Biomedical subjects

D Dagan

Publications and source records attributed to D Dagan.

At least 19 recordsLinked to original sources

PubMed2004

Single-trabecula building block for large-scale finite element models of cancellous bone.

Recent development of high-resolution imaging of cancellous bone allows finite element (FE) analysis of bone tissue stresses and strains in individual trabeculae. However, specimen-specific stress/strain analyses can include effects of anatomical variations and local damage that can bias the interpretation of the results from individual specimens with respect to large populations. This study developed a standard (generic) 'building-block' of a trabecula for large-scale FE models. Being parametric and based on statistics of dimensions of ovine trabeculae, this building block can be scaled for trabecular thickness and length and be used in commercial or custom-made FE codes to construct generic, large-scale FE models of bone, using less computer power than that currently required to reproduce the accurate micro-architecture of trabecular bone. Orthogonal lattices constructed with this building block, after it was scaled to trabeculae of the human proximal femur, provided apparent elastic moduli of approximately 150 MPa, in good agreement with experimental data for the stiffness of cancellous bone from this site. Likewise, lattices with thinner, osteoporotic-like trabeculae could predict a reduction of approximately 30% in the apparent elastic modulus, as reported in experimental studies of osteoporotic femora. Based on these comparisons, it is concluded that the single-trabecula element developed in the present study is well-suited for representing cancellous bone in large-scale generic FE simulations.

Animals↗

PubMed2002

Retrospective analysis of the first clonal outbreak of nalidixic acid-resistant Shigella sonnei shigellosis in Israel.

Reported here is a retrospective molecular analysis of the isolates recovered from the first outbreak of nalidixic acid (NA)-resistant Shigella sonnei shigellosis to occur in Israel. The outbreak affected 94 children. In the retrospective analysis, a total of 13 NA-resistant isolates and five NA-susceptible isolates recovered during the outbreak period were examined. Restriction fragment length polymorphism profiles obtained by digestion with BamHI, PvuI, HinfI or SmaI yielded identical profiles for all 18 isolates. All NA-resistant strains had an identical plasmid profile, but this profile differed from that displayed by the susceptible strains. In all of the NA-resistant strains a 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83-->Phe. In this outbreak, the isolates of NA-resistant Shigella sonnei belonged to a single clone and NA resistance was associated with a point mutation in the gyrA gene.

Adolescent↗

PubMed1999

Regulation of plasma membrane blebbing by the cytoskeleton.

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events.

Acrylamide↗

PubMed1999

A dynamically regulated 14-3-3, Slob, and Slowpoke potassium channel complex in Drosophila presynaptic nerve terminals.

Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

14-3-3 Proteins↗

PubMed1998

Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation.

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.

Aging↗

PubMed1998

Expression of two inward rectifier potassium channels is essential for differentiation of primitive human hematopoietic progenitor cells.

A potassium inward rectifier (K(ir)) current was previously shown by us to be induced in primitive hematopoietic progenitor cells, stimulated with the combination of interleukin-3 (IL-3) and stem cell factor (SCF). Biophysical features of whole cell currents implicated the involvement of more than one K(ir) channel type. Employing IL-3 + SCF stimulated human cord blood CD34+38- cells, we isolated and characterized different components of this current. Reverse transcription-polymerase chain reaction (RT-PCR) subcloning identified the expression of a strongly rectifying K(ir) channel (K(ir) 4.3) as well as a weakly rectifying K(ir) channel (K(ir) 1.1) in these cells. Inhibition of the expression of each of the channels suppressed progenitor cell generation by IL-3 and SCF-stimulated CD34+38- cells in 7-day suspension cultures. The variable expression of two essential inward rectifying potassium channels early in the course of hematopoietic progenitor cell differentiation may play a potentially important role in potassium homeostasis in these cells.

ADP-ribosyl Cyclase↗

PubMed1998

Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel.

Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins.

Amino Acid Sequence↗

PubMed1998

Microglia generate external proton and potassium ion gradients utilizing a member of the H/K ATPase family.

