Fine structural localization of acetylcholinesterase in electroplaque of the electric eel.

The electroplaques composing the electric organ of the eel, Electrophorus electricus, have been utilized for the dual purpose of demonstrating the subcellular sites of acetylcholinesterase activity and as a model for comparison of the several cytochemical methods available. Fresh tissue and tissue fixed by immersion in formalin, hydroxyadipaldehyde, or glutaraldehyde was reacted with the Cu-thiocholine method, the Cu-ferrocyanide thiocholine method, or the thiolacetic acid (TAA) method using Pb, Ag, or Au as capture reagents. Controls were obtained by omission of substrate, or by addition to complete media of varying concentrations of different cholinesterase inhibitors. Reactions were run at 0-5 degrees C at a pH range of 5.0-7.1 for 0.25 to 120 min. Regardless of the capture metal, the localization obtained with TAA as substrate was identical with that observed with acetylthiocholine, the majority of precipitate being deposited on or near the external innervated surface of the plaque and within the tubulovesicular organelles opening onto the innervated surface. Both of the thiocholine methods and the Pb-TAA method showed reaction product in synaptic vesicles of the nerve endings innervating the plaque which was uninhibitable by 10(-4)M physostigmine. All methods also showed some inhibitor-sensitive deposition of reaction product in the mucoid material forming the immediate extracellular environment of the innervated surface.

Ultrastructural localization of phospholipid synthesis in the rat trigeminal nerve during myelination.

A method for the ultrastructural localization of acyltransferase enzymes involved in phospholipid metabolism has been applied to the developing rat trigeminal nerve. Determination of acyltransferase levels in the nerve indicated that a peak of activity occurs at the 8th day after birth with gradual declines of activity up to 15 days. Morphological surveys and determinations of cholesterol levels suggested that heavy myelin formation occurs in the nerve during this latter period. Fixed nerves incubated in a medium for localization of acyltransferases indicated deposition of reaction product associated with Golgi cisternae, intracellular smooth vesicles, and the plasma membrane of the Schwann cell in the incipient stages of myelin formation. Golgi-derived vesicles appeared to move toward the Schwann cell surface and fuse with the plasma membrane. Activity continued to be detectable in the plasma membrane of the internal mesaxon as long as cytoplasm was evident and mature myelin membrane was not yet formed. Cells in which myelin formation appeared advanced showed little or no enzyme marker. Consistent with cytochemical observations were biochemical determinations of acyltransferases which showed high levels of the enzymes in microsomes, while no activity could be detected in the myelin fraction. Acyltransferase reaction product was also observed in the Golgi apparatus of ganglion cell bodies, axoplasmic smooth vesicles, and the axolemma. Localization of acyltransferase enzymes in Schwann cells, ganglion cell bodies, and axons during development of the nerve is discussed in relation to membrane biogenesis in the nervous system.

Fine structural localization of acyltransferases. The monoglyceride and -glycerophosphate pathways in intestinal absorptive cells.

A study of the fine structural localization of the acyltransferases of the monoglyceride and alpha-glycerophosphate pathways for triglyceride synthesis in the intestinal absorptive cell is reported. Glutaraldehyde-fixed tissue was found to synthesize diglyceride and triglyceride from monopalmitin and palmityl CoA, and parallel morphological studies showed the appearance of lipid droplets in the smooth endoplasmic reticulum of the absorptive cell. Glutaraldehyde-fixed tissue also synthesized triglyceride from alpha-glycerophosphate, although this enzyme system was more susceptible to fixation than the monoglyceride pathway acyltransferases. Cytochemical methods for the localization of free CoA were based (a) on the formation of the insoluble lanthanium mercaptide of CoA and (b) on the reduction of ferricyanide by CoA to yield ferrocyanide which forms an insoluble precipitate with manganous ions. By these methods the monoglyceride pathway acyltransferases were found to be located mainly on the inner surface of the smooth endoplasmic reticulum. The alpha-glycerophosphate pathway acyltransferases were localized mainly on the rough endoplasmic reticulum. Activity limited to the outer cisternae of the Golgi membranes occurred with both pathways. The possible organization of triglyceride absorption and chylomicron synthesis is discussed in view of these results.

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS : I. The Site of Activity of Acyltransferases Involved in Synthesis of the Membrane Phospholipid.

The localization of acyltransferases involved in acylation of alpha-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.

Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in cerebral cortex.

The distribution of cyclic 3',5'-nucleotide phosphodiesterase activity in rat cerebral cortex was determined by cytochemical methods. The detectable phosphodiesterase activity was localized in postsynaptic (dendritic) nerve endings, most of it immediately adjacent to the synaptic membrane. Most of the postsynaptic nerve endings showed phosphodiesterase activity.

Hormone-sensitive adenyl cyclase: cytochemical localization in rat liver.

An electron microscopic procedure has been developed, using rat liver, for the localization of hormone-sensitive adenyl cyclase. Isoproterenol-sensitive adenyl cyclase is located almost exclusively in the parenchymal cells. In contrast, glucagon-sensitive adenyl cyclase is located primarily in the reticulo-endothelial cells but is also present in parenchymal cells. Sodium fluoride-sensitive adenyl cyclase is found in both cell types.

