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Biomedical subjects

Y Ben-Shaul

Publications and source records attributed to Y Ben-Shaul.

At least 19 recordsLinked to original sources

Adaptive acetylcholinesterase splicing patterns attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice.

Balanced dopaminergic cholinergic interactions are crucial for proper basal ganglia function. This is dramatically demonstrated by the worsening of Parkinson's disease symptoms following acetylcholinesterase (AChE) inhibition. Typically, in the brain, the synapse-anchored synaptic AChE (AChE-S) variant is prevalent whereas the soluble readthrough AChE (AChE-R) variant is induced in response to cholinesterase inhibition or stress. Because of the known functional differences between these variants and the fact that AChE-R expression is triggered by various stimuli that themselves are often associated with Parkinson's disease risk, we hypothesized that the splice shift to AChE-R plays a functional role in Parkinsonian progression. After establishing that Paraoxon-induced AChE inhibition indeed aggravates experimental Parkinsonism triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, we tested the roles of individual AChE variants by exposing transgenic mice overexpressing either the AChE-S or AChE-R variant to MPTP. Differential reductions of tyrosine hydroxylase levels in the striatum and substantia nigra indicated that transgenic AChE-R expression confers resistance as compared with the parent FVB/N strain. In contrast, AChE-S overexpression accelerated the MPTP-induced damage. Survival, behavioral measures and plasma corticosterone levels were also compatible with the extent of the dopaminergic damage. Our findings highlight the functional differences between individual AChE variants and indicate that a naturally occurring stress or AChE inhibitor-induced splicing shift can act to minimize dopaminergic cholinergic imbalances. We propose that inherited or acquired alternative splicing deficits could accelerate Parkinsonism and that, correspondingly, adaptive alternative splicing events may attenuate disease progression.

Acetylcholine↗

Unsupervised spike detection and sorting with wavelets and superparamagnetic clustering.

This study introduces a new method for detecting and sorting spikes from multiunit recordings. The method combines the wavelet transform, which localizes distinctive spike features, with superparamagnetic clustering, which allows automatic classification of the data without assumptions such as low variance or gaussian distributions. Moreover, an improved method for setting amplitude thresholds for spike detection is proposed. We describe several criteria for implementation that render the algorithm unsupervised and fast. The algorithm is compared to other conventional methods using several simulated data sets whose characteristics closely resemble those of in vivo recordings. For these data sets, we found that the proposed algorithm outperformed conventional methods.

Action Potentials↗

Trial to trial variability in either stimulus or action causes apparent correlation and synchrony in neuronal activity.

In this report we show that the observed inter-neuronal correlation reflects a superposition of correlations associated with the intrinsic correlation between neurons, and correlations associated with variability in the stimuli presented to, or the actions performed by, the subject. We argue that the effects of either stimulus or action variability on the observed correlation, though generally ignored, can be substantial. Specifically, we demonstrate how observed correlations are effected by trial to trial variability in either stimulus or action. In addition, assuming that all relevant stimuli and actions are known, we outline a method for eliminating their effects on the observed correlation. It is also shown that tuning of correlations to a stimulus or an action might be a direct consequence of variability in that stimulus or action, even in the absence of any modulation of direct inter-neuronal interaction. The effects of stimulus and action variability should therefore be carefully considered when designing and interpreting experiments involving multi-neuronal recordings.

Action Potentials↗

The cytoskeletal network controls c-Jun expression and glucocorticoid receptor transcriptional activity in an antagonistic and cell-type-specific manner.

The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Müller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.

Animals↗

Co-localization of the insulin receptor, jun protein and choline acetyltransferase in embryonic chick retina.

Previous work demonstrated that the availability of insulin to the embryonic chick retina at a critical developmental stage stimulated the activity of the acetylcholine synthetic enzyme, choline acetyltransferase (ChAT) and that this increase required the AP-1 transcription factor, c-jun. Here it is shown that immediately following a 2-5 min exposure to insulin there is, in the amacrine and ganglion cells of the chick embryo retina, a transient increase in the level of jun protein followed by a long-lasting increase in ChAT. These and previous results show that insulin receptor activation is necessary for the characteristic retina developmental increase in ChAT protein and that this increase is preceded by a transient increase in the synthesis of the transcription factor c-jun in the same retina cells. The data demonstrate an intracellular signal transduction pathway from the developmentally-activated insulin receptor through c-jun to ChAT and cholinergic differentiation.

Animals↗

Developmental localization of retina cognin synthesis by in situ hybridization.

