Search PubMed⌕ Search

Biomedical subjects

M Linial

Publications and source records attributed to M Linial.

At least 37 records · Page 2Linked to original sources
PubMed1997

Acceleration of neuronal maturation of P19 cells by increasing culture density.

P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The mature neurons are capable of neurotransmitter release, and from functional synapses. We have previously shown that high culture density suppresses the cholinergic phenotype of P19 neurons. Here we demonstrate that increasing culture density accelerates the maturation of P19 neurons in a continuous manner. This is manifested by several criteria: increased efficiency of evoked [3H]aspartate release; decreased level of basal release; up-regulation of synaptic vesicle proteins; increased neurite outgrowth rate; and earlier segregation of axons and dendrites. While glutamate release is enhanced in dense cultures, the efficiency of [3H]GABA release is hardly affected, suggesting that P19 GABAergic neurons are not affected by culture density. The acceleration of neuronal maturation in dense cultures is also exhibited by the ability of dense, but not sparse cultures to release [3H]aspartate at an earlier day of differentiation. Furthermore, density effects are monitored already a few hours after plating the cultures, when neurite length in dense cultures is several fold higher than in sparse cultures. This indicates that commitment to a faster and coordinated maturation process occurs already very early during P19 neuronal differentiation.

Animals↗
PubMed1997

Differential expression pattern of Rab-GDI isoforms during the parotid gland secretion cycle.

Rab GDP dissociation inhibitor (GDI) plays an important role in regulating the GDP/GTP cycle of small GTP binding proteins of the Rab family. It also regulates their association to membranes. The small family of Rab-GDI consists of several closely related isoforms, the functional differences between which are still unknown. Here we show that multiple GDI isoforms are expressed in rat parotid gland and that the individual GDI isoforms have a characteristic expression both at the RNA and at the protein level, during the parotid secretory cycle. GDIalpha, the major isoform in brain, is expressed throughout the secretory process and is equally distributed between cytoplasmic and membranous fractions. In contrast, an isoform related to, but different from GDIbeta is found predominantly in the cytoplasmic fraction and its expression is detected only after beta-adrenergic stimulation of the gland, at the end of the secretion phase, when exocytosis is already completed. The induction of such a GDI isoform at the beginning of the recovery stage correlates with the expression pattern of Rab1 and Rab5, but not Rab2 and Rab4. Our results suggest different functional roles for multiple GDI isoforms along the secretion and recovery phases in rat parotid gland.

Adrenergic beta-Agonists↗
PubMed1997

Global self-organization of all known protein sequences reveals inherent biological signatures.

A global classification of all currently known protein sequences is performed. Every protein sequence is partitioned into segments of 50 amino acid residues and a dynamic programming distance is calculated between each pair of segments. This space of segments is initially embedded into Euclidean space. The algorithm that we apply embeds every finite metric space into Euclidean space so that (1) the dimension of the host space is small, (2) the metric distortion is small. A novel self-organized, cross-validated clustering algorithm is then applied to the embedded space with Euclidean distances. We monitor the validity of our clustering by randomly splitting the data into two parts and performing an hierarchical clustering algorithm independently on each part. At every level of the hierarchy we cross-validate the clusters in one part with the clusters in the other. The resulting hierarchical tree of clusters offers a new representation of protein sequences and families, which compares favorably with the most updated classifications based on functional and structural data about proteins. Some of the known families clustered into well distinct clusters. Motifs and domains such as the zinc finger, EF hand, homeobox, EGF-like and others are automatically correctly identified, and relations between protein families are revealed by examining the splits along the tree. This clustering leads to a novel representation of protein families, from which functional biological kinship of protein families can be deduced, as demonstrated for the transporter family. Finally, we introduce a new concise representation for complete proteins that is very useful in presenting multiple alignments, and in searching for close relatives in the database. The self-organization method presented is very general and applies to any data with a consistent and computable measure of similarity between data items.

Amino Acid Sequence↗
PubMed1997

An optical submicrometer calcium sensor with conductance sensing capability.

The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.

Animals↗
PubMed1997

Culture density regulates both the cholinergic phenotype and the expression of the CNTF receptor in P19 neurons.

