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Biomedical subjects

D Muller

Publications and source records attributed to D Muller.

At least 163 records · Page 9Linked to original sources

Long-term potentiation, protein kinase C, and glutamate receptors.

Among the various molecular events that have been proposed to contribute to the mechanisms of long-term potentiation (LTP), one of the most cited possibilities has been the activation of protein kinase C (PKC). Here we review various aspects of the cellular actions of PKC activation and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of synaptic responses. We discuss the implications of these effects for interpretations of the role of PKC in the mechanisms of LTP induction and maintenance.

Animals↗

A simple method for organotypic cultures of nervous tissue.

Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.

Animals↗

Early and late results of surgery for Wolff-Parkinson-White syndrome.

Surgical treatment for accessory atrioventricular connections was performed in 60 patients with Wolff-Parkinson-White syndrome between 1981 and 1986. The initial procedure successfully divided 69 of 73 pathways identified preoperatively, including 39 of 40 left free wall pathways, 23 of 24 posteroseptal pathways, six of seven right free wall pathways, and one of two anteroseptal pathways. Three additional pathways were identified for the first time during follow-up. The primary procedure was successful in curing 53 (88%) of the 60 patients and in dividing 69 (91%) of the total of 76 pathways. Subsequent procedures increased the overall clinical cure rate to 97%. The surgery was performed with low morbidity and no perioperative or late mortality. Patients were followed-up for a mean of 6.4 years (range four to nine years). No patient showed clinical or electrocardiographic evidence of recurrence of a pathway which had been divided surgically. We conclude that regardless of pathway site, surgical treatment carries a low risk and has a high probability of avoiding lifelong antiarrhythmic therapy.

Adolescent↗

Detection of mRNAs encoding collagenase I and stromelysin 2 in carcinomas of the head and neck by in situ hybridization.

The mRNAs encoding 2 metalloproteinases, stromelysin 2 and collagenase I, have been detected by in situ hybridization in 26 carcinomas of the head and neck. 23 tumors of 26 expressed these mRNAs. Collagenase mRNAs were present in individual invasive cancer cells and in tumor cells at the periphery of poorly differentiated clusters (4 cases). Numerous stromal cells, principally fibroblasts were labeled (18 cases). Stromelysin mRNAs have been localized in tumor cells frequently arranged along disrupted basement membranes (8 cases). Many stromal cells in close contact to cancer cells also expressed the stromelysin mRNAs (17 cases). Normal residual cells were never labeled. These observations plead for the role of stromelysin produced by both stromal and tumor cells in the breakdown of basement membranes and the involvement of both collagenase and stromelysin in stromal invasion in carcinomas of the head and neck in vivo.

Blotting, Northern↗

Double-coated silica supports for high-performance affinity chromatography of proteins.

In order to prepare easily derivatizable supports for high-performance affinity chromatography (HPAC), the advantages of traditional polysaccharide-based supports were combined with the excellent mechanical properties of silica by coating the porous silica beads with a double layer of polysaccharide. The starting material was preliminarily impregnated with dextran or agarose, substituted with a calculated amount of positively charged diethylaminoethyl (DEAE) functions, in order to neutralize the cation-exchange capacity. These silica beads were then recoated by a second coupling with a native dextran or agarose so the DEAE functions introduced by the first coating could be overlayed and the coating state of the silica beads was further improved. The passivation of silica was confirmed by eluting standard proteins on the double-coated silica supports in high-performance size-exclusion chromatography. The elution of an acidic biopolymer, heparin, on different coated silica supports under gradient conditions demonstrated the major improvement of the native polymeric overlayer on the ionic properties of the support. These double-coated silica supports can also easily be activated by classical activation methods and coupled with active ligands (protein A and heparin). The active supports grafted by protein A were used in HPAC of human immunoglobulin G. The double-coated silica supports grafted by heparin were used in HPAC of human alpha-thrombin and human antithrombin III and for the purification of bovine thrombin from a commercial crude thrombin preparation.

Animals↗

Specific adsorption of serine proteases on coated silica beads substituted with amidine derivatives.

Amidine derivatives interact with serine proteases, the inhibition being due to interactions between amidine functions and the active sites of the enzymes. Five different types of amidine (substituted or unsubstituted) were coupled to coated silica beads, which had previously been coated with DEAE-dextran to minimize the non-specific interactions due to silanol groups. Coated silica functionalized with substituted amidines shows a strong affinity towards human plasmin. This affinity is probably due to hydrophobic interactions between the substituted amidine and the human plasmin structure. Coated silica grafted by p-aminobenzamide gives a specific interaction with human plasmin. The importance of ionic strength and the steric conformation of the ligand is discussed. This support was used to purify thrombin from crude preparations by high-performance affinity chromatography.

