Search PubMed⌕ Search

Biomedical subjects

M Henkart

Publications and source records attributed to M Henkart.

At least 19 recordsLinked to original sources
PubMed1987

Inhibition of NK and ADCC activity by antibodies against purified cytoplasmic granules from rat LGL tumors.

Highly purified preparations of cytoplasmic granules from transplantable rat large granular lymphocyte (LGL) tumor lines (rat natural killer (RNK) tumors) were used to immunize rabbits. Antibodies from these animals gave two precipitin lines with granule extracts in Ouchterlony experiments. They reacted with at least four different bands on nitrocellulose blots of SDS gels of LGL granule proteins. By immunofluorescence, specifically adsorbed antigranule antibodies did not recognize LGL or T cell surface antigens but reacted with the cytoplasmic granules in permeabilized RNK tumor cells as well as with normal rat LGL. These same antisera showed little or no reactivity with a panel of other cells, including peripheral blood T cells, thymocytes, macrophages, and EL-4 tumor cells. F(ab')2 preparations of these antigranule antibodies completely blocked granule-mediated lysis of both SRBC and nucleated targets, while control F(ab')2 preparations from rabbits immunized with EL-4 granules or TNP-KLH showed no significant inhibition of this cytolytic activity at the same antibody concentration. Anti-granule F(ab')2 preparations specifically inhibited (greater than 75%) rat natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in a dose-dependent manner but did not effect cytotoxic T cell activity. Pretreatment of either effectors or targets by these antibodies had no effect. Anti-granule F(ab')2 preparations, at concentrations showing strong inhibition of lysis, did not inhibit the binding of LGL to YAC-1 or Ab-coated P815 targets. These results demonstrate that a granule component(s) is necessary for the lytic activity of LGL in both NK and ADCC and provide the first direct evidence that a secretory event involving these granules is part of the lytic process.

Animals↗
PubMed1987

Secretory processes in lymphocyte function.

The secretion of immunoglobulin by plasma cells has been considered a classical example of the "non-regulated" pathway of protein secretion, in which newly synthesized protein is processed by the Golgi, packaged into small vesicles, and immediately secreted without intracellular storage. In the case of lymphokine secretion by T lymphocytes, it is generally not clear whether this non-regulated pathway is also being used, as opposed to the "regulated" pathway which has been proposed to operate in the cytotoxic lymphocyte mechanism. In this case, as in mast cells and endocrine cells, proteins are synthesized and then stored in cytoplasmic granules. The secretion is triggered (regulated) by a membrane receptor-ligand interaction, which for the cytotoxic lymphocytes is part of the target cell binding process. In cytotoxic T lymphocytes, this secretion process can be measured by following the appearance of a granule serine protease in the medium, and it has been shown to be triggered by target cells or by immobilized antibodies which bind the T cell receptor complex. In addition to cytotoxic lymphocytes, cloned T helper cells contain this serine protease in cytoplasmic granules with a low internal pH. Helper lymphocytes secrete this enzyme in response to (1) soluble antigen which has been processed by cells bearing the appropriate MHC antigens; (2) immobilized antibodies against the T cell receptor complex; (3) a combination of phorbol ester and calcium ionophore. Thus in both helper and cytotoxic lymphocytes, the regulated pathway of protein secretion clearly operates after triggering by the T cell antigen receptor.

Animals↗
PubMed1984

Reconstitution of the receptor for immunoglobulin E into liposomes. Reincorporation of purified receptors.

Mast cells and related cells have on their surface receptors that bind immunoglobulin E (IgE) with high affinity and which, when aggregated, trigger exocytosis. We recently demonstrated that when these receptors are solubilized with mild detergents, their subunits dissociate unless an appropriate lipid:detergent ratio is maintained. The conditions required to maintain the receptors' integrity appeared to parallel those previously determined as necessary to obtain adequate incorporation of unpurified IgE-receptor complexes from detergent extracts into liposomes. We now show that purified IgE-receptor complexes having the full complement of subunits become preferentially inserted into liposomes. If the receptor subunits are chemically cross-linked to each other, at least some of such receptors can be incorporated, even though lipid is omitted during their purification. The findings suggest that the IgE-binding alpha subunit of the receptor is anchored to the bilayer by means of one or both of the other subunits.

Animals↗
PubMed1984

Role of microtubules in the organization and localization of the Golgi apparatus.