Retina cognin (R-cognin) is a 50 kDa protein involved in cell recognition and neuronal differentiation during development of the embryonic chick retina. Initial characterization of a partial cDNA encoding R-cognin revealed a striking similarity to the cDNA encoding protein disulfide isomerase (PDI), a 57 kDa multifunctional protein. The exact nature of the relationship between R-cognin and PDI is not known; however, both proteins appear to be encoded by the same gene. In the present study, we developed cRNA probes to examine the expression of R-cognin and PDI transcripts in embryonic chick retina and liver. In the retina, the amount of transcript decreased with embryonic age, in parallel to a similar decrease in R-cognin protein. In the liver, where PDI is prominently expressed, the amount of transcript was not developmentally regulated. The spatial and temporal pattern of expression of the R-cognin-encoding retinal transcript was examined by in situ hybridization. R-cognin mRNA was expressed in cells across the retina early in retinogenesis, but became restricted to the cells of the inner retina later in development. This pattern of expression was the same as the developmental pattern of R-cognin protein [Dobi et al., Invest. Ophthalmol. Vis. Sci. 27, (1986) p. 323-329], thus, demonstrating that this secreted protein functions at the surface of the cells where it is transcribed.

Animals↗

Causal role for jun protein in the stimulation of choline acetyltransferase by insulin in embryonic chick retina.

Previous work showed that the availability of insulin to the embryonic chick retina at a critical developmental stage stimulated the activity of the acetylcholine synthetic enzyme, choline acetyltransferase (ChAT) (R. E. Hausman et al., 1991, Dev. Brain Res. 59, 31-37). Here we show that a 2- to 5-min exposure to insulin results in a greater than 24 hr elevation in ChAT protein. Immediately following exposure to insulin there is a transient increase in the level of jun protein followed by an increase in ChAT. The stimulation of ChAT protein is not the result of an overall stimulation of protein synthesis as other proteins are not affected. Exposure of the cells to antisense oligonucleotide to jun, but not to sense oligonucleotide, reduces the increase in both jun and ChAT. These and previous results suggest that insulin is necessary for the characteristic increase in ChAT protein during retina development and that this increase requires the transient synthesis of jun.

Animals↗

Influence of metabolic inhibitors on the degradation of tight junctions in HT29 cells.

The human colon adenocarcinoma cell line HT 29 grows in culture without tight junctions (TJ). Tight junction strands of the fascia occludens type can be induced by treatment with proteases and are subsequently degraded during a period of about 3 h. Experiments using a variety of metabolic inhibitors such as 2-deoxyglucose, 2,4-dinitrophenol, and CCCP show that the degradation of TJ is retarded under conditions of ATP depletion. Thus it appears that the removal of TJ from the cell surface is an energy-dependent process. Moreover, DNP can specifically inhibit the degradation of TJ even in the absence of ATP depletion. The possible involvement of a proton gradient in the mechanism of TJ degradation is discussed.

2,4-Dinitrophenol↗

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.

Antipain↗

A morphological study of a human adenocarcinoma cell line (HT29) differentiating in culture. Similarities to intestinal embryonic development.

HT29 cells, a human adenocarcinoma cell line, when grown in Dulbecco's modified Eagle's medium (DMEM), form a multilayer of morphologically undifferentiated and unpolarized cells. However, when DMEM is replaced by RPMI medium, after 1-4 passages, a large amount of intracellular (ICL) and intercellular (ITCL) or secondary lumina (SL) are observed. These are detected in the light microscope and appear in the electron microscope as spherical structures embedded inside a multilayer of cells and bordered with microvilli. After 4-15 passages in RPMI, the cells retain the same pattern of cell growth but in addition exhibit apical brush-border microvilli and reveal a well developed belt of tight junctions. After 15 passages a single layer of polarized cells is clearly observed and a large number of 'domes' appeared. These results show that each of these culture types mimics morphologically specific stages described during intestinal ontogenesis between the 9th and the 16th week in the human embryo.

Adenocarcinoma↗

The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line.

The human colonic cancer cell line HT29, when grown in DMEM, forms a morphologically unpolarized cell culture in which the cells are covered with irregular microvilli and devoid of belt zonula occludens type tight junctions. However, by modifying the culture medium and growing the cells in RPMI, a different morphology was obtained. A large number of intracellular luminae appeared and at late confluency 90-95% of cells exhibited an apical brush border after four subsequent passages. Junctional complexes and a well developed zonula occludens were revealed under the apical brush border. Immuno-electron microscopical localization of specific markers, sucrase isomaltase (SI), secretory components (SC) and beta 2 microglobulin (beta 2M) showed that SI was limited to the apical surface, whereas 2M and SC were located at the basolateral surfaces. These results indicate that modification of culture conditions affects the ability of HT29 cells to express epithelial cell polarity.