An ion selective electrode in self-referencing mode was employed to detect ionic concentration gradients at the vicinity of microglia isolated from newborn rat brains. At 5 mM extracellular potassium concentration, a gradient of -9.43+/-4.2 microM (n=48) was recorded dissipating over a distance of 10 microm from the outer surface of the cell membrane. Pharmacological studies indicated that neither the Na+/K+-ATPase nor the inward rectifier potassium channel makes significant contributions to generation of this gradient. The recorded potassium gradient was found to be augmented by increase in extracellular potassium or proton concentrations and could be inhibited by Omeprazole (10 microM) and by the specific H+/K+-ATPase blocker SCH28080 (1 microM). These, along with the coexistence of a gradient of excess of protons, strongly suggest that a K/H ATPase is the major generator of both the potassium and the proton gradients. The Kd of the glial transporter for K ions is an order of magnitude higher (3.7 mM) than that of the epithelial H+/K+-ATPase. This is a first report of an H+/K+ transporter in microglia cells with a Kd in the physiological range of [K+]out. Implications of the H+/K+-ATPase on potassium homeostasis in microglia under high extracellular potassium and low pH, as found at the site of brain injury, are discussed.

Animals↗

PubMed1997

Expression of voltage-gated potassium channels decreases cellular protein tyrosine phosphorylation.

Protein tyrosine phosphorylation by endogenous and expressed tyrosine kinases is reduced markedly by the expression of functional voltage-gated potassium (Kv) channels. The levels of tyrosine kinase protein and cellular protein substrates are unaffected, consistent with a reduction in tyrosine phosphorylation that results from inhibition of protein tyrosine kinase activity. The attenuation of protein tyrosine phosphorylation is correlated with the gating properties of expressed wild-type and mutant Kv channels. Furthermore, cellular protein tyrosine phosphorylation is reduced within minutes by acute treatment with the electrogenic potassium ionophore valinomycin. Because tyrosine phosphorylation in turn influences Kv channel activity, these results suggest that reciprocal modulatory interactions occur between Kv channel and protein tyrosine phosphorylation signaling pathways.

Cells, Cultured↗

PubMed1997

Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel.

The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src-induced suppression of current is accompanied by a robust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src-induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src-induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.

DNA, Complementary↗

PubMed1996

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors.

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.

Antigens, CD↗

PubMed1995

A novel cGMP-activated Cl- channel in renal proximal tubules.

Cl- channels activated by natriuretic peptides were detected in cultured rat proximal convoluted tubule (PCT) cells with the use of patch-clamp methodology. Bath application of atrial natriuretic peptide (ANP) activates a 150-pS Cl- channel with the open probability (Po) of the channel increasing from 0.0008 +/- 0.0003 to 0.021 +/- 0.008. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a membrane-permeable analogue of cGMP, increased channel activity in the on-cell mode. In inside-out patches the channel was activated by cGMP in a dose-dependent manner. Channel activity decreased after washing out and increased on reapplication of cGMP. A similar activation was observed also in presence of either of two protein kinase inhibitors, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride or KT5823, or a phosphatase inhibitor. Bath application of urodilatin mimicked the action of ANP. Po of the channel was found to be independent of both voltage and Ca2+, and gating activity could be blocked by the stilbene, 4,4-dinitrostilbene-2,2-disulfonic acid. These results demonstrate a Cl- conductance in PCT cells modulated by ANP and urodilatin via their second messenger, cGMP.

Animals↗

PubMed1994

Diverse modulations of chloride channels in renal proximal tubules.

Cl- selective channels were detected and characterized in apical membranes of cultured rat renal proximal convoluted tubule cells (PCT) using patch-clamping methods. Subpopulations of Cl- channels modulated by cyclic nucleotides, Ca2+, or voltage were identified. Two different 30-pS, voltage-independent, Cl- channels modulated by adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ were seen most frequently. The cAMP-dependent channel was activated by membrane-permeable analogues of cAMP, dibutyryl-cAMP or 8-bromo-cAMP. Catalytic subunit of protein kinase A (PKA) applied to detached inside-out patches, activated the channel as well, suggesting activation via phosphorylation. Channel activity was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, by 4,4-dinitrostilbene-2,2-disulfonic acid, and by SCN-. Permeability sequence for different halides was Cl- > I > F with a Cl(-)-to-cation permeability ratio (PCl/Pcation) of 7:1. The Ca(2+)-sensitive channel was not activated by cAMP nor by PKA. A third anionic selective channel encountered infrequently is voltage dependent and has a unitary conductance of 145 pS, with a PCl/Pcation value of 9:1. This diversity of Cl- channels may underlie the rich repertoire of physiological functions attributed to Cl- channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Lysis of blood platelets by human osteosarcoma cells in vitro.