Normal interphase PtK2 and A549 cells display long microtubules radiating from the microtubule-organizing center (MTOC) to the plasma membrane. Both MTOC and Golgi apparatus are contained in the same perinuclear area. Treatment of cells with 1 microM colcemid for 2 h results in microtubule depolymerization and fragmentation of the Golgi apparatus into elements scattered throughout the cytoplasm. Both normal microtubules and the Golgi apparatus assemble again following removal of colcemid. Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'(alpha, beta-methylene)diphosphate [pp(CH2)pG], into interphase cells growing in normal medium results in the formation of microtubule bundles resistant to colcemid and prevents the fragmentation of the Golgi apparatus. Injection of pp(CH2)pG into cells incubated with colcemid results in substitution of tubulin ribbons for microtubules and has no effect on the Golgi-derived elements scattered throughout the cytoplasm. Removal of colcemid 1 h after the injection of pp(CH2)pG results in polymerization of large numbers of short, single randomly oriented microtubules, whereas the Golgi apparatus remains fragmented. Treatment of cells with 10 microM taxol for 3 h results both in polymerization of microtubule bundles without relation to the MTOC in the cell periphery and fragmentation of the Golgi apparatus. The Golgi-derived fragments are present exclusively in regions of the peripheral cytoplasm enriched in microtubules. The codistribution of microtubules and Golgi elements can be reversed in taxol-treated cells by injection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin. Cells incubated with colcemid after treatment with taxol have large numbers of Golgi-derived elements in close association with colcemid-resistant microtubule bundles. Incubation of cells with 50 microM vinblastine for 90 min results in microtubule dissembly, formation of tubulin paracrystals, and fragmentation of the Golgi apparatus into elements without relation to the tubulin paracrystals.

Alkaloids↗
PubMed1984

Potassium current in clonal cytotoxic T lymphocytes from the mouse.

The electrical properties of the cell membrane of clonal cytotoxic T lymphocytes in the mouse were studied by using the whole cell variation of the patch electrode voltage-clamp technique. Outward currents were activated with an exponential time course of several milliseconds time constant when the membrane potential was made more positive than -50 to -40 mV. This current is not activated as a result of Ca2+ entry. The estimated reversal potential of the current indicates that the current is predominantly carried by K+. The activation kinetics depend only on membrane potential, not on [K+]0. The amplitude of the current decreases exponentially with time constants of several hundred milliseconds during a maintained voltage pulse, due mainly to a decrease in conductance. Recovery from inactivation roughly followed a single exponential time course with a time constant of tens of seconds; this time constant depended upon not only the membrane potential but also the amount of initial inactivation. The current is suppressed by quinidine and tetraethylammonium, their half-suppression concentrations being 23 microM and 14 mM respectively. An increase of the outward current is suggested to be associated with the lethal hit of the cytotoxic reaction.

Action Potentials↗
PubMed1983

Clathrin-induced pH-dependent fusion of phosphatidylcholine vesicles.

Interaction of clathrin coat protein with dioleoyl-phosphatidylcholine (DOPC) vesicles at pH 6.5 and below results in the formation of stable vesicle-clathrin complexes (Steer, C. J., Klausner, R. D., and Blumenthal, R. (1982) J. Biol. Chem. 257, 8533-8540). In this report we show by gel chromatography and sedimentation analysis that the interaction of clathrin coat protein with unilamellar dioleoyl phosphatidylcholine vesicles at pH = 6.0 results in the formation of larger structures. As shown by electron microscopy and an increase in trapped volume of both sucrose and inulin those larger structures represent fused bilayers. We examined the mixing of membrane lipid as a result of membrane fusion using resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle membrane. At a protein:lipid ratio of 1:500 there was 50% vesicle-vesicle fusion, at pH 6.0, as indicated by the change in efficiency of energy transfer between the fluorescent probes. Fusion was completed within 60 s. A number of other proteins (ovalbumin, rabbit IgG, trypsin, pronase, calmodulin, tubulin, synexin, bovine serum albumin) at 10-fold or higher concentrations, did not induce fusion of dioleoyl phosphatidylcholine vesicles, either at pH 7.4 or at pH 6.0. This system provides a model for pH-dependent and protein-mediated fusion of uncharged lipid bilayers.

Animals↗
PubMed1983

Role of microtubules in the distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies.

Immunofluorescence microscopy reveals that both microtubule organizing center (MTOC) and Golgi apparatus are contained in the same perinuclear area of A549 cells in interphase. The cells display long microtubules stretching radially from the MTOC to the plasma membrane. Treatment of cells with taxol results in polymerization of microtubules without relation to the MTOC and formation of microtubule bundles predominantly localized in the cell periphery. After incubation with taxol, the Golgi apparatus is fragmented and is conspicuously present in areas of the cytoplasm enriched in microtubules. Incubation of cells with Colcemid results in complete depolymerization of microtubules and fragmentation of the Golgi into elements randomly distributed throughout the cytoplasm. Cells treated with taxol before being incubated with Colcemid contain large numbers of Golgi-derived elements in close association with Colcemid-resistant microtubules. Microtubule depolymerization by vinblastine also is followed by fragmentation of the Golgi apparatus. These Golgi-derived elements show no association with the atypical polymers of tubulin induced by vinblastine. The codistribution of Golgi-derived elements with taxol-induced microtubule bundles can be reversed by microinjection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin.

Alkaloids↗
PubMed1982

Aggregation and calcium-induced fusion of phosphatidylcholine vesicle-tubulin complexes.

