfected cells. We conclude that STOP must play an important role in the generation of microtubule cold stability and in the control of microtubule dynamics in brain.

Amino Acid Sequence↗
PubMed1996

cDNA cloning and characterization of an atrophin-1 (DRPLA disease gene)-related protein.

Dentatorubral and pallidoluylsian atrophy (DRPLA) is a progressive neurological disorder characterized by neuronal degeneration, especially in the cerebellar dentate nucleus. DRPLA is caused by an unstable expansion of a CAG trinucleotide repeat coding for glutamine in a gene of unknown function, termed atrophin-1, located on chromosome 12. To gain additional understanding of atrophin-1, we have isolated a second member of the atrophin-1 gene family by screening rat cDNA libraries. The 1006-amino-acid product of this gene, which we have termed rat atrophin related protein(rARP), does not contain a glutamine repeat, but it does contain two regions of alternating acidic and basic amino residues similar to those found in atrophin-1. rARP is widely expressed as both a 7.4- and a 9.4-kb message, with enrichment in cerebellum and testis. Like atrophin-1, the rARP in vitro translation product migrates more slowly on SDS-polyacrylamide gel electrophoresis than predicted by molecular weight. We conclude that, at least in the rat, polyglutamine is not an essential feature of the atrophin family of genes.

Amino Acid Sequence↗
PubMed1996

DRPLA gene (atrophin-1) sequence and mRNA expression in human brain.

Dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease) is one of five disorders currently known to result from expansion of a CAG trinucleotide repeat encoding glutamine. The reported full length cDNA sequence encodes a serine repeat and a region of alternating acidic and basic amino acids, as well as the glutamine repeat. We now report the nucleic acid and deduced amino acid sequences of the open reading frame of this gene, obtained from a series of independently isolated and sequenced cDNA clones. Eight nucleotide differences from the originally published sequence result in a change of 34 amino acids, most prominently in the region of alternating acidic and basic residues. Northern analysis and in situ hybridization indicate that atrophin-1 mRNA is expressed in multiple brain regions. The level of mRNA expression as determined by in situ hybridization in a DRPLA-diseased brain is indistinguishable from the level observed in a matched control brain. These results indicate that the correlation between atrophin-1 expression and regions of pathology in DRPLA is at best partial, and that the expanded allele does not cause a major loss of mRNA expression. The pathology of the disorder may therefore arise from the altered structure and function of the abnormal protein.

Amino Acid Sequence↗
PubMed1996

Chemical induction of mitotic checkpoint override in mammalian cells results in aneuploidy following a transient tetraploid state.

Populations of tetraploid cells are found in a variety of human tumours where they may act as precursors of aneuploidy and tumorigenesis. Here we demonstrate the drug induction of tetraploid cells at mitosis by interference with cell cycle checkpoints and the coordination of mitotic events. Tetraploid cells result from mitotic exit in the absence of either chromosome segregation or cytokinesis. One class of agents that induces tetraploidy causes override of cell cycle checkpoints that require metaphase chromosome alignment as a pre-condition for initiating exit from mitosis. As a result cells exposed to such drugs progress partially through mitosis, but exit without chromosome segregation or cytokinesis. Inhibitors of microtubule assembly comprise a second class of agents that induce tetraploidy. Many cell types are incapable of maintaining indefinite mitotic arrest in the presence of microtubule inhibitors and finally exit mitosis without microtubule dependent chromosome segregation. Inhibitors of topoisomerase II represent a third class of drugs that induce tetraploidy at mitosis. By inhibiting DNA decatenation required for sister chromatid separation at the onset of anaphase such drugs block chromosome segregation. When topoisomerase II activity is inhibited, cells nonetheless reform nuclei and exit from mitosis without chromosome segregation. Finally, inhibition of cleavage furrow formation by agents such as cytochalasins represents a fourth mechanism of tetraploidization at mitosis. We find that when Chinese Hamster Ovary cells become tetraploid, regardless of which mechanism induces this state, they are genetically unstable and become aneuploid at the subsequent mitosis. In conclusion, the failure of mitotic checkpoint function can generate gross aneuploidy from which cells with an advantage for tumor growth may be selected.

2-Aminopurine↗
PubMed1996

Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific-related mortality of transformed cells.

Taxol (paclitaxel) induces a microtubule hyperassembled state, and effectively blocks cells in mitosis. Here we report that Taxol also induces a stable late-G1 block in nontransformed REF-52 and WI-38 mammalian fibroblast cells, but not in T antigen-transformed cells of the same parental lineage. G1 arrest is characterized by partially dephosphorylated pRb, and inactive cdk2 kinase. Nontransformed cells recover normally from Taxol arrest. In contrast, T antigen transformed cells continue inappropriately past both G1 and G2-M in the presence of Taxol, and undergo a rapid death upon release. These results demonstrate a microtubule sensitive step in G1 regulation of nontransformed fibroblast cells. Also, Taxol selectively induces death of transformed cells, possibly because they slip the Taxol-dependent G1 arrest, as well as G2/M arrest, which are both specific to nontransformed cells.