The in vitro interactions between human osteosarcoma (HOS) cells and platelets were studied in real time using video-enhanced microscopy. Interference reflection techniques showed that platelets were lysed within minutes after contacting HOS cells that had been treated with interferon-gamma. Untreated HOS cells lysed platelets less efficiently. Platelet lysis depended on platelet-tumor cell contact and on extracellular Ca2+. A number of possible mechanisms were excluded. Lysis of platelets in proximity to tumor cells can provide these with growth factors and thereby contribute to the metastasis-enhancing effect of platelets.

Blood Platelets↗

PubMed1993

Properties and modulation of a calcium-activated potassium channel in rat olfactory bulb neurons.

1. Single calcium-activated potassium channels (KCa channels) were recorded from membrane patches of rat olfactory bulb neurons in culture. Only one kind of KCa channel was seen, and it was present in approximately 50% of detached patches. 2. This channel, like maxi-KCa channels of other tissues, had a single-channel conductance of 270 pS, a reversal potential (Erev) of 0 mV in symmetrical K+, and was highly selective for K+ over Na+ and Cl-. 3. The KCa channel was blocked by d-tubocurarine (d-TC) on the cytoplasmic side, and charybdotoxin (CTX) on the extracellular side. This pharmacology is identical to that of one type of KCa channel from rat brain, observed previously in artificial bilayers and called the type 1 KCa channel. 4. The probability that the channel was in the open state (Po) increased with membrane depolarization. The position of the Po versus transmembrane voltage (Vm) curve was shifted by changes in [Ca2+]i so that the channel was open more often in higher [Ca2+]i. The gating kinetics resembled those of the type 1 KCa channel observed in bilayers. 5. Po was increased after superfusion of the cytoplasmic membrane surface with the active catalytic subunit of cyclic AMP-dependent protein kinase (PK-A), together with MgATP. Phosphorylation altered the distribution of channel closed times but had little effect on open times. The results suggest that phosphorylation is an important molecular mechanism in modulating the activity of this KCa channel from mammalian brain.

Animals↗

PubMed1992

Na(+)-activated K+ channels are widely distributed in rat CNS and in Xenopus oocytes.

We recorded the activity of K+ channels activated by sodium (KNa channels) in two widely used preparations, primary cell cultures prepared from neocortex, cerebellum, midbrain, brainstem and spinal cord, and Xenopus oocytes. KNa channels from all regions shared an absolute dependence on [Na+], had conductances of 140-170 pS in symmetrical 150 mM K+ and exhibited characteristic substates. The role of this channel must now be considered in terms of its widespread distribution.

Animals↗

PubMed1992

Directional control of neurite outgrowth from cultured hippocampal neurons is modulated by the lectin concanavalin A.

Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclockwise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar alpha-methyl-D-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counterclockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth.

Actins↗

PubMed1992

Release of endosomal content induced by plasma membrane tension: video image intensification time lapse analysis.

Rhodamine-labeled vinculin microinjected into chicken embryo fibroblasts and rhodamine-labeled alpha 2-macroglobulin added to the fibroblast culture medium were sequestered in endocytotic vesicles and digested. When a sealed microcapillary coated with fibronectin or polylysine was attached to the fibroblasts and pulled at speeds of 100-200 microns/h, stretching the plasma membrane, a variable fraction of the endosomes released the rhodamine label. Release from individual vesicles was rapid, reaching completion in less than 30 to 120 s. The microfilament disrupting agent cytochalasin B prevented release, as did the microtubule stabilizing drug taxol. Colcemide, which disrupts microtubules, did not inhibit the release. Release was dependent on extracellular calcium, as it was prevented by 10 mM EGTA in the incubation medium. We postulate that opening of vesicular channels consequent to centripetal transmission of tension generated in the plasma membrane along microfilaments may be a mechanism of release of endosomal content.

Alkaloids↗