Search PubMed⌕ Search

Biomedical subjects

D C Merz

Publications and source records attributed to D C Merz.

At least 19 recordsLinked to original sources
PubMed1995

Tyrosine phosphorylation during synapse formation between identified leech neurons.

1. We have examined whether tyrosine phosphorylation is required for synapse formation between identified neurons from the central nervous system of the leech in culture. 2. Within a few hours of contact with the cell body of the serotonergic Retzius neuron (R cell), the soma of the postsynaptic pressure-sensitive neuron (P cell), but not the R cell, could be labelled intracellularly with an antibody against phosphotyrosine residues. The labelling seemed specific for P cells contacted by R cells, as it was greatly reduced in pairs of either R or P cells and in single cells. Genistein (20 microM) and lavendustin A (10 microM), selective inhibitors of tyrosine kinases, blocked the labelling of contacted P cells, whereas their ineffective analogues (genistein and lavendustin B) had no effect on labelling. 3. R cell contact also induced the loss of an extrasynaptic, depolarizing response (due to modulation of cation channels) to serotonin (5-HT) in the P cell within a few days of juxtaposing cell bodies and within an hour of contact with growth cones. Treatment of the neurons with the tyrosine kinase inhibitors (but not the ineffective analogues) prevented the loss of the depolarizing response and of single cation channel modulation by 5-HT. 4. R cells formed inhibitory, Cl(-)-dependent synapses with P cells. Synapse formation was prevented by the tyrosine kinase inhibitors but not by their ineffective analogues. These compounds had no obvious effect on neurite outgrowth or cell adhesion. We conclude that tyrosine phosphorylation is a signal during the formation of this synapse.

Animals↗
PubMed1995

Signalling synapse formation between identified neurons.

We have investigated the signals between identified leech neurons during the formation of specific synapses in culture. At an inhibitory serotonergic synapse between two well-studied neurons, the postsynaptic cell has an additional (extrasynaptic) excitatory response to 5-HT which may underly a form of activity-dependent modulation. Thus, the presynaptic neuron must select which 5-HT response will be activated and which will be excluded at its synapses. The selection of these responses preceded synapse formation and was specifically induced at sites of contact with the presynaptic neuron, this not being observed for other cell pairings. Aldehyde-fixed presynaptic cells were equally effective, unless pre-treated with trypsin or wheat germ agglutinin, suggesting that contact with a specific cell-surface glycoprotein induced this physiological change in 5-HT sensitivity. The mechanism underlying the selective loss of the extrasynaptic response has been examined by single channel recording. Cation channels in the postsynaptic neuron were modulated by protein kinase C (PKC) upon binding of 5-HT to a 5-HT2 receptor. However, at sites of contact with the presynaptic neuron, the channels were no longer sensitive to PKC. Furthermore, when cation channels from uncontacted neurons were inserted or 'crammed' into contacted neurons, they were rapidly rendered insensitive to PKC, demonstrating a cytoplasmic signal for the uncoupling of channel modulation. Interestingly, the cytoplasm of contacted postsynaptic neurons showed immunoreactivity for tyrosine phosphorylation: exposure of the neurons to specific inhibitors of tyrosine kinases prevented tyrosine phosphorylation, the loss of cation channel modulation and synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Segmental specialization of calcium-activated potassium conductances in an identified leech neuron.

1. Retzius (R) neurons of the fifth and sixth segmental ganglia of the leech, called R(5,6) neurons are specialized to innervate the adjacent reproductive organs and are morphologically and functionally distinct from R neurons of standard ganglia [R(x) cells]. In this study the electrical properties of the R(x) and R(5,6) neurons were compared under current-clamp and voltage-clamp conditions. 2. The action-potential waveforms of R(x) and R(5,6) cells were similar except for the presence in the R(5,6) cells of a long afterhyperpolarization (AHP) following action potentials arising from the resting membrane potential but not from more depolarized potentials. Its role may thus be to inhibit firing of the R(5,6) neurons at rest or in response to weak depolarizing stimuli. 3. In the presence of the Ca2+ channel blocker Cd2+, the long AHP of the R(5,6) was abolished, and the action potentials of all R cells were identical. 4. Under voltage clamp, current kinetics and densities were similar between R(x) and R(5,6) cells for Ca2+ currents, delayed and inward rectifier K+ currents, and a rapid Ca(2+)-activated K+ current (IKc) that is common to the two cell types. The R(5,6) cells, however, expressed a second Ca(2+)-activated K+ current that was not observed in the R(x) cells. This current, called IKAHP, activated and inactivated more slowly than IKC, with a time course similar to that of the AHP observed under physiological conditions. 5. Neither IKC nor IKAHP was blocked by high concentrations of charybdotoxin or apamin, which block vertebrate Ca(2+)-activated K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1994

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons.