The P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The cells mature into functional neurons, as determined by their ability to release neurotransmitters in a Ca(2+)- and depolarization-dependent manner. P19 neurons in culture represent a mixed population in terms of their neurotransmitter phenotype. The cholinergic phenotype of these neurons is modulated by culture density. Cholinergic markers, such as the vesicular acetylcholine transporter, acetyl cholinesterase, and choline acetyltransferase, are expressed in about 85% of the cells in sparse cultures and are largely suppressed at high cell densities. In contrast, glutamate release is enhanced in dense P19 neuronal cultures. The factor mediating the density effect is concentrated exclusively on the cell membrane of P19 neurons and not on the nonneuronal cells, which also differentiate from P19 embryonal carcinoma cells. This membrane-associated component retains its functionality, even after membrane fixation. The downregulation of the cholinergic properties in dense cultures is paralleled by a downregulation of the alpha subunit of the ciliary neurotrophic factor (CNTF) receptor. Thus, it is suggested that the membrane-associated factor, which mediates the density effect, downregulates the cholinergic phenotype by inhibiting the responsiveness of these neurons to CNTF. We further suggest that the P19 cell line can serve as a model system for the study of neurotransmitter phenotype acquisition and plasticity throughout neuronal differentiation.

Acetylcholine↗
PubMed1997

SNARE proteins--why so many, why so few?

Both trafficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a small number of proteins, i.e., the exocytic core complex. In nerve terminals, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP-25 at the plasma membrane. In this survey, different secretory systems that depend on the exocytic core proteins are considered. The possibility that specificity in membrane recognition and fusion is achieved by the numerous variants of proteins of the exocytic core is discussed. Variability of the core complex proteins is determined by the complexity of gene families, isoform-specific localization, and posttranslational modifications. Basic biochemical properties depend on specific isoforms, and the possible protein-protein interactions are determined, in turn, by the compatibility of different isoforms. A correlation between specific variants and distinct biochemical or cellular properties is shown. The outcome of this survey is that heterogeneity in secretion may be dictated by the large number of possible combinations of variants of only a few proteins.

Animals↗
PubMed1997

Expression and localization of synaptotagmin I in rat parotid gland.

Synaptotagmins are a gene family of membrane proteins with distinct expression patterns. Synaptotagmin I is an abundant protein of the synaptic vesicle membrane and was implicated as the Ca2+ sensor in fast responding synapses. Yet, its precise role along the synaptic vesicle life cycle is not fully understood. In this report we show that synaptotagmin I is not exclusively confined to neuronal and neuroendocrine systems, rather, it is also expressed in the exocrine system of the parotid gland. The gene for synaptotagmin I was isolated and sequenced from rat parotid cDNA. The identity of synaptotagmin I protein was further confirmed by several independent antibodies. The protein is exclusively found in the membranous fraction of purified granules, similarly to VAMP-2, another major integral membrane protein of synaptic vesicles. Synaptotagmin I represents 0.4% of the total membrane protein mass of the granule. Using immunoelectron microscopy the two proteins were also localized primarily to the granules' membranes. These findings suggest that synaptotagmin I which regulates Ca(2+)-dependent neurotransmitter release also plays a role which is common to all secretory organelles-neuronal, endocrine and exocrine. A role for synaptotagmin I in integrating signals with protein secretion in the parotid gland is suggested.

Animals↗
PubMed1995

alpha-latrotoxin is a potent inducer of neurotransmitter release in Torpedo electric organ--functional and morphological characterization.

In this report we show that alpha-latrotoxin from black widow spider venom is a potent activator of neurotransmitter release in synaptosomes from the Torpedo electric organ. Binding of the purified toxin (5 nM) to the synaptosomal fraction occurs already at 4 degrees C and is dependent on the presence of divalent ions. However, neurotransmitter release commences only after temperature elevation (22 degrees C) and is completed within 2 min. The effect of alpha-latrotoxin on release is achieved at 1 nM and is already saturated at 5 nM. The release is stimulated by the presence of Ca2+ ions. Activation of release by alpha-latrotoxin is accompanied by morphological changes in electric organ synaptosomes. The synaptosomes swell, resulting in a 55% increase in section area. Moreover, the number of synaptic vesicles per unit area decreases about three-fold, and rows of docked synaptic vesicles are rarely detected as opposed to control synaptosomes. These morphological changes indicate that the massive release is mainly due to synaptic vesicle fusion. alpha-Latrotoxin binding sites are highly concentrated in the innervated face of the electrocytes. Immunoelectron microscopy on electric organ sections reveals alpha-latrotoxin binding sites over the entire plasma membrane at release sites and facing Schwann cells surrounding Torpedo nerve terminals. Surprisingly, a high concentration of binding sites is also found at structures surrounding branching unmyelinated axons. This staining is in close proximity to Schwann cell envelopes and to the basal lamina around axonal tips. The mode of action of alpha-latrotoxin in view of the localization of its binding sites is discussed.