The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy was used to determine the oxidation states of osmium compounds present in erythrocyte ghost preparations and related systems treated with osmium tetroxide. Osmium tetroxide and cholesterol, codeposited at -100 degrees C, began to react at -70 degrees C, and Os(VI) was formed. Similarly, Os(VI) was detected for the known cholesterol-osmate ester prepared and purified chemically. However, osmium tetroxide applied in phosphate buffer (pH 7.2) gave rise to large proportions of Os(IV) and Os(III) species in addition to Os(VI) compounds. Egg phosphatidylcholine likewise produced a mixture of Os(VI), Os(IV), and Os(III), but dipalmitoyl phosphatidylcholine failed to give significant amounts of osmium containing products under identical conditions. Glutaraldehyde gave a mixture of compounds with the same osmium oxidation states when allowed to react with aqueous osmium tetroxide. Unfixed and glutaraldehyde-fixed erythrocyte ghosts also produced mixtures of Ss(VI), Os(IV) and Os(III) under conditions identical to those of normal tissue processing. Additionally, the mixture of adducts initially formed by treatment with osmium tetroxide was further reduced by dehydration of the tissue with ethanol, rpesulting in a final mixture which was 50-60% Os(III). The results support a scheme for the reaction os osmium tetroxide with tissues in which the initial reaction site is the double bonds of unsaturated lipids to form Os(VI) derivatives. Subsequent hydrolysis and further reduction yield complexes of Os(IV) and Os(III). A mixture of these three states is present in membrane specimens during microscopic observation. Os(VI) and Os(IV) could be present as osmate esters and osmium dioxide, respectively; Os(III) could be present as an oxo- or amino complex(es). The photoelectron spectrum of intact erythrocyte ghosts can be synthesized from the spectra of phospholipid and cholesterol only, suggesting the predominance of the reaction with lipids in the fixation process.

A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.

The presence of Fe(CN)6(-4) provides sequential, one-electron reduction pathways for OSO4. An equilibrium is established containing OSO4, Fe(CN)6(-4), Fe(CN)6(-3), OSO2(OH)4(-4), and labile cyano-bridged OS-Fe species containing Os in nominal oxidation states of VIII, VII, and VI. These osmium complexes are chelated by appropriately placed donor atoms in the macromolecular tissue matrix, and chelation facilitates the reduction of osmium in situ to lower oxidation states (predominantly IV) that are relatively nonlabile. The greater reactivity and concentration of the Os(VII and VI) intermediates in this system leads to more Os deposition than OsO4 alone; the chelation is responsible for the immobilization of Os and the observed staining pattern in electron micrographs. Chemical data from model systems and electron micrographs of tissue are presented in support of this mechanism.

Cytochemical demonstration of sodium, potassium-adenosine triphosphatase by a hemepeptide derivative of ouabain.

A cytochemical probe for the ultrastructural localization of the NaKATPase was devised, which utilizes the biological affinity of the noncompetitive inhibitor, ouabain (ouab) to which was coupled a hemepeptide (H11P) which possesses peroxidatic activity. The conjugate, ouab-H11P, had an apparent Ki of approximately 8 x 10(-7) M. When reacted with fixed tissue from the salt gland of osmotically stressed ducklings, the NaKATPase was localized to the basal and lateral infoldings of the plasma membranes of secretory epithelial cells. Reaction product consisted of fine textured deposits distributed in focal patches on the outer aspects of the membrane. Apical membranes were negative, as were intracellular membrane components. Preincubation of tissue with unlabeled ouabain or binding of ouab-H11P in the presence of 10 mM K+, no ATP and no Mg++, resulted in the absence or diminution of reaction product.

A review of child psychotherapy research since 1963.

Reports on individual nonbehavioral child and adolescent psychotherapy since 1963 are reviewed. Inclusion criteria required some minimal contrasting group. Forty-three studies were assessed for basic methodological adequacy and main findings. The authors conclude that summary impressions from this body of literature cannot be made due to the magnitude of the flaws in basic psychotherapy research methodology. Suggestions are made regarding the future of child and adolescent psychotherapy research.

Psychopathology and developmental delay in homeless children: a pilot study.

The authors report a survey of 50 parent-child pairs from homeless families housed in New York City hotels. The purpose of the survey was to determine the extent of emotional or behavioral disturbances and of developmental delays in homeless children aged 4 through 10 years, the presence of depression or a history of depression or other psychiatric problems in the parents of these children, and to determine whether the children and adults had mental health needs. The results indicate that nearly all of the children showed some difficulties. Sixty-one percent of the children had receptive verbal functioning at or below the first percentile for age, 29% were functioning at the fifth percentile for age in psychomotor ability, and 38% exhibited emotional and behavioral problems. Twenty-eight percent of the parents exhibited evidence of mild to severe depression; a smaller percentage admitted to past psychiatric problems.

Biochemical and morphometric studies of the relationship of acetylcholine synthesis and vesicle numbers after stimulation of frog neuromuscular junctions: the effect of a choline-O-acetyltransferase inhibitor.

The present study compares choline acetylase (ChAc) activity with morphometric determinations of synaptic vesicles at the neuromuscular junctions of frog pectoralis muscle subjected to high and low frequency stimulation in the presence or absence of NVP, a ChAc inhibitor. Muscles stimulated at 10/sec for 20 min with one hour rest, in the presence of NVP, showed an approximately 50% reduction in synthesis of Ach, and a 50--60% reduction in the numerical density of synaptic vesicles relative to controls preparations. Similar nerve muscle preparations stimulated at 2/sec for one hour without rest, in the presence of NVP, also showed approximately 50% less synthesis of Ach and vesicle numbers 43% lower than that seen in control muscles treated in the same way in the absence of drug. The results indicate a correlation between the inhibitory effect of NVP on ChAc and the numbers of vesicles present within terminals. In addition, stimulation of nerve terminals, with or without NVP, produced a significant increase in the numerical density of synaptic vesicles over respective unstimulated controls. These and other results are discussed in relation to current hypotheses concerning recirculation of synaptic vesicles.