Adsorption↗

High-performance affinity chromatography of basic fibroblast growth factor on polystyrene sulphonate resins modified with serine.

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor, so it was usually purified by affinity chromatography on a heparin-grafted support. It was previously observed that, among several modified polystyrene that exhibit anticoagulant heparin-like properties, insoluble polystyrenes modified by chlorosulphonation substituted with serine (PSSer) develop specific interactions with bFGF in solution. These PSSer resins were used as stationary phases in high-performance affinity chromatography in order to separate the radiolabelled growth factor from a bovine brain crude extract. The growth factor is strongly bound to the solid phase, as demonstrated by the adsorption isotherms. It can be adsorbed on the resin at low ionic strength and be desorbed by raising the salt concentration in the eluent. The effects of flow-rate, of the initial buffer and of the slope of the salt gradient on the adsorption and on the desorption of bFGF were investigated in order to study the thermodynamic and the kinetic parameters of the interactions and to define the optimum conditions for a fast and efficient separation of the growth factor.

Animals↗

Synaptic modulation of N-methyl-D-aspartate receptor mediated responses in hippocampus.

Low magnesium medium and the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) were used to analyze the effect of several manipulations on the component of excitatory postsynaptic potentials (EPSPs) mediated by activation of NMDA receptors in area CA1 of hippocampal slices. The D-AP5 sensitive component of synaptic responses was characterized by a marked sensitivity to changes in extracellular magnesium and calcium concentrations. In both cases the changes in D-AP5 sensitive responses were considerably larger than those in non-NMDA-dependent potentials. Similarly, frequency facilitation, which is due to a transient increase in release, was accompanied by a greater enhancement of NMDA than non-NMDA receptor-mediated components. The degree of paired-pulse facilitation observed with D-AP5 sensitive responses was magnesium-dependent between concentrations of 0.05 and 0.5 mM, an effect not seen with control potentials. Intracellular injections of hyperpolarizing current pulses differentially affected NMDA and non-NMDA receptor-mediated components. Taken together, these results indicate that changes in the amount of transmitter release may affect to a greater degree NMDA than non-NMDA receptor-mediated components of synaptic responses, probably because of the voltage-sensitive blockade by magnesium of the NMDA receptors. In contrast, induction of long-term potentiation (LTP) by high frequency stimulation produced a larger increase in non-NMDA as opposed to NMDA receptor-dependent responses, a result that does not support the idea that an increase in transmitter release is responsible for LTP.

2-Amino-5-phosphonovalerate↗

Mutations in the alpha 1 domain of a class I gene define residues important for specific allorecognition.

Our strategy to use saturation mutagenesis to produce an unbiased collection of major histocompatibility class I mutants has resulted in unpredicted mutant phenotypes. First, we have shown data supporting our earlier work of the Dp20(Y27N) mutant. Allorecognition is altered at the clonal level while no variation in lymphocytic choriomeningitis virus (LCMV)-restricted recognition is observed. The defect does not destroy the integrity of this class I protein on the basis of three observations: (i) LCMV self-restricted recognition is not impaired, (ii) beta 2 microglobulin still associates with Dp20(Y27N) at the cell surface, and (iii) this mutant can stimulate a primary MLR. Thus, we believe Dp20(Y27N) specifically affects allorecognition, perhaps by altering self peptide associations. The Dp14(A11V;E32Q) mutant appears to interact with T cell receptors (TCR) from a cloned cytotoxic T lymphocyte, but is altered in inducing a wild type signal into the responding cell. This is presumably due to decreased interaction at the cell surface between Dp14(A11V;E32Q) and wild type-specific TCR such that variations are detected in how a cell perceives extracellular signals. Analysis of additional mutants suggests that mutant Dp163(N66S) alters the binding site for monoclonal antibodies 7-16.10 and 135, while leaving unaltered the binding site for monoclonal antibodies 34-1.2 and 11-20.3. This maps the residue responsible for 7-16.10 and 135 binding to the region of Dp163(N66S).

Animals↗

Endo-exocytotic images and changes in synaptic transmission induced by diamide at a cholinergic junction.