Animals↗
PubMed1995

Nucleotide binding by the synapse associated protein SAP90.

The rat synapse associated protein SAP90 is a member of a superfamily of potential guanylate kinases localized at cell-cell contact sites. This superfamily includes the synapse associated protein SAP97, a close relative of SAP90, the Drosophila tumor suppressor gene product dlg-Ap, the mammalian zonula occludens proteins ZO-1 and ZO-2 and the erythrocyte protein p55. Here we show that SAP90 specifically binds GMP in the micromolar range while binding to ATP, GDP and ADP is at a much lower affinity (10-25 mM), whether or not binding is detected for other guanine and adenine nucleotides. No guanylate kinase activity of SAP90 was detected under our experimental conditions. The importance of the GMP binding capacity per se and an evolutionary role for conserving of the guanylate kinase domain in this superfamily are discussed.

Adenosine Triphosphate↗
PubMed1995

Cholinergic properties of neurons differentiated from an embryonal carcinoma cell-line (P19).

P19 is a mouse-derived embryonal carcinoma cell-line capable of differentiation toward ectodermal, mesodermal and endodermal lineages. Following treatment with retinoic acid these cells differentiate into neurons, astrocytes and fibroblast-like cells. We induced P19 differentiation under conditions which lead to a homogeneous neuronal culture (> 95% neurons). Under these conditions, most cells (approximately 85%) express high levels of the cholinergic markers acetyl cholinesterase and choline acetyltransferase while approximately 10% of cells express the GABAergic marker glutamic acid decarboxylase. While the proportion of the GABAergic neurons is constant at different culture conditions, the cholinergic phenotype is suppressed at high cell densities. The cholinergic nature of P19 neurons is also evident in their ability to form contacts with a muscle cell-line--C2. At day 10 of differentiation cells are capable of depolarization-dependent acetylcholine release. The release is Ca2+ dependent, and drops to baseline levels at 0.5 mM Ca2+. The cells also respond to sub-nM levels of alpha-latrotoxin by acetylcholine release. All major proteins implicated in synapse functionality are expressed prior to day 10 at both at RNA and protein levels. However, the expression pattern of each gene is unique. The genes include cytoskeletal proteins, synaptic vesicle proteins and terminal specific proteins. We suggest that this cell-line can serve as an in-vitro model system for the study of neuronal phenotype acquisition. Under our conditions, the P19 cells can also provide a system in which to study the differentiation of functional cholinergic neurons.

Acetylcholine↗
PubMed1995

The effect of calcium levels on synaptic proteins. A study on VAT-1 from Torpedo.

In this study we compare major synaptic proteins from Torpedo electric organ to their homologues from mammalian brain. Most of these proteins are members of small gene families. We demonstrate a high degree of evolutionary conservation of most synaptic proteins. However, in the electric organ each gene family is represented only by a single member. We focus on VAT-1, a major protein of the vesicle membrane in Torpedo. VAT-1 is located on the synaptic vesicle membrane and is highly concentrated on the plasma membrane following the application of alpha-latrotoxin. Taking advantage of the relative simplicity of Torpedo synapses, we performed an in vitro study on the properties of VAT-1 affected by changes in Ca2+ levels. VAT-1 is a low affinity Ca2+ binding protein whose ability to bind Ca2+ resides mainly, but not entirely, on the carboxy-terminal domain of the protein. In the presence of Ca2+, the protein is organized in a high molecular mass complex, which is destabilized by depleting Ca2+. This effect occurs only by chelating Ca2+ ions, but not with other divalent ions. VAT-1 is not complexed to any of the proteins which were implicated in the docking/fusion complex such as VAMP, synaptophysin or syntaxin, regardless of Ca2+ levels. Dependence of the stability of protein complexes on Ca2+ levels is also demonstrated on Torpedo n-Sec1. The possible physiological implications of such Ca2+ dependence are discussed.