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R-P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin.

Animals↗
PubMed1994

Segmental specificity of neuronal recognition during synapse formation between identified leech neurons.

An early event in the formation of inhibitory synapses between the serotonergic Retzius (R) neuron and the pressure-sensitive (P) neuron of the leech is the elimination of an extrasynaptic, depolarizing response to 5-HT in the P cell from sites of contact. This effect is induced specifically by contact with the R neuron but not with other identified leech neurons, including other serotonergic neurons that do not form chemical synapses with the P cell. In the reproductive (fifth and sixth) segmental ganglia, the R cells (R(5,6)) exhibit functional and morphological differences with the standard R cells (R(x)) in the other 19 segmental ganglia. In the study presented here, we tested the specificity of P cell recognition of R cells by examining whether there were differences between the R(x) and R(5,6) cells with respect to synapse formation with the P cell. R(5,6) neurons did not innervate P cells in the fifth and sixth ganglia (P(5,6)) in vivo or in vitro, nor did they form synapses with P cells from standard ganglia (P(x)) in vitro except on rare occasions, after long periods in culture. In contrast to the effects of R(5,6) neurons, R(x) neurons readily innervated both P(x) and P(5,6) cells in vitro, suggesting that the lack of an R(5,6)-P(5,6) synapse reflects differences in the presynaptic rather than the postsynaptic cell. In contrast to the R(x) cell, contact with the R(5,6) neurons had no significant effect on the responses of the P cell to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cell-specific contact selects transmitter responses in an identified leech neuron.

Serotonergic Retzius (R) neurons of the leech form a Cl-dependent synapse with pressure-sensitive (P) neurons both in vivo and in vitro. However, P cells show an extrasynaptic, cationic response to application of 5-hydroxytryptamine (5-HT) which is reduced upon contact between the neurons in culture. We have examined the cellular specificity of the selection of 5-HT responses in the P cell by pairing it in culture with a variety of identified neurons. Non-synaptic sensory cells, non-serotonergic pre- and postsynaptic partners and serotonergic neurons that do not form chemical synapses with the P cell failed to alter its responses to 5-HT. The selective reduction of the extrasynaptic response to 5-HT in the P cell therefore appears to be induced specifically by contact with its only known serotonergic partner during neuronal recognition leading to synapse formation.

Animals↗
PubMed1991

Distinct receptors, second messengers and conductances underlying the dual responses to serotonin in an identified leech neurone.

1. Pressure-sensitive mechanosensory (P) neurones of the leech Hirudo medicinalis produce two responses to serotonin (5-HT): activation of a Cl- conductance and of a non-selective monovalent cation conductance. The effects of channel blockers, the receptor pharmacology and the second-messenger dependence of these responses were studied in voltage-clamped P cells in culture. Antagonists were applied by superfusion and agonists by pressure ejection. 2. Zn2+ (100 mumol l-1) and H+ (pH 6.5 and lower) reversibly reduced the Cl- conductance activated by 5-HT. The cation conductance was impermeant to calcium ions and was reduced by micromolar concentrations of the Na+ channel inhibitors amiloride and 3,4-dichlorobenzamil. 3. High concentrations of antagonists or agonists of 5-HT1 receptors and an antagonist of 5-HT3 receptors had no effect on either response of P cells to 5-HT. Micromolar concentrations of ketanserin or cyproheptadine, which selectively antagonize 5-HT2 receptors, reduced the cation but not the Cl- conductance. From these results, the receptor underlying the cation conductance appears to be of the 5-HT2 subtype, whereas the receptor activating the Cl- conductance does not fit within the mammalian classification scheme. 4. Brief (less than 500 ms) application of membrane-permeant agonists of the second messenger cyclic AMP elicited a Cl- conductance, whereas antagonists of cyclic-AMP-dependent protein kinase A reversibly suppressed the Cl- conductance elicited by 5-HT and by cyclic AMP agonists. Compounds affecting other second messenger pathways were without effect on the Cl- conductance. It therefore appears that the Cl- conductance is activated by cyclic-AMP-dependent protein kinase A. 5. Cyclic nucleotide agonists and antagonists were without effect on the cation conductance. However, brief application of phorbol esters, which activate protein kinase C, elicited an amiloride-sensitive cation current. An inhibitor of protein kinase C reduced the cation conductance activated by 5-HT and by phorbol esters. Therefore, the cation conductance appears to depend on activation of protein kinase C. 6. We conclude that 5-HT activates two types of receptor coupled to separate ionic channels via different second messenger pathways in P cells. A receptor that is distinct from the mammalian subtypes activates Cl- channels via cyclic-AMP-dependent protein kinase A. 5-HT2 receptors appear to activate cation channels by means of protein kinase C.

