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Biomedical subjects

M W Cohen

Publications and source records attributed to M W Cohen.

At least 37 records · Page 2Linked to original sources
PubMed1990

Selenium and immune cell functions. I. Effect on lymphocyte proliferation and production of interleukin 1 and interleukin 2.

The dietary intake of selenium (Se) has been shown to influence the development and expression of various biologic processes. This study examined the immunologic competence of lymphocytes from C57BL/6J mice maintained for 8 weeks on Se-deficient (0.02 ppm Se), normal (0.20 ppm Se, as sodium selenite), or Se-supplemented (2.00 ppm Se) Torula yeast-based diets. The ability of the cells to recognize alloantigens, to proliferate in response to stimuli, and to produce interleukin 2 (IL-2) was determined. Se deficiency significantly inhibited the ability of the lymphocytes to proliferate in response to allogeneic stimulation in the mixed lymphocyte reaction or to mitogen stimulation by phytohemagglutinin, whereas Se supplementation significantly enhanced both responses. In contrast, the amounts of IL-2 and interleukin 1 (IL-1) produced by lymphocytes and macrophages, respectively, removed from Se-deficient or Se-supplemented animals did not differ significantly from the amounts of IL-2 and IL-1 produced by cells removed from animals maintained on the control diet. These results suggest that the mechanism(s) responsible for the observed effects of Se on lymphocyte proliferation are independent of the levels of IL-2 or IL-1.

Animals↗
PubMed1990

Selenium and immune cell functions. II. Effect on lymphocyte-mediated cytotoxicity.

Selenium (Se) is an essential nutritional factor with a chemopreventive potential. This study examined the ability of C57BL/6J mice, maintained for 8 weeks on Se-deficient (0.02 ppm Se), normal (0.20 ppm Se), or Se-supplemented (2.00 ppm Se) Torula yeast-based diets, to generate cytotoxic lymphocytes (CTL) and to destroy tumor cells. CTL were generated in vivo by intraperitoneal immunization with P815 cells and in vitro by allogeneic stimulation of cells from animals maintained on a normal diet in media supplemented with 1 x 10(-9) to 1 x 10(-6) M Se (as selenite). Lymphocytes from animals maintained on the Se-supplemented diet had a greater ability to destroy tumor cells than lymphocytes from animals maintained on the normal diet, whereas Se deficiency reduced the cytotoxicity. The effects on cytotoxicity were accompanied by parallel changes in the levels of lymphotoxin produced. The greatest enhancement of tumor cytodestruction occurred with supplementation of 1 x 10(-7) M Se, whereas with 1 x 10(-6) M there was inhibition of the cytotoxic responses. The stimulatory effect of Se occurred during the phase of CTL generation rather than during the lytic phase of cytotoxicity. These results indicated that Se supplementation enhances CTL generation and the ability of a host to destroy malignant cells, whereas Se deficiency has the opposite effect.

Animals↗
PubMed1990

Intracellular and surface acetylcholine receptors during the normal development of a frog skeletal muscle.

125I-alpha-bungarotoxin (125I-alpha BT) was used to measure the pool sizes of surface and intracellular acetylcholine receptors (AChRs) in the myotomal muscle of Xenopus laevis over a developmental period (stages 23-48; 1.03-7.5 d) which ranged from initial to mature stages of neuromuscular synaptogenesis. The surface pool increased progressively throughout development. The intracellular pool increased more slowly and also underwent a transient decrease. Linear regression indicated that AChRs begin to appear intracellularly and in the surface membrane at embryonic ages of 13.2 and 18.5 hr, respectively. The findings also suggest that newly synthesized AChRs contribute much more to the intracellular pool than do AChRs internalized from the surface membrane and that the rates of supply and/or intracellular resident times of these 2 sources of intracellular AChRs change during the course of normal development. Carbamylcholine, even at concentrations 10-fold greater than needed to block completely 125I-alpha BT binding to surface AChRs, blocked specific intracellular binding by only 80%. Considered in the light of previous studies on cell cultures, these results suggest that 20% of the intracellular sites are on alpha-subunits not yet assembled into pentameric AChRs. Light microscope radioautography revealed an essentially uniform distribution of intracellular AChRs along the length of the muscle cells. It is concluded that during the normal development of Xenopus myotomal muscle the accumulation and maintenance of AChRs in the postsynaptic membrane occurs in the absence of any preferential concentration of intracellular AChRs in the subsynaptic region.

