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 immunochemical determination of (Na+ + K+)-ATPase content in subcellular fractions prepared from the avian salt gland: evidence for enzyme heterogeneity.

Subcellular membrane fractions were prepared from the salt glands of osmotically-stressed ducklings. Two fractions were characterized biochemically with respect to (Na+ + K+)-ATPase, alkaline phosphodiesterase I, succinate dehydrogenase, esterase, and galactosyltransferase activities and immunochemically with respect to (Na+ + K+)-ATPase. The ratios of the estimates of the (Na+ + K+)-ATPase contents obtained biochemically and immunochemically from the two fractions differed by more than 2 X. The results are consistent with the presence of at least two molecular species of (Na+ + K+)-ATPase, unevenly distributed between the two fractions.

Characterization and use of polyclonal antibody to Na+,K+-ATPase: immunocytochemical localization in salt glands of the duck.

The amount of Na+,K+-ATPase of the avian salt gland increased concomitantly with plasma membrane surface area during salt feeding of ducklings (adaptation), and both enzyme content and membrane surface area decreased upon return to fresh water (deadaptation). In a further study of the enzyme, a marker for plasma membrane biogenesis, polyvalent antibodies were raised to the denatured alpha-subunit of the purified ATPase. Antisera did not inhibit enzymatic activity but immunoprecipitated the phosphorylated intermediate of the alpha-subunit. Furthermore, the alpha-subunit, which was not glycosylated, was immunoprecipitated from homogenates of tissue slices metabolically labelled with [35S]-methionine, using antisera raised against either duck salt gland or dog kidney alpha-subunit. The former antisera also recognized the alpha-subunit in the brain, heart, kidney, liver, intestine and skeletal muscle of the duck. Immunocytochemistry with the antisera raised to the duck salt gland alpha-subunit revealed reaction at basolateral as well as apical plasma membrane in the duck salt gland principal cells, with essentially no deposits on peripheral cells, fibroblasts, erythrocytes, endothelial cells and neural elements. Within the principal cells, immunolabelling was also detected on small vesicles, multivesicular bodies and lysosomes; deposits on extracellular debris and vesicles in the lateral and lumenal spaces were also apparent. The labelling patterns were qualitatively but not quantitatively similar in salt glands of control, adapted and deadapted ducklings, and are discussed in the context of a model for plasma membrane biogenesis and turnover in which degradative events may play a major role.

Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings.

During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.

Partial immunochemical identity between (Na+ + K+)-ATPase and a membrane component extractable with chloroform: methanol.

An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.

Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and y-1: II. Cytochemical localization of acyltransferase activities.

We have cytochemically localized the acyltransferase activities in the alga Chlamydomonas reinhardtii. Glycerol 3-phosphate (G3P) acyltransferase and lysophosphatidate (LPA) acyltransferase activities were cytochemically assayed utilizing biochemically optimized reaction conditions (Jelsema et al., 1982). The cytochemical assays clearly demonstrate that in the wild-type cells (137+) and the y-1 mutant, both enzymes were present in the photosynthetic membranes, envelope, and pyrenoid tubules of the chloroplast. Activity was also localized to the Golgi apparatus and its associated vesicles. Additionally, LPA acyltransferase activity was found associated with the outer mitochondrial membranes. The cytochemical data from this study confirm the biochemical data obtained using purified chloroplast membranes (Jelsema et al., 1982) and establishes the presence of these glycerolipid-synthesizing enzymes in the photosynthetic membranes of the chloroplast. These findings support the earlier reports of the presence and activity of lipid-synthesizing enzymes in the chloroplast, and also is in agreement with the postulated role of the pyrenoid tubules in the synthesis of the thylakoid membranes. This report differs in that these lipid-synthesizing enzymes have been localized to the chloroplast photosynthetic membranes themselves as well as exhibiting significant levels of activity for both enzymes in the Golgi apparatus. During light-induced chloroplast biogenesis in the yellow mutant of C. reinhardtii (y-1), the photosynthetic membranes appear to be the primary site of acyltransferase activity, suggesting that in situ synthesis of the membrane lipids during this period of rapid membrane formation is the primary mode for the synthesis of the thylakoid lipids. That these intrinsic thylakoid enzyme activities are not exclusively a function of the growth phase of the cell, but are found in mature chloroplast of the 137+ cells as well, further supports the conclusion that the photosynthetic membranes of the chloroplast have the capacity for synthesis of their own membrane lipids.

Membrane lipid metabolism in Chlamydomonas reinhardtii 137+ and Y-1: I. Biochemical localization and characterization of acyltransferase activities.