Carcinoma↗

Effect of protein synthesis inhibitors on formation and degradation of tight junctions in HT 29 adenocarcinoma cells.

The human colon adenocarcinoma cell line HT 29 grows in DMEM virtually without tight junctions (TJ). Fascia occludens type TJ can be induced in these cells by treatment with a variety of proteases or with hypertonic ammonium sulfate solution. The induced formation of TJ is not affected by pretreatment of the cells with cycloheximide or puromycin. The induced TJ are almost completely degraded within 2 h at 37 degrees C both in the absence and presence of the inhibitors studied. With ammonium sulfate as the initial inducing agent, it was possible to induce a second round of TJ formation as early as 2 h after the initial treatment, i.e., immediately after the degradation of the TJ formed in the first round. The same result was obtained in cells treated with cycloheximide. Similar results were also obtained when TJ were initially induced by a very mild trypsin treatment. However, if the initial induction involved a more rigorous proteolytic treatment, the cells needed a recovery period of several h before TJ could be induced again. Under these conditions, recovery from the protease treatment was impaired by the addition of protein synthesis inhibitors at any time prior to complete recovery. It follows that proteolytic treatment of cells not only induces TJ formation but also destroys cell surface proteins which must be available for the formation of TJ strands. It seems possible that these proteins mediate cell adhesion events which may be a prerequisite for, but not a part of the actual TJ formation.

Adenocarcinoma↗

Rapid formation of tight junctions in HT 29 human adenocarcinoma cells by hypertonic salt solutions.

The human colon adenocarcinoma cell line HT 29 grows virtually without tight junctions (TJ) under standard culture conditions. Earlier studies have shown that focal TJ (fasciae occludentes) can be rapidly assembled in this cell line under the influence of various proteases. Here we show that focal TJ can be induced in this cell line by a brief treatment with appropriate salt solutions. Induction by ammonium sulfate in Hanks' buffer reached a maximum value after 15 to 30 min. The amount of TJ increased with the salt concentration and reached a plateau value at a concentration of 160 mM ammonium sulfate. The amount and complexity of TJ induced by ammonium sulfate were similar to those in experiments using trypsin as inducing agent as shown by morphometric analysis. At 0 degrees C, no TJ were formed under the influence of the salt. A comparative study of TJ induction using a variety of inorganic and organic salts gave the following results. All alkali sulfates induced TJ, although with different yield. Both calcium and magnesium chloride were potent inducers. Ammonium and sodium salts encompassing a variety of anions covered a wide range from maximum induction (sulfate, citrate) to almost complete absence of induction (nitrate). Sodium chloride did not induce any TJ. It follows that the induction of TJ is a specific effect of individual ionic components of the solution as opposed to a general effect of osmolarity and ionic strength. The data suggest tentatively that antichaotropic but not chaotropic ions have the potential to trigger the formation of TJ in this experimental system.

Adenocarcinoma↗

Proteinase-induced formation of focal tight junctions in HT 29 adenocarcinoma cells does not require extracellular calcium.

The human adenocarcinoma cell line HT 29 grows virtually without tight junctions, but the formation of focal tight junctions can be induced by brief treatment with proteinases. The freeze-fracture morphology of proteinase-induced tight junctions is not affected by treatment with EGTA or EDTA over a period of 30 min. The induction of tight junctions by trypsin or pronase can proceed in the presence of 3 mM EGTA or EDTA. Neither the formation nor the structure and complexity of the induced tight junctions is affected by the chelators. It follows that no extracellular divalent cations are required for the induced formation and the structural integrity of focal tight junctions in HT 29 cells.

Adenocarcinoma↗

ATP requirement for induced tight junction formation in HT 29 adenocarcinoma cells.

Tight junctions (TJ) of the fascia occludens type can be rapidly assembled in the human colon adenocarcinoma cell line HT 29 under the influence of trypsin or ammonium sulfate. We have studied the influence of the metabolic inhibitors, dinitrophenol (DNP) and deoxyglucose (DG), on the induced formation of TJ in this cell line. A reduction of the ATP level by DG treatment to 20% of control values did not affect the amount and complexity of induced TJ fibrils. However, under conditions of severe ATP depletion obtained by DNP, the velocity of TJ formation was substantially reduced, and the arrangement of the TJ fibrils as observed by freeze-fracture electron microscopy showed characteristic changes.

2,4-Dinitrophenol↗

Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation.

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacillus subtilis↗

Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line.

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.

Adenocarcinoma↗