Fluorescein-concanavalin A conjugates distinguish between normal and malignant human cells: a preliminary report

A method is described for using a fluorescein isothiocyanate concanavalin A conjugate to stain human cell membranes in formalin fixed paraffin embedded tissue. 57 neoplastic and normal tissue sites were examined. In 54 malignant tumours, bright green fluorescence was confined to the cell membranes while in 23 benign tumours and normal tissue sites, the membranes were unstained or showed a diminished level of fluorescence. The distinction between malignant and hyperplastic or normal cells was clear cut and definite.     

Fluorescein-concanavalin A conjugates distinguish between normal and malignant human cells: A preliminary report

Summary A method is described for using a fluorescein isothiocyanate concanavalin A conjugate to stain human cell membranes in formalin fixed paraffin embedded tissue. 57 neoplastic and normal tissue sites were examined. In 34 malignant tumours, bright green fluorescence was confined to the cell membranes while in 23 benign tumours and normal tissue sites, the membranes were unstained or showed a diminished level of fluorescence. The distinction between malignant and hyperplastic or normal cells was clear cut and definite.     

Striated inclusions and defective mitochondria in the restricted form of the ‘amycelial’ mutant ofNeurospora crassa

Summary The cytoplasm of the restrictive form of the amycelial mutant ofNeurospora crassa, growing on sucrosemedium, contains paracrystalline, microfilamentous inclusions and mitochondria with internal membranous whorls lacking in acetate-grown cultures.The support of the Fonds national suisse de la recherche scientifique is gratefully acknowledged.     

Permeability of cellophane membranes to parotid proteins during dialysis

Summary Pooled parotid saliva was dialyzed in cellophane membranes against water for periods of up to 1 week and loss of proteins was monitored by acrylamide gel-electrophoresis. A gradual loss of cationic proteins was observed whereas anionic proteins were not appreciably affected. Loss of the cationic proteins could be greatly reduced by performing dialyses against dilute electrolyte solutions rather than water. These effects were attributed primarily to electrostatic charges associated with the dialysis membranes.The assistance of Mr E. Pederson and Dr G. Clark is gratefully acknowledged. Supported by Naval Medical Research and Development Command Project MR005.19 6048, Bethesda, Maryland. The opinions expressed herein are those of the authors and are not be construed as reflecting the views of the Navy Department or the Naval Service at large. The use of commercially available products does not imply endorsement of these products or preference to other similar products on the market.Deceased.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.     

Fructan and its relationship to abiotic stress tolerance in plants

Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation.     

Effect of ionic environment on densities of membrane-associated particles in presynaptic membranes observed in freeze-fractured synaptosomes.

The effects of high-K and high-Ca in the incubation medium on membrane-associated particles of the presynaptic membranes were examined. There was a marked increase in the density of protoplasmic fracture face after incubation in the high-K or high-Ca medium.     

Effect of ionic environment on densities of membrane-associated particles in presynaptic membranes observed in freeze-fractured synaptosomes

Summary The effects of high-K and high-Ca in the incubation medium on membrane-associated particles of the presynaptic membranes were examined. There was a marked increase in the density of protoplasmic fracture face after incubation in the high-K or high-Ca medium.     

Molecular machinery mediating vesicle budding,docking and fusion

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.     

Cytochemical localization of surface carbohydrates on mycoplasma membranes

Summary Surface carbohydrate structures, containinga-D-glucosyl or sterically closely related residues, were visualized on mycoplasma membranes by a cytochemical staining procedure with concanavalin A and iron-dextran complexes.Supported by a grant from the Deutsche Forschungsgemeinschaft.     

Intracellular sterol trafficking

Summary Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.     

Intracellular sterol trafficking

Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.     

Ultrastructural localization of photosystem I and II active sites in chloroplasts in situ]

Activities related to photosystem I and photosystem II are cytochemically localized at the electron microscope level by using suitable artificial electron donors and acceptors with, or without, factors uncoupling the light-induced electron flow between the two systems. Photo-oxidation of DAB, insensitive to DCMU, is linked to photosystem I activity. It occurs within the grana and stroma membranes of the chloroplasts. Photo-reduction of TC-NBT sensitive to DCMU, is a photosystem II mediated reaction. It is trapped within the grana membranes.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Aluminium in repair membranes and Al,Ca and Pi in the haemolymph of Al-injected shell-repairing snails (Helix pomatia L.)

Summary In AlCl₃-injected shell-repairing snails,Helix pomatia L., the Al-associated decrease of the weights of the shell-repair membranes was unrelated to the Al-concentration in the membranes. In the haemolymph the concentration of Al was related to the dose of injected Al, while the concentration of Ca was increased by the highest Al-dose only. No phosphate was detected in either controls or Al-injected snails. It is concluded that Al inhibits the growth of the CaCO₃-crystals by mechanisms other than incorporation in, or adsorption to, the crystals.     

Zytoplasmatische Feinstrukturen der Thrombozyten des Haushuhns

Summary Thrombocytes of clinical healthy, laying hybrid-hens were studied by using the electron microscope EM 9. In the cytoplasm of these cells osmiophilic granules of different stages were found, also lamellary bodies with or without vacuoles. A single lamella consists of myelin-membranes. Furthermore linear, branched out or circular membranes, which consist of 3 stratums, were observed in the cytoplasm. Ring-shaped microtubules lay close by the plasma membrane.     

Amino acid incorporation by human milk fat globules membranes (author's transl)]

Human milk fat globule membranes (MFGM) can incorporate radioactive 14C amino acids in a hot trichloracetic acid-insoluble material. Aspecific adsorption and bacterial contamination are unlikely. The products of protein synthesis were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by action of proteolytic enzymes. Various inhibitors of protein synthesis were assayed. Fragments of rough endoplasmic reticulum or mitochondria could be involved in this incorporation.     

The role of distinct membrane components of the enzyme in the ouabain sensitivity of Na+/K+ ATPase]

Purified plasma membranes from a cell line derived from the murine plasmocytoma MOPC 173 exhibited a 50% inhibition of the Na+K+ ATPase by 120 muM ouabain. EDTA treatment of such membranes induced a drop in the ouabain concentration needed for 50% inhibition to 0.4 muM. Supernatants obtained after EDTA treatment were able to complement with Ca(++) + Mg++ the washed EDTA treated membranes which recover their original resistance.     

Role of laminin-nidogen complexes in basement membrane formation during embryonic development

Laminin and nidogen (entactin) are major glycoprotein components of basement membranes. At least seven different isoforms of laminin have been identified. Laminin and nidogen form high affinity complexes in basement membranes by specific binding between the laminin γ1 chain and the G3 globule of nidogen. Additional interactions between nidogen and collagen IV, perlecan and other basement membrane components result in the formation of ternary complexes between these matrix components. Nidogen is highly susceptible to proteolytic cleavage, and binding to laminin protects nidogen from degradation. Nidogen is considered to have a crucial role as a link protein in the assembly of basement membranes. Basement membrane components are synthesized at high levels during tissue growth and development, and sites of morphogenesis correlate with localized remodelling of basement membranes. The formation of distinct basement membrane matrices in the developing embryo is influenced by the laminin isoforms produced and by whether laminin and nidogen are co-expressed and secreted as a complex or are produced by cooperation between two cell layers. The potential roles of laminin-nidogen complexes, cell-matrix interactions, and other intermolecular interactions within the matrix in basement membrane assembly and stability are discussed in this review.