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J S Morrow

Publications and source records attributed to J S Morrow.

At least 19 recordsLinked to original sources
PubMed1992

Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.

We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.

Amino Acid Sequence↗
PubMed1992

Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle.

Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing 'bodies' within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.

Animals↗
PubMed1991

Actin and tubulin binding domains of synapsins Ia and Ib.

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1991

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

Amino Acid Sequence↗
PubMed1991

Diagnostic tests for choreoacanthocytosis.

Two patients with striatal atrophy and a clinical syndrome consistent with choreoacanthocytosis had normal dried blood smears but their red cells demonstrated an abnormal sensitivity to various conditions known to promote discocyte-echinocyte transformation. Dilution in normal saline, in vitro aging, and contact with glass caused a great proportion of these patients' red cells to develop multiple spiny or rounded projections. Under identical conditions, such shape changes did not occur in normal patients or in those with Huntington's disease. Scanning electron microscopy showed that the age-induced increase in acanthocytic-appearing cells could be reversed with chlorpromazine. These data suggest that the red cells from these patients with striatal degeneration are deficient in their ability to preserve normal shape in the face of echinocytic stress and that this observation has diagnostic and, possibly, pathophysiologic significance.

Acanthocytes↗
PubMed1990

Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions.

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)- succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of beta spectrin within a sequence homologous to the actin-binding region of alpha actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.

Actins↗
PubMed1990

PATHMASTER: modelling differential diagnosis as "dynamic competition" between systematic analysis and disease-directed deduction.

PATHMASTER is an expert system under development to assist in teaching histopathologic differential diagnosis. The system incorporates two "orthogonal" representations of the knowledge required to perform differential diagnosis. One representation groups histopathologic features around the anatomic structures of liver tissue, the second representation groups the features around the diseases in which they occur. Using these two representations, PATHMASTER models the process of diagnosis as a "dynamic competition" between systematic analysis and disease-directed deduction. By varying two parameters of PATHMASTER's underlying mathematical model, the interplay between these factors can be varied.

Algorithms↗
PubMed1990

Calmodulin and calcium-dependent protease I coordinately regulate the interaction of fodrin with actin.

The calcium-dependent proteolysis of fodrin has been implicated in the regulation of secretion, neutrophil and platelet activation, and long-term potentiation in neurons. In vitro studies indicate that calcium-dependent protease I (calpain I) cleaves fodrin in the middle of the alpha subunit and in the COOH-terminal third of the beta subunit. Cleavage at the beta site requires calmodulin, which binds with high affinity to a single site in the alpha subunit. In vitro binding assays, nondenaturing gel electrophoresis, and velocity sedimentation identify a linkage between calcium-dependent protease I proteolysis of fodrin and the ability of calmodulin to regulate the self-association of fodrin and its interaction with actin. Three functional states appear to exist: (i) intact fodrin, which constitutively forms tetramers and binds F-actin; (ii) alpha-cleaved fodrin, which loses its ability to self-associate and bind F-actin in the presence of calmodulin; and (iii) alpha,beta-cleaved fodrin, a form that is incompetent to establish tetramers or bind actin. Because actin binding and fodrin self-association occur at opposite ends of the molecule, whereas calmodulin binds at its center, these results indicate that long-range interactions exist within fodrin. They also offer an example of how two calcium-dependent regulatory processes may act synergistically to reversibly regulate a linkage between the membrane and the cytoskeleton.

Actins↗
PubMed1989

Calmodulin regulates fodrin susceptibility to cleavage by calcium-dependent protease I.

The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin.

Animals↗
PubMed1989

Functional diversity among spectrin isoforms.

The purpose of this review on spectrin is to examine the functional properties of this ubiquitous family of membrane skeletal proteins. Major topics include spectrin-membrane linkages, spectrin-filament linkages, the subcellular localization of spectrins in various cell types and a discussion of major functional differences between erythroid and nonerythroid spectrins. This includes a summary of studies from our own laboratories on the functional and structural comparison of avian spectrin isoforms which are comprised of a common alpha subunit and a tissue-specific beta subunit. Consequently, the observed differences among these spectrins can be assigned to differences in the properties of the beta subunits.

Humans↗
PubMed1989

Contributions of the beta-subunit to spectrin structure and function.

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.

Actins↗
PubMed1989

Automated bibliographic retrieval based on current topics in hepatology: hepatopix.

The Hepatopix computer program helps the physician go from a computerized clinical record directly to a computerized search of the medical literature. The program uses a hierarchical list of current (key) topics in hepatology to offer "intelligent" searches. Each topic has associated "selection logic" and a tested Medline search. Starting with a liver biopsy case record, Hepatopix evaluates the selection logic to determine which topics may be pertinent to the case (based on clinical findings, lab tests, and critical words or phrases in the summary). The physician then picks those topics which are interesting enough to warrant a literature search. The citations are retrieved using search strategies stored for each topic and presented. Hepatopix is operational with over 200 topics in the realm of liver neoplasms, operating on liver biopsy case summaries from the Klatskin Database of Liver Biopsies. Besides demonstrating clinical case-directed bibliographic retrieval, it demonstrates the utility of a "key topics" list as a bridge between medical databases.

Bibliography of Medicine↗
PubMed1989

Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in Madin-Darby canine kidney cells and in intact renal tubule cells.

In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537). The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of approximately 1 ankyrin to 4 Na,K-ATPase's, with a kD of 2.6 microM. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) 125I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of approximately 240 kD that is associated with both erythrocyte and kidney membranes that binds 125I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.

Animals↗
PubMed1989

An unusual beta-spectrin associated with clustered acetylcholine receptors.

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane is an early event in the formation of the neuromuscular junction. The mechanism of clustering is still unknown, but is generally believed to be mediated by the postsynaptic cytoskeleton. We have identified an unusual isoform of beta-spectrin which colocalizes with AChR in AChR clusters isolated from rat myotubes in vitro. A related antigen is present postsynaptically at the neuromuscular junction of the rat. Immunoprecipitation, peptide mapping and immunofluorescence show that the beta-spectrin in AChR clusters resembles but is distinct from the beta-spectrin of human erythrocytes. alpha-Spectrin appears to be absent from AChR clusters. Semiquantitative immunofluorescence techniques indicate that there are from two to seven beta-spectrin molecules present for every clustered AChR, the higher values being obtained from rapidly prepared clusters, the lower values from clusters that require several minutes or more for isolation. Upon incubation of isolated AChR clusters for 1 h at room temperature, beta-spectrin is slowly depleted and the AChR redistribute into microaggregates. The beta-spectrin that remains associated with the myotube membrane is concentrated at these microaggregates. beta-Spectrin is quantitatively lost from clusters upon digestion with chymotrypsin, which causes AChR to redistribute in the plane of the membrane. These results suggest that AChR in clusters is closely linked to an unusual isoform of beta-spectrin.

Actins↗
PubMed1989

Pain therapy for pancreatic carcinoma with neurolytic celiac plexus block.

The past year's experience of the Pain and Regional Anesthesia Service at Hartford Hospital with neurolytic celiac plexus blockade for pancreatic cancer has been reviewed. The series includes eight patients with intractable pain from pancreatic cancer as well as two patients with pain from other abdominal malignancies. Nearly all patients noted good to excellent analgesia for up to 11 months. The only complication noted was one episode of mild, transient hypotension. Neurolytic celiac plexus blockade is a safe and effective means to relieve the terminal pain associated with not only pancreatic cancer but also other abdominal malignancies.

Autonomic Nerve Block↗