Animals↗
PubMed1986

Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies.

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.

Antibodies, Monoclonal↗
PubMed1983

Conversion of nonfusing mumps virus infections to fusing infections by selective proteolysis of the HN glycoprotein.

Mumps virus strains differ in their ability to induce cell fusion following an infection: strains with active neuraminidase (NANase) fail to cause cell fusion, while strains with less active NANase cause cell fusion. When chymotrypsin is added to infected cells, cell fusion is amplified in a concentration-dependent manner for all mumps virus strains. Virions produced in such infections do not express HN glycoprotein-associated activities. Chymotrypsin treatment of purified mumps virus in vitro results in sequential cleavage of the HN glycoprotein without affecting F glycoprotein structure. Initially, HN is cleaved into two glycopolypeptides, HNc1 (32K) and HNc2' (41K), with concomitant loss of hemagglutinating and NANase activities, and infectivity. Further incubation with chymotrypsin causes complete degradation of HNc1 and digestion of HNc2' to HNc2 (13K-19K). Both HNc2' and HNc2 contain the [3H]palmitic acid label found in the HN polypeptide, which suggests that these fragments are associated with the viral membrane. Analyses of infected cells and released virions indicate that chymotrypsin acts similarly on HN exposed at the cell surface. Exogenous NANase does not abolish the protease-augmented cell fusion, though it does reduce cell fusion of untreated fusing strain infections. These results confirm that mumps virus HN glycoprotein is critically linked to cell fusion cytopathology and show that cryptic cell fusion activity in nonfusing strain infections can be unmasked by the proteolytic removal of the HN glycoprotein.

Animals↗
PubMed1983

Biosynthesis of mumps virus F glycoprotein: non-fusing strains efficiently cleave the F glycoprotein precursor.

Mumps virus infection of the CV-1 cell line results either in no cytopathic effect or extensive cell fusion, depending upon the infecting mumps virus strain. Growth cycle analyses indicated that both types of infection were the result of multiple cycle replication of mumps virus. Intracellular virus-specific polypeptide synthesis was examined by pulse- and pulse-chase-labelling with radioactive amino acids and sugars. The major polypeptides seen on SDS-polyacrylamide gels were NP (69 000 mol. wt.), P (45 000 mol. wt.) and M (40 000 mol. wt.); a non-structural polypeptide (22 000 mol. wt.) was also present in infected cell lysates. The HN (74 000 to 79 000 mol. wt.) glycopolypeptide was detected in [3H]glucosamine- and [3H]mannose-labelled infected cells. A 65 000 mol. wt. species that had incorporated these precursors was seen in pulse-labelled infected cell lysates, and this glycopolypeptide vanished during the chase interval with the concomitant appearance of two glycopolypeptides (59 000 mol. wt. and 14 000 to 15 000 mol. wt.) which represented the F1 and F2 subunits of the F glycoprotein. Immunological data confirmed the relatedness of the 65 000 mol. wt. glycopolypeptide to the F glycoprotein and identified it as the precursor F0. The F0 precursor glycopolypeptide was seen in cells infected with both fusing and non-fusing strains, and F0 was processed completely to F glycoprotein for all infections. Thus, the lack of cell fusion after infection with certain mumps strains is not the consequence of incomplete processing of the F0 precursor.

Animals↗
PubMed1982

Differentiation of mumps virus strains with monoclonal antibody to the HN glycoprotein.

A hybridoma cell line secreting antibody of the immunoglobulin G3 isotype with kappa light chains and with activity against the HN glycoprotein of the Kilham strain of mumps virus was established. The antibody exhibited structural homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a microheterogeneous isoelectric spectrum characteristic of an antibody of monoclonal origin. The specificity of the monoclonal antibody, shown by immunoprecipitation performed with radiolabeled virus and infected cell lysates, was for the larger mumps virus glycoprotein. In functional assays the antibody inhibited the hemagglutinating and neuraminidase activities and neutralized the infectivity of the homologous Kilham strain of virus and clearly differentiated this strain from two heterologous strains, Enders and O'Take. The antibody was markedly less effective with the O'Take strain than with either the Kilham or Enders strain in inhibiting both hemagglutination and neuraminidase activity against the macromolecular substrate fetuin. The inhibition of the neuraminidase activity of the Kilham strain was independent of substrate size, the antibody inhibiting the hydrolysis of both fetuin and the trisaccharide neuraminlactose. By contrast, the antibody did not inhibit the hydrolysis of neuraminlactose by the two heterologous mumps strains. These results provide the first demonstration of antigenic differences between mumps virus strains and highlight the utility of monoclonal antibody in analyzing the structural basis underlying functional activities of the HN glycoproteins.