Animals↗
PubMed1987

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures.

The capacity of individual muscle cells and neurons to establish synaptic specializations along the entire extent of their neurite-muscle contacts was assessed in culture. Spinal cord neurons derived from embryos of Xenopus laevis were plated at low density in cultures of Xenopus myotomal muscle cells in order to obtain isolated motor units whose neuron and muscle cells were not contacted by any other neuron. These isolated motor units were examined for localization of acetylcholine receptors (AChRs) after staining with fluorescent alpha-bungarotoxin and, in some cases, for localization of a synaptic vesicle antigen by immunofluorescence. The neurite-muscle contacts formed by competent neurons exhibited discontinuous sites of AChR localization occupying about 25% of the contact length as compared with 2% for incompetent neurons. Competent neurons, unlike incompetent ones, established these neurite-associated receptor patches (NARPs) on virtually all the muscle cells they contacted and functionally innervated them. These and other observations on the distribution of NARPs throughout the isolated motor units indicate that the capacity of competent neurons to establish NARPs extends to the limits of growth of most if not all of their neurites, that this capacity is least in the most proximal portions of initial neuritic segments, and that the overall capacity of muscle cells to generate NARPs can be saturated by long lengths of neurite-muscle contact. The results also suggest that even in the absence of competitive interactions between neurons there are spatial discontinuities in neuritic action and/or muscle response during the establishment of NARPs. Synaptic vesicle antigen patches (SVAPs) occurred along the neuritic arbor of all neurons, but their distribution in competent neurons (those which established NARPs) and in incompetent ones differed. For competent neurons the percentage of neurite length occupied by SVAPs was 4.8-fold greater on muscle cells than off, whereas the corresponding value for incompetent neurons was only 1.5-fold. This large preferential localization of SVAPs along neurite-muscle contacts of competent neurons was further associated with a colocalization of SVAPs and NARPs that was greater than predicted by chance. These results suggest that muscle cells are much more effective in influencing the distribution of synaptic vesicles along the neuritic arbor of competent neurons than along the arbor of incompetent neurons and that this influence is greatest at sites of postsynaptic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Localization of acetylcholine receptors and cholinesterase on nerve-contacted and noncontacted muscle cells grown in the presence of agents that block action potentials.

The possible role of nerve activity in triggering changes in the localization of acetylcholine receptors (AChRs) and cholinesterase (ChE) on nerve-contacted Xenopus muscle cells has been assessed. The localization of these molecules was examined on nerve-contacted and noncontacted muscle cells in cultures of spinal cord and myotomal muscle derived from Xenopus embryos. Sites of high AChR density were revealed by staining with fluorescent alpha-bungarotoxin and sites of ChE localization were revealed histochemically. Localization of AChRs and ChE at sites of nerve-muscle contact occurred when the culture medium contained 1.2 micron tetrodotoxin (TTX), 1.2 micron TTX, 10 mM magnesium, and no calcium salts, 1.2 micron TTX and 2 mM manganese, or 106 mM potassium methyl sulfate instead of sodium chloride. The nerve-contacted muscle cells in each of these modified culture media also exhibited a reduced incidence of AChR and ChE patches away from the site of contact. It is concluded that the neural factor(s) that triggers the local and remote changes in AChR and ChE distribution can be supplied to the neurites and externalized in the absence of nerve impulses, and that the nerve and muscle cells can interact even when they are largely depolarized.

Action Potentials↗
PubMed1984

In vivo development of cholinesterase at a neuromuscular junction in the absence of motor activity in Xenopus laevis.