Insertion of tubulin into the bilayer of dipalmitoyl phosphatidylcholine vesicles at the phase transition results in the formation of stable vesicle-tubulin complexes (Klausner, R. D., Kumar, N., Weinstein, J. N., Blumenthal, R., and Flavin, M. (1981) J. Biol. Chem. 256, 5879-5885). These complexes aggregated when maintained below phase transition for 10-20 min. Addition of millimolar concentrations of Ca2+, Mn2+, Zn2+, and Co2+, but not Mg2+, caused the vesicle-tubulin complexes to fuse into larger structures as shown by (a) electron microscopy, (b) increased trapped volume, and (c) changes ion resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle. There was no loss of internal aqueous contents from the vesicle-tubulin complexes during Ca2+-induced fusion. Anti-tubulin drugs had no effect on the aggregation or fusion, and vesicle-bound tubulin did not associate with microtubules when tubulin was assembled in vitro. Trypsin-treated vesicle-tubulin complexes were incapable of supporting Ca2+-induced fusion. This system provides a model for Ca2+-induced and protein-mediated nonleaky fusion of uncharged lipid bilayers.

Animals↗
PubMed1980

Identification and function of intracellular calcium stores in axons and cell bodies of neurons.

In many nerve cells and axons there are specialized appositions (subsurface cisterns) between the surface membrane and endoplasmic reticulum (ER) that closely resemble appositions between the sarcoplasmic reticulum (SR) and surface membrane of muscle (triads, diads). Their presence suggests that the function of the ER of neurons may be similar to the function of the SR of muscle. The squid axon, when loaded with calcium by physiological means and prepared for electron microscopy by rapid freezing and freeze-substitution, contains electron-opaque deposits in the ER as well as in mitochondria. These deposits contain Ca as identified by electron probe X-ray microanalysis. Lightly loaded axons contain smaller amounts of deposits. The ER of axons not specifically loaded with Ca contain small amounts of electron-opaque material while the mitochondria generally do not. Ca-containing deposits were found in the ER and certain other organelles of neurons of Aplysia and ganglion cells and neurites of rat sympathetic ganglia in culture when they were prepared by a modified fixation procedure. Thus, the ER of neurons probably is a Ca-sequestering compartment. Some indirect evidence suggests that Ca may be released from intracellular stores in response to surface membrane stimuli.

Animals↗
PubMed1978

Formation of synapses between cells of a neuroblastoma X glioma hybrid clone and mouse myotubes.

Synapses form between cells of a neuroblastoma X glioma hybrid clone and cultured mouse skeletal myotubes. The synapses are cholinergic, and the acetylcholine release mechanism is dependent on calcium ions. The transmitter output of the synapses is low, with considerable variability in the latency and amplitude of the postsynaptic responses to presynaptic action potentials. The fine structure of physiologically identified functional junctions was examined electron microscopically. Small (50 nm) clear vesicles were seen presynaptically and there were areas with a wide (approx. 50 nm) gap containing basement membrane-like material between the pre- and postsynaptic cells. In addition, in some regions there was a densely staining material lining the muscle membrane and some suggestion of infolding of the muscle membrane. In none of the cases, however, have areas been found where small, clear vesicles cluster around pre- and postsynaptic membrane densities. Thus, functional synapses can occur in the absence of the highly organized synaptic structure seen at mature synapses.

Action Potentials↗
PubMed1977

Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties.

1. Reliable methods for establishing fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) cells in long term (greater than 1 mo) dissociated cell cultures are described. These cells have been studied by morphologic and intracellular electrophysiologic techniques. 2. Cells studied electrophysiologically can be relocated after preparation for electron microscopy and examined in thin sections. The electron microscope shows that the surface membranes of these cells were directly accessible to the culture medium. The surfaces of SC cells were studded with synaptic boutons, whereas the DRG cell surfaces generally had none. 3. Current-voltage relationships and linear electrotonic properties of the neurons are described. Delayed and anomalous rectification were seen in both cell types. The length of SC cell dendrites was about one characteristic electrotonic length, while little or no contribution of the relatively sparse DRG cell processes was seen in the transient responses of the DRG cells. 4. Postspike and posttetanic hyperpolarizations in DRG cells were due to a surface membrane conductance increase; this was probably primarily an increase in K+ conductance. Post-activation hyperpolarization in SC cells was primarily due to activation of an electrogenic Na+ pump.

Animals↗
PubMed1976

Localization of calcium binding sites associated with the calcium spike in barnacle muscle.

La ion behaves as a competitive inhibitor of Ca ions on the calcium spike in the giant muscle fiber of the barnacle, Balanus nubilus. La-treated muscle fibers, in which the rate of rise of the spike was diminished to a known degree, have been examined with the electron-microscope. In such fibers dense particles are seen in association with the surface membrane and external lamina of the cell. La particles are not seen in association with fibers that have been allowed to recover from La inhibition before fixation. The number of La particles seen in association with the muscle fiber increases with increasing La concentration when the Ca and Mg concentrations are held constant and decreases with increasing Ca and Mg concentration when the La concentration is held constant. The results suggest that the La visible in the electron-microscope under the conditions of these experiments is bound to a class of sites similar to those involved in the Ca spike.

Animals↗
PubMed1976

Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle and neurons.

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate and Purkinie cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.

Animals↗