Small tissue fragments excised from the electric organ of Torpedo marmorata were treated with diamide, a penetrating thiol oxidizing agent, until synaptic transmission was blocked. At this stage, we found an unexpected number of exo-endocytotic images in the presynaptic plasmalemma. Omega-shaped profiles, some of them coated, were seen in thin sections of fixed tissue and pits opened in the P-face of the presynaptic membrane in freeze-fracture replicas from rapidly-frozen preparations. Diamide-treated specimens were frozen at 1 ms time intervals before, during and after a single electrical stimulus. This stimulation did not result in a further increase in the density of presynaptic pits, not in any change affecting the density or size distribution of intramembrane particles. This result is in contrast with what is observed in untreated specimens where transmission of a nerve impulse is accompanied by a momentary rise in the number of large particles. The density of synaptic vesicles--especially that of a subpopulation of small size vesicles--transiently increased within the first 2 h of diamide treatment. During the first stages of intoxication, diamide prolonged the time course of postsynaptic potentials--both spontaneous and evoked--probably by altering the gating properties of receptors (acetyl-cholinesterase activity was not impaired). Later on, all evoked responses were blocked. The spontaneous transmitter release greatly increased, first in the form of quantal miniature potentials. These then subsided whereas a class of very small potentials was generated at a high frequency. Also under the action of diamide, calcium progressively accumulated in the tissue but the number of synaptic vesicles containing calcium deposits was reduced. It is concluded that diamide causes a marked increase in the number of exo-endocytotic images in the presynaptic membrane, suppresses quantal but not subquantal release, and interferes with calcium sequestration in and extrusion from terminals.

Animals↗

Characterization of outer membrane proteins from Actinobacillus actinomycetemcomitans.

Outer membranes were prepared from whole cells of various strains of Actinobacillus actinomycetemcomitans and analysed by SDS-polyacrylamide gel (12.5%) electrophoresis (SDS-PAGE). In all strains four common major outer membrane proteins (OMPs) with molecular masses of 30, 34, 36 and 39 kDa could be distinguished. Heating the OMP preparation of strain Y4 at 60, 70, 90 and 100 degrees C produced a band of 30 kDa, which gradually lost its intensity from 70 degrees C onwards concomitantly with the development of two new protein bands of 34 and 36 kDa. Furthermore, the 36 kDa OMP appeared susceptible to proteolysis by trypsin; degraded products apparently produced a new electrophoretic band of 27 kDa. Y4-derived OMP fractions were solubilized with a Triton-SDS mixture to investigate the presence of peptidoglycan-associated proteins. The 39 kDa OMP was found to be peptidoglycan-associated.

Actinobacillus↗

Protein kinase C activity is not responsible for the expression of long-term potentiation in hippocampus.

Long-term potentiation (LTP) in hippocampus has been proposed to result from a tonic activation of protein kinase C. This hypothesis predicts that stimulation of the kinase would produce a smaller change in response size on potentiated versus control pathways and, conversely, that inhibition of the kinase would reduce potentiated inputs to a greater degree than control responses. We tested these predictions using phorbol esters to activate and using the antagonist H-7 to inhibit protein kinase C; we found that the actions of these drugs on synaptic transmission were not affected by prior induction of LTP. Both compounds, however, significantly decreased the contribution of N-methyl-D-aspartate receptors to synaptic potentials, a result that accounts for the suppressive effects of these compounds on LTP formation. Thus protein kinase C is probably not involved in the expression of LTP but may play a role in the receptor-mediated events participating in its induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Continuing diet trends in men: the Baltimore Longitudinal Study of Aging (1961-1987).

Due to increasing evidence for the role of nutrition as a cause of illness and of the efforts of health agencies to induce Americans to change their diets, it is important to monitor populations for changes in their nutrient intakes. Seven-day diet records were collected from 105 free-living men in the 1960s, 1970s, and 1980s. Age range was initially 27 to 65 and finally 50 to 88 years. The men were divided into age groups so that longitudinal, time series, and cross-sectional analyses could distinguish among differences due to aging, cohort, and secular change. Longitudinal decreases over three decades of time include height, intakes of kcal/day, kcal/kg, kcal from fat, and cholesterol (all p less than .0001). Longitudinal increases over time include body weight, intakes of kcal from alcohol (p = .002), kcal from carbohydrate, and polyunsaturated to saturated fatty acid ratio (P/S) (all p less than .0001). Values that correlated cross-sectionally with age in the 1960s were height, kcal/day, kcal/kg, kcal from protein, and P/S ratio; height, intakes of kcal from protein, and fiber in the 1970s; and body weight, height, intakes of kcal from carbohydrate and fiber in the 1980s (all p less than .05). Protein consumption remained constant while carbohydrate increased as time passed. The more striking secular changes were in type and amount of fat consumed (42% to 34% of kcal, a 37% decline in cholesterol and a 72% increase in P/S ratio). Voluntary changes in the diets of these community-dwelling men indicate that, at any age, men can make beneficial changes in intake of nutrients that affect health.