Animals↗
PubMed1995

Bacterial neurotoxins--a thousand years later.

Clostridium bacteria are responsible for the neuroparalysis in tetanus and in botulism by producing potent neurotoxins. Here we review the current developments in understanding the toxins' mode of action by deciphering the molecular basis for their function. The active forms of tetanus and botulinum neurotoxins block neurotransmitter release via a zinc-dependent protease activity. All known tetanus and botulinum toxins cleave only three key components in the synaptic vesicle docking and fusion protein complex. While tetanus and botulinum types B, D, F and G cleave VAMP/synaptobrevin, an integral membrane protein of the synaptic vesicles, two other synaptic proteins from the plasma membrane, SNAP-25 and syntaxin, are cleaved by botulinum types A and E and botulinum type C, respectively. We discuss the mechanism by which the proteolytic activity of these toxins causes a block in vesicle fusion.

Animals↗
PubMed1994

Proline clustering in proteins from synaptic vesicles.

Synaptic vesicle proteins share a specialized and common fate throughout the life cycle of the vesicle, and thus may need to respond to some common signals. It is therefore expected that these proteins will share some common motifs. However, sequence comparison among many of these proteins has not revealed any obvious motifs. Such a motif may be formed by the relative abundance of proline residues, which are not randomly distributed along the sequence but rather are clustered at the cytoplasmatic face of vesicular proteins. We propose that proline clusters serve as structural spacers between functional domains as well as potential target sites for protein-protein interactions. In view of the proline-rich nature of SH3 binding proteins, some of the proline-rich synaptic vesicle proteins may also participate in SH3 binding. Such binding may modulate certain signalling pathways in nerve terminals. Surprisingly, the consensus sequence between the proline clusters of synaptic vesicle proteins is found in a large family of abundant proline-rich proteins of the secretory organelles of the parotid exocrine gland.

Amino Acid Sequence↗
PubMed1993

VAT-1 from Torpedo electric organ forms a high-molecular-mass protein complex within the synaptic vesicle membrane.

VAT-1 is an abundant 41-kDa protein from Torpedo cholinergic synaptic vesicles. Most of VAT-1 immunoreactivity (70%) is localized to the synaptic vesicle membrane while the rest (30%) copurifies with larger membranous fragments. VAT-1 forms a high-molecular-mass complex within the synaptic vesicle membrane. The Stokes radius of the VAT-1 complex is 4.85 nm and the sedimentation coefficient is 8.0 x 10(-13) S. Using these values, the calculated apparent mass of the VAT-1 complex is 176 kDa and the friction coefficient is consistent with that for a globular protein. Electrophoresis of solubilized synaptic vesicle proteins following cross-linking resulted in a 40-kDa ladder which was detected by VAT-1 antibodies. This is in accord with VAT-1 protein complex being composed primarily of VAT-1 subunits. The hydrodynamic characteristics of the VAT-1 protein complex suggest that it is composed of three or four VAT-1 subunits. Synaptophysin, an abundant component of Torpedo synaptic vesicle membranes, which has a similar apparent size as VAT-1, is not part of the VAT-1 protein complex. Interactions between the subunits within the protein complex do not depend on disulfide bonds or on lowering the ionic strength. However, partial dissociation of VAT-1 subunits from the complex occurs by chelating calcium ions.

Animals↗
PubMed1993

The protein VAT-1 from Torpedo electric organ exhibits an ATPase activity.

VAT-1 is an abundant protein in Torpedo electric organ which copurifies with a major ATPase activity from synaptic vesicles. VAT-1 was expressed in E. coli and the product was purified and analyzed. The protein binds specifically to an ATP column and displays an ATPase activity as measured by the kinetics of [32P]phosphate release. The activity is dependent on divalent ions, with both Mg2+ and Ca2+ supporting the reaction. The apparent Km for ATP is 18 microM. This ATPase activity is not affected by known inhibitors of the vesicular V- and P-type ATPases such as vanadate and N-ethylmaleimide. We suggest that VAT-1 activity may affect ATP-dependent reactions in Torpedo nerve terminals, such as phosphorylation and dephosphorylation of proteins.

Adenosine Triphosphate↗