Antibodies, Monoclonal↗
PubMed1982

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy.

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.

Animals↗
PubMed1981

Functions of surface glycoproteins of myxoviruses and paramyxoviruses and their inhibition.

Two glycoproteins, HN and F, are present on the surface of paramyxoviruses. HN has receptor-binding amd neuraminidase activities. F is involved in viral penetration, cell fusion and haemolysis and is activated by proteolytic cleavage by a host enzyme into two disulphide-bonded subunits (f1 and F2). The ability of the virus to initiate infection and undergo multiple cycle replication depends on the presence of an activating protease in the host; thus cleavage of F is a major determinant of pathogenesis. The new N-terminus generated on F1 by cleavage is involved in biological activity, and the amino acid sequence of this region of F1 by cleavage is involved in biological activity, and the amino acid sequence of this region of F1 is hydrophobic and highly conserved among para-myxoviruses. In an attempt to design specific inhibitors, oligopeptides and analogous to this region were synthesized and found to be highly active, specific inhibitors of viral penetration, cell fusion and haemolysis. Inhibition is amino-acid-sequence-specific and affected by peptide length, steric configuration and addition of groups to the n-terminal and C-terminal amino acids. Replication of influenza virus was also specifically inhibited by oligopeptides resembling the N-terminus of the HA2 polypeptide. Like that of F1 protein the N-terminus of HA2 is generated by a proteolytic cleavage that activates infectivity. These results have provided information on the action of proteins in viral penetration and membrane fusion and they suggest a possible new approach to chemical inhibition of viral replication. Studies with specific antibodies to each of the paramyxovirus glycoproteins have shown that antibodies to the F protein are essential for effective prevention of the spread of infection. Antibodies to the HN protein, although capable of neutralizing released virus, do not prevent spread to adjacent cells through membrane fusion mediated by the F protein. These findings have implications for the design of effective vaccines against paramyxoviruses and also provided additional insight into the mechanisms involved in the atypical and severe infections observed in individuals who received inactivated paramyxovirus vaccines and were later infected.

Amino Acid Sequence↗
PubMed1981

The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis.

The F glycoprotein of paramyxoviruses is responsible for cell fusion and hemolysis and for virus penetration via fusion of viral and cell membranes. These functions are activated by specific proteolytic cleavage of an inactive precursor (F0) into two disulfide-linked polypeptides (F1 and F2). The susceptibility of the F0 protein to cleavage by a host protease is a major determinant of virus host range and virulence. Synthetic oligopeptides that mimic the N-terminal region of the F1 polypeptide are specific inhibitors of paramyxoviruses, and oligopeptides that mimic the N-terminus of the HA2 polypeptide of influenza virus, also generated by cleavage, specifically inhibit that virus. Antibodies to F protein prevent the spread of paramyxovirus infection via membrane fusion, but antibodies to HN protein do not, although they neutralize released virus. These results and previous findings that formalin-treated virus does not induce antibodies to F protein provide an explanation for atypical measles. The HN protein has both receptor-binding and neuraminidase activities, and Cl- inhibition of neuraminidase may modulate these antagonistic activities. Studies in patients with subacute sclerosing panencephalitis (SSPE) suggest that there is a host restriction of synthesis of the M protein of measles virus in brain cells which is involved in the abortive, persistent infection that causes SSPE.

Antibodies, Viral↗
PubMed1980

Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection.

The effects of monospecific antibodies to the viral glycoprotein with hemagglutinating and neuraminidase activity (HN) and the viral glycoprotein with membrane-fusing activity (F) of the paramyxovirus simian virus 5 (SV5) on the spread of infection in two cell types have been investigated. In CV-1 cells, infection can spread by either released progeny virus adsorbing to and infecting other cells, or by fusion of an infected cell with an adjacent cell as a result of the cell-fusing activity of the F glycoprotein. In these cells, antibodies specific for the HN glycoprotein prevented the dissemination of infection by released infectious virus, but spread by cell fusion was not inhibited. Antibodies to the F glycoprotein completely prevented the spread of infection in these cells. In Madin-Darby bovine kidney cells, which are relatively resistant to SV5-induced fusion, antibodies to either the HN or F glycoproteins were capable of preventing the dissemination of infection. These results indicate that effective immunological prevention of the spread of paramyxovirus infection requires the presence of antibodies that inactivate the F glycoprotein. This requirement for anti-F antibodies has obvious implications for the design of effective paramyxovirus vaccines and provides an explanation for previous failures of formalin-inactivated paramyxovirus vaccines as well as additional insight into the possible immunopathological mechanisms involved in the atypical and severe infections that have occurred in individuals who received inactivated paramyxovirus vaccines and were subsequently infected by the virus.

Animals↗