Animals↗
PubMed1996

cDNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21: expression, chromosomal localization and analysis of a highly polymorphic (CAG)n trinucleotide repeat.

The two most consistent features of the diseases caused by trinucleotide repeat expansion-neuropsychiatric symptoms and the phenomenon of genetic anticipation-may be present in forms of dementia, hereditary ataxia, Parkinsonism, bipolar affective disorder, schizophrenia and autism. To identify candidate genes for these disorders, we have screened human brain cDNA libraries for the presence of gene fragments containing polymorphic trinucleotide repeats. Here we report the cDNA cloning of CAGR1, originally detected in a retinal cDNA library. The 2743 bp cDNA contains a 1077 bp open reading frame encoding 359 amino acids. This amino acid sequence is homologous (56% amino acid identify and 81% amino acid conservation) to the Caenorhabditis elegans cell fate-determining protein mab-21. CAGR1 is expressed in several human tissues, most prominently in the cerebellum, as a message of approximately 3.0 kb. The gene was mapped to 13q13, just telomeric to D13S220. A 5'-untranslated CAG trinucleotide repeat is highly polymorphic, with repeat length ranging from six to 31 triplets and a heterozygosity of 87-88% in 684 chromosomes from several human populations. One allele from an individual with an atypical movement disorder and bipolar affective disorder type II contains 46 triplets, 15 triplets longer than any other allele detected. Though insufficient data are available to link the long repeat to this clinical phenotype, an expansion mutation of the CAGR1 repeat can be considered a candidate for the etiology of disorders with anticipation or developmental abnormalities, and particularly any such disorders linked to chromosome 13.

Amino Acid Sequence↗
PubMed1996

Occupational therapy task observation scale (OTTOS): a rapid method for rating task group function of psychiatric patients.

OBJECTIVE: We designed a simple rating instrument, the Occupational Therapy Task Observation Scale (OTTOS), to facilitate evaluation and documentation of patient performance during occupational therapy task groups and to improve the communication between occupational therapists and other treatment team members. METHOD: After analysis of other rating instruments, a preliminary version of OTTOS was designed, extensively field tested, and further modified. Interrater reliability was determined, and validity was tested against three established rating instruments: the Bay Area Functional Performance Evaluation, the Comprehensive Occupational Therapy Evaluation Scale, and the Milwaukee Evaluation of Daily Living Skills. RESULTS: The final version of OTTOS contains two parts, 10 items for evaluation of specific task functions and 5 items for rating general behavior. Field use demonstrated that the scale successfully tracked changes in functional capacity and included most facets of patient function evaluated during task groups. Use of OTTOS required minimal training, and scoring required less than 2 min for each patient. The correlation between the scores of experienced occupational therapists was high (.92 for the total scores). The correlation between OTTOS and the other rating instruments ranged from .880 to .340; the highest correlations, as expected, were with test subscales that most closely resembled OTTOS: CONCLUSION: Preliminary data indicated that OTTOS offers a reliable and valid method for rapidly rating the function of patients with psychiatric illness attending task groups. In addition, use of OTTOS improved the communication between occupational therapists and other health care providers, facilitated the education of occupational therapy students, and met the documentation requirements of third-party payers.

Humans↗
PubMed1995

Carbamazepine induction of apoptosis in cultured cerebellar neurons: effects of N-methyl-D-aspartate, aurintricarboxylic acid and cycloheximide.

We have previously demonstrated that carbamazepine (CBZ) at concentrations above the therapeutic range is toxic to cultured cerebellar granule cells. Here, we ask whether the effect of CBZ involves neuronal apoptosis or necrosis. Treatment of cultured cerebellar granule cells with CBZ for 3 days resulted in a concentration-dependent fragmentation of DNA revealed as a laddered pattern in agarose gel electrophoresis, a phenomenon characteristic of apoptosis. Pretreatment of cells with N-methyl-D-aspartate (NMDA) blocked CBZ-induced DNA fragmentation and neurotoxicity as assayed by loss of mitochondrial activity with MTT or by [3H]ouabain binding to Na+/K(+)-ATPase. Aurintricarboxylic acid (ATA), a polyanionic dye, also markedly suppressed DNA fragmentation and cell death detected by morphological examination. A considerable level of DNA ladder formation was detected in untreated cells and this basal DNA fragmentation was also blocked by NMDA and ATA. Moreover, NMDA and ATA prevented CBZ-induced chromatin condensation as revealed by DNA binding with the fluorescent dye Hoechst 33258. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, prevented CBZ-induced cell death detected morphologically and attenuated CBZ-induced neurotoxicity assessed by mitochondrial activity and [3H]ouabain binding assays. Taken together, our results suggest that CBZ-induces death of cerebellar granule cells by an apoptotic process that is sensitive to NMDA, ATA and cycloheximide.

Animals↗