Embryos of Xenopus laevis were selected prior to the onset of innervation and were raised for 2 days in the anaesthetic tricaine methanesulphonate (200 micrograms/ml). The gross development of these tricaine-reared animals appeared normal despite the absence of spontaneous motor activity and the lack of motor responses to prodding with a pin. Motor activity quickly appeared when the anaesthetic was withdrawn. Intracellular recording from the myotomes of intact, tricaine-maintained animals failed to reveal any spontaneous muscle action potentials. Synaptic potentials increased in frequency and amplitude upon withdrawing tricaine, but resting potentials remained unchanged. Cholinesterase activity, detected histochemically, was observed at the ends of the myotomes, the main site of innervation. The intensity of the histochemical reaction product at these sites appeared to be about as great in the myotomes of tricaine-reared animals as in control myotomes. Miniature end-plate currents (m.e.p.c.s), examined by focal external recording, declined with a time constant of 2.9 +/- 0.2 ms (mean +/- S.E. of mean) in the myotomes of tricaine-reared animals (stages 40-41). The time constants in the myotomes of control animals were 1.8 +/- 0.1 ms at stages 40-41 and 8.7 +/- 0.7 ms at stages 24-26 (shortly after the onset of innervation). The anticholinesterase neostigmine doubled m.e.p.c. time constants in the myotomes of tricaine-reared animals as well as in control myotomes at stages 40-41. It is concluded that motor activity is not required for the in vivo development of physiological levels of synaptic cholinesterase in Xenopus myotomal muscle.

Aminobenzoates↗
PubMed1983

Developmental changes in the distribution of acetylcholine receptors in the myotomes of Xenopus laevis.

The acquisition and distribution of nerve fibres and of acetylcholine (ACh) receptors were examined in the myotomes of Xenopus laevis during normal development. This muscle is well-suited for investigating temporal relationships during neuromuscular synaptogenesis because the age of the Xenopus embryo at the onset of innervation can be assessed with an accuracy of about one hour. Myotomal nerve fibres were visualized after staining them with nitroblue tetrazolium and ACh receptors were examined after exposure to alpha-bungarotoxin labelled with 125I or fluorescent dye. Nerve fibres were seen in the myotomes of some embryos as early as stage 19 (20 . 75 hr) and in virtually all embryos by stage 24 (26 . 25 hr). From the outset they were located mainly at the ends of the myotomes, but some myotomes also exhibited nerve fibres in more central regions. ACh receptors were already present in myotomes by stage 19 (20 . 75 hr) and initially had a widespread, uniform distribution. The density of extrajunctional ACh receptors increased until stage 36 (50 hr) and then declined less than 3-fold over the next 10 days of development. Discrete patches of high ACh receptor density began to appear at the ends of the myotomes at stage 22 (24 hr) and were seen in almost all embryos by stage 26 (29 . 5 hr). ACh receptor patches were also seen in central regions of some myotomes and these were usually aligned in patterns which resembled the course of nerve fibres. The present findings suggest that myotomal muscle cells in Xenopus embryos begin to acquire ACh receptors shortly before the arrival of nerve fibres and that discrete patches of ACh receptors begin to form at presumptive synaptic sites on the average about 3 hr after the arrival of the nerve fibres. The latter delay is considerably shorter than that in developing rat muscle. The temporal and spatial relationships between nerve fibres and the development of ACh receptor patches in Xenopus myotomes in vivo are consistent with findings in Xenopus cell cultures which indicate that nerve fibres can rapidly induce ACh receptor localization at sites of nerve--muscle contact.

Acetylcholine↗
PubMed1982

Cholinesterase localization at sites of nerve contact on embryonic amphibian muscle cells in culture.

Cholinesterase (ChE), detected histochemically, was found to be localized at many sites of nerve-muscle contact in cultures of spinal cord and muscle cells derived from Xenopus laevis embryos. Such contacts were often characterized by a corresponding localization of acetylcholine receptors and by synaptic ultrastructure, including aggregates of clear vesicles in the nerve fibre and an 80-100 nm wide intercellular cleft. The ChE reaction product was localized in the cleft. When cultures were grown in the presence of curare many of the nerve-contacted muscle cells still exhibited ChE at the sites of contact. It is concluded that ChE accumulates at synaptic contacts in these cultures even in the absence of muscle action potentials and contraction.

Animals↗
PubMed1982

Influence of nerve on the formation and survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus muscle cells in culture.