Adult↗

Type A botulinum toxin disorganizes quantal acetylcholine release and inhibits energy metabolism.

The physiological, morphological and biochemical effects of type A Botulinum toxin (BoTX) were analysed in the electric organ of Torpedo, a modified neuromuscular system. The quantal content of the postsynaptic potential, or electroplaque potential (EPP), was reduced by BoTX but the quantum size remained unchanged till complete failure of the neurally evoked transmission. BoTX also suppressed the occurrence of spontaneous electroplaque potentials (MEPPs) of a quantal size but potentials of a smaller amplitude still kept on occurring in the intoxicated synapses. BoTX inhibited the evoked release of acetylcholine (ACh; biochemically measured) but the rate of spontaneous ACh release transiently increased during the period when evoked release went down. On the other hand, there were no significant change of ACh content, of ACh turnover, of ACh repartition in the vesicular and free compartments, or in the number of synaptic vesicles. Surprisingly, the amount of ATP was reduced to 50% in BoTX treated tissue at the time of transmission failure; also the level of creatine phosphate (CrP) was lowered to less than 20% and the rate of activity of creatine kinase was reduced. It was concluded that, electrophysiologically, BoTX affects synaptic transmission in a very similar way in the electric organ and in the neuromuscular junctions. On the other hand, the shortage of ATP supply found in the present study may play a role in the pathophysiology of intoxication and should be taken into account in investigations designed to see whether BoTX affects various phosphorylations in cholinergic nerve terminals.

Acetylcholine↗

The role of cell adhesion and migration in the in vitro invasiveness of mouse adrenal carcinoma cells.

Using the Matrigel invasion assay, we have examined the role of cell adhesion and migration in the invasiveness of two cell lines, DM and HSR, derived from the Y1 mouse adrenocortical tumor. The DM cells were metastatic and more invasive (10-fold) than the nonmetastatic HSR cells. The difference in invasiveness could not be ascribed to different levels of secreted type IV collagenolytic activity since HSR cells secreted higher levels of activity. Cells from the metastatic DM line showed greater motility to both laminin and fibronectin when compared to the HSR line. Furthermore, both Matrigel and laminin promoted the attachment and spreading of DM cells but they had little effect on the adhesion of the HSR cells. Electron microscopic examination revealed an increased ruffling of the cell membrane in the metastatic DM line. These studies suggest a role for cell adhesion and migration on the invasion of Matrigel by malignant tumor cells.

Adrenal Gland Neoplasms↗

Developmental changes in synaptic properties in hippocampus of neonatal rats.

The properties of synaptic responses in area CA1 of hippocampus were analyzed in slices prepared from 7-9 and 12-15 day old neonate rats. As expected from earlier work, only slices of two-week-old animals showed a consistent degree of long-term potentiation (LTP) in response to patterned high frequency stimulation. Several other synaptic properties were found to change during this developmental period. Inhibitory responses were absent in 7-9 day old but not in 12-15 day old neonates. Paired-pulse facilitation and the calcium sensitivity of postsynaptic responses were considerably reduced in 7-9 as compared to 12-15 day old rats. However, phorbol esters and 4-aminopyridine treatment still produced a strong facilitation of field potentials. The N-methyl-D-aspartate (NMDA) component of responses to single pulse stimulation in low magnesium medium was found to be larger in slices of 7-9 than 12-15 day old or adult animals. At the two time periods examined, trains of high frequency stimulation applied in the presence of regular magnesium elicited an NMDA dependent response. It is concluded that the differences in synaptic properties observed between 7-9 and 12-15 day old neonates may not account for the absence of LTP in the younger animals.

Action Potentials↗

Coated silica supports for high-performance affinity chromatography of proteins.

Polymer-coated silica supports are potentially good stationary phases for high-performance affinity chromatographic separations of proteins. Silica beads have been coated with a polysaccharide (dextran or agarose), substituted by a calculated amount of positively charged diethylaminoethyl functions in order to neutralize the negatively charged silanol groups of silica and to facilitate the formation of a hydrophilic polymeric layer on the inorganic surface. The silica-based supports were prepared in two steps. First, the silica was impregnated with a solution of diethylaminoethylated polymer, and then the coating polymer was crosslinked in order to avoid leakage of the polymeric layer. The supports present minimal non-specific interactions with proteins, as tested by high-performance size-exclusion chromatography. These coated silica supports were coupled with active ligands, such as protein A, concanavalin A and heparin, by conventional coupling methods. The resulting affinity stationary phases were tested by the elution of proteins in order to study their performance in high-performance affinity chromatography.

Antithrombin III↗