The acyltransferases involved in the synthesis of the chloroplast membrane glycerolipids were analysed biochemically in dark-grown and greening Chlamydomonas reinhardtii y-1 as well as in the synchronous wild-type algae (strain 137+) and wild-type membranes. Using oleoyl-CoA as a substrate, three acyltransferase enzyme activities were detected. Glycerol-3-phosphate (glycerol-3-P) acyltransferase exhibited a pH optimum of 8.0 and was inhibited by addition of N-ethylmaleimide (MalNEt). Lysophosphatidate (PtdLys) acyltransferase exhibited a pH optimum of 7.0 and was not affected by the addition of MalNEt. From preliminary analyses, the activity at pH 5.5 appeared to be associated with dihydroxyacetone phosphate acyltransferase activity. Both glycerol-3-P and PtdLys acyltransferases were analysed further and found to be present in dark-grown and light-induced y-1 cells as well as in synchronous 137+ cells and their photosynthetic membranes. Both enzyme activities were enriched at least 10-fold in the photosynthetic membranes of 137+ chloroplasts relative to the activities present in the whole cells. This enrichment is indicative of their intrinsic localization in the thylakoids, suggesting that the photosynthetic membranes exhibit a greater degree of autonomy with respect to the synthesis of their membrane lipids than previously reported. A role for glycerol-3-P and PtdLys acyltransferases in the synthesis of the chloroplast membrane lipids is suggested further by the increases in both enzyme activities coincident with and preceding thylakoid biogenesis following light induction of dark-grown y-1 cells. Increased acyltransferase activity preceded the increase in the chlorophyll content of greening y-1 cells, which is a generally accepted marker for thylakoid synthesis. The increase in the PtdLys acyltransferase activity upon light-induction of the y-1 cells was both more immediate and more dramatic than the increase in glycerol-3-P acyltransferase activity. PtdLys acyltransferase activity was negligible in dark-grown cells and the dramatic increase upon light induction may be important in the subsequent initiation of chloroplast membrane lipid synthesis. On the basis of the localization of acyltransferase enzyme activities to the photosynthetic membranes of 137+ cells and the increase in acyltransferase activity both preceding and occurring in concert with thylakoid synthesis, we propose a direct role for the photosynthetic membranes in the synthesis of their membrane lipid components.

Neurochemical characterization of an antimony-choline analog in rat cortical synaptosomes.

An analog of choline, in which nitrogen was replaced by antimony, was neurochemically characterized in rat cortical synaptosomes. It was found to be a substrate for several cholinergic enzymes, transported by a Na+-dependent, hemicholinium-3-sensitive process, acetylated, and subsequently released by depolarization in a calcium-dependent manner. Sb-choline also completed with choline for Na+-dependent uptake and for acetylation by [14C]acetyl-CoA in synaptosomes. These results suggest that Sb-choline and its acetylated product should be useful substrates for the x-ray microanalytical localization of cholinergic pools in intact nerve terminals.

Organotypic cultures of the avian salt gland: biosynthesis of membrane proteins.

The salt glands of control and salt-stressed ducklings were dissociated with collagenase to produce cell aggregates or 'minilobules' which were cultured. The fine-structural organization of freshly isolated and cultured (up to 72 h) aggregates were examined and showed surprisingly intact fine-structural organization with minimal changes from untreated glands. Incorporation of [3H]leucine into total protein, membrane protein and immunoprecipitable Na,K ATPase was determined in cultures at various time points, by pulse or pulse-chase experiments. Incorporation of label was linear for 4 h in protein, but higher in cultures of salt-stressed glands than in controls. Na,K ATPase was synthesized throughout the time of the experiment, the rate being highest during the first 4 h, reaching a plateau by 24 h. Up to 10% of the total label was present in Na,K ATPase. These results are discussed with reference to the use of minilobule culture to analyse further synthesis and assembly during biogenesis and control of these processes.

The use of a hidden metal-capture reagent for the measurement of Na+--K+-ATPase activity: a new concept in cytochemistry.

The original lead-trapping method for demonstrating Na+--K+-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K+-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na+--K+-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.

Enucleation of differentiated murine erythroleukemia cells in culture.

Friend murine leukemia cells induced to undergo erythrocytic differentiation by dimethyl sulfoxide give rise to progeny resembling ortho- or polychromatic normoblasts, which usually do not complete the maturation process to yield forms analogous to erythrocytes. Treatment of these differentiated cells with cytochalasin B can lead to a high proportion (i.e., 80-85%) of enucleated cells in vitro. Nuclear extrusion in cells induced to differentiate by dimethyl sulfoxide and subsequently treated with cytochalasin B began within 24-36 hr of exposure to the antibiotic, with the appearance of a pre-enucleated stage in which the cell nucleus became pycnotic and eccentrically located. Maximum enucleation occurred after 72-96 hr of exposure to cytochalasin B and was significantly enhanced when dimethyl sulfoxide was included in the culture medium during the period of treatment with cytochalasin B. Enucleation appeared to take place only in differentiated progeny, because nondifferentiated cells treated with cytochalasin B alone yielded a population of multinucleated cells. The findings indicate that highly tumorigenic nondifferentiated Friend erythroleukemia cells can be converted in high yield to mature enucleated forms that are unable to proliferate in vitro.

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.

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.

Ouabain binding during plasma membrane biogenesis in duck salt gland.

The conditions necessary for optimal ouabain binding in the avian salt gland were examined. Binding was enhanced by ATP and Mg2+ and was decreased by K+, but was unaffected by added Na+. Both maximal binding and complete inhibition of Na, K-ATPase activity were obtained at 1 X 10(-6) M ouabain. Half maximal binding and half maximal inhibition of Na, K-ATPase activity were obtained at 1.7 X 10(-7) M ouabain. Ouabain binding increased in parallel with increasing specific activity of the Na, K-ATPase duringsalt-induced salt gland specialization. The ratio of Na, K-ATPase activity to ouabain-binding sites remained constant during the salt stress as well as after removal of the salt diet. Autoradiography indicated binding to partially and fully differentiated secretory cells of the salt gland. The ouabain binding assay appeared to be a more useful indicator of membrane amplification than Na, K-ATPase activity since it is rapid, essentially irreversible, less sensitive to tissue fixatives, and quantitatively measured the number of enzyme molecules.