Embryonic Xenopus muscle cells grown in culture develop discrete patches of high acetylcholine receptor (AChR) density. By following identified muscle cells after staining with fluorescent alpha-bungarotoxin, we have found that many of these AChR patches survive in a fixed position for several days. For AChR patches on the lower surface of the cell (the surface apposed to the culture dish), more than 60% of those which were followed beginning on day 2 survived for a further 4 days. The survival rate was greater when patches were followed from day 3 or later and was almost as high for AChR patches on the upper surface. New AChR patches also formed on all of the muscle cells. When muscle cells were cultured together with spinal cord cells, nerve-muscle contacts developed with a characteristic localization of AChRs along the path of contact. AChR patches did not form elsewhere on these contacted cells. Nerve-contacted muscle cells examined 2 to 3 days after adding spinal cord cells to established (2- to 5-day-old) muscle cultures also exhibited a marked reduction of AChR patches away from the site of contact. This reduction was not due to the nerve having contacted pre-existing AChR patches. Rather, the findings indicate that contact by an appropriate nerve inhibits the formation of AChR patches elsewhere on the contracted muscle cells and reduces the survival of pre-existing AChR patches. Nerve contact also inhibited the formation of cholinesterase (ChE) patches remote from the site of contact and appeared to cause some reduction in the survival of pre-existing ChE patches. Spontaneous twitching was not observed in these experiments, thereby indicating that the remote effects of nerve contact were not mediated by muscle action potentials or contraction. Such remote influences of the nerve may play a role in determining the pattern of innervation on individual muscle cells.

Animals↗
PubMed1982

Increased adhesiveness at sites of high acetylcholine receptor density on embryonic amphibian muscle cells cultured without nerve.

In culture, myotomal muscle cells from Xenopus laevis embryos develop discrete patches of high acetylcholine receptor (AChR) density. To examine the relative adhesiveness of these sites, muscle cells having AChR patches on their lower surface (apposed to the culture dish) were identified and were then treated with dibucaine or potassium-Ringer in order to cause the cells to round up. More than 90% of these cells remained attached at an AChR patch after rounding up, and this was the case even when the cells had a single patch on their lower surface. When the cells were torn away from the culture dish by mechanical agitation, small cellular fragments still remained firmly attached to the dish and many of these fragments contained an AChR patch. It is concluded that AChR patches on the lower surface of the cell are often located at sites of increased adhesiveness to the culture dish. The findings are discussed in terms of the formation and maintenance of AChR patches.

Animals↗
PubMed1981

Localization of cholinesterase at sites of high acetylcholine receptor density on embryonic amphibian muscle cells cultured without nerve.

Myotomal muscle cells, derived from Xenopus embryos and grown in culture without nerve, develop discrete sites of cholinesterase (ChE) activity on their surface. The spatial relationship of these ChE patches to surface patches of acetylcholine receptors (AChRs) has been examined in the present study by a combination of ChE histochemistry and fluorescent staining of the receptors. ChE patches and AChR patches developed as early as the 1st day in culture and exhibited a high incidence of spatial overlap. The frequency of overlap varied with the age of the culture and ranged from 50 to 98% for patches on the lower cell surface (facing the floor of the culture dish) and from 28 to 79% for patches on the upper cell surface. The high incidence of overlap cannot be explained on the basis of a random distribution of patches since both types of patch occupied less than 3% of the cell surface. ChE and AChR patches also developed when cultures were grown in a serum-free medium as well as when cultures were prepared from young embryos in which muscle innervation had not yet begun. At some patches, the surface membrane was invaginated and at these invaginations, there was also a high incidence of overlap between the ChE and AChR stains. It is concluded that the mechanisms involved in the localization of AChRs and ChE on the surface of Xenopus myotomal muscle cells tend to be closely linked and operate even in the absence of innervation, previous contact by nerve, or electrical and contractile activity. Considered together with previous ultrastructural observations, the present findings suggest that these cells develop elaborate postsynaptic-like specializations even in the absence of neural factors.

Animals↗
PubMed1980

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type.

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent alpha-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.

Animals↗