The pharmacist's role in the recognition and management of insomnia.

Pharmacists, in collaboration with physicians, are in a unique position to assist the many patients who complain of disturbed sleep and/or impaired daytime functioning. The aim of this article are to familiarize physicians with the role that is and can be played by the pharmacist and to reinforce for practicing pharmacists their multifaceted approach to the insomniac patient. Suggestions are made for pharmacist involvement in patient evaluation, education, and monitoring, and for other possible roles of the pharmacist in dealing with the insomniac patient and the appropriate use of hypnotics.


The Drosophila ncd microtubule motor protein is spindle-associated in meiotic and mitotic cells.

The nonclaret disjunctional (ncd) protein is required for normal chromosome distribution in oocytes and early embryos. Mutants of ncd cause frequent nondisjunction and loss of chromosomes, suggesting a role for the protein in spindle function or chromosome movement in meiosis and early mitosis. The ncd protein contains a region of predicted sequence similarity to the microtubule motor protein, kinesin. In vitro motility assays have demonstrated that ncd is a motor that unexpectedly moves toward the minus ends of microtubules, opposite to the direction of kinesin movement. Using antibodies directed against nonconserved regions of the protein, we have localized the ncd motor protein to the meiotic and early mitotic spindle, and to spindles in a mitotically dividing cultured cell line. Its presence in the spindle of meiotic and mitotic cells implies a role for the protein as a spindle motor. The motor may play an essential role in establishing spindle bipolarity in meiosis.


The pebble gene is required for cytokinesis in Drosophila.

Cytokinesis is developmentally controlled during Drosophila embryogenesis. It is omitted during the initial nuclear division cycles. The nuclei of the resulting syncytium are then cellularized at a defined stage, and cytokinesis starts in somatic cells with mitosis 14. However, cytokinesis never occurs in somatic cells of embryos homozygous or transheterozygous for mutations in the pebble gene. Interestingly, the process of cellularization, which involves steps mechanistically similar to cytokinesis, is not affected. Moreover, all the nuclear aspects of mitosis (nuclear envelope breakdown, chromosome condensation, spindle assembly and function) proceed normally in pebble mutant embryos, indicating that pebble is specifically required for the coordination of mitotic spindle and contractile ring functions. The pebble phenotype is also observed, but only with very low penetrance, during the early divisions of the germ line progenitors (the pole cells). alpha-Amanitin injection experiments indicate that these early pole cell divisions, the first cell divisions during embryogenesis, do not require zygotic gene expression. These divisions might therefore rely on maternally contributed pebble function. The maternal contribution from heterozygous mothers might be insufficient in rare cases for all the pole cell divisions.


Kinetochore formation and behaviour following premature chromosome condensation.

The potential for interphase centromeres to support kinetochore formation following premature chromosome condensation (PCC) has been investigated. We show that the centromere remains competent to initiate kinetochore formation throughout the cell cycle. PCC-kinetochores display a typical trilaminar morphology, associate with microtubules and show movement towards the centrosome. Indirect immunofluorescence studies illustrate that the centromere/kinetochore region of prematurely condensed chromosomes associates with proteins that are normally found within this region in both a cell cycle-dependent and an independent manner.


Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells.

We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying mono- and two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.


Dolichol delays G1-arrest for one cell cycle in human fibroblasts subjected to depletion of serum or mevalonate.

It is well-established that some product(s) or metabolite(s) of mevalonate is (are) critical for growth of mammalian cells. In the search for this (these) compound(s) it seems meaningful to distinguish between compounds needed for cell cycle progression in proliferating cells and compounds needed for growth activation of arrested cells. By using time-lapse video recording we have studied the possible regulatory role of cholesterol, dolichol and mevalonate in the cell cycle of human diploid fibroblasts (HDF). HDF, which are serum-dependent, were rapidly growth-arrested in the first part of G1 upon removal of serum factors. They also responded to mevinolin (an HMG CoA reductase inhibitor) by a similar G1-block, indicating that a mevalonate-derived product is involved in the G1-located cell cycle control of HDF. Interestingly, dolichol counteracted the G1-block caused by both types of treatment. Hence, the early G1-cells could traverse the remainder of the cell cycle and divide despite depletion of serum or mevalonate. We also demonstrated that addition of dolichol resulted in a significant decrease in the rate of protein degradation. This protein stabilizing effect may constitute the mechanism by which dolichol delays the G1-arrest of HDF.


Very low cadmium concentrations stimulate DNA synthesis and cell growth.

Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death. On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.


Cell phenotype, binding affinity and promoter structure modulate transactivation by HNF1 and LAP.

To evaluate the importance of the transcription factors known to bind to the albumin promoter as well as the parameters involved in their activity, we have used cotransfections with an albumin promoter-cat plasmid combined with expression vectors driving the expression of cDNAs coding for liver-enriched factors known to interact with this promoter. We describe the characteristics of a set of clones of hepatic origin: well differentiated, partial variants or pleiotropic dedifferentiated variants. These lines have been characterized for the accumulation of RNAs corresponding to each of the albumin promoter-binding factors. Only HNF1, and to a lesser extent C/EBP, show differences depending upon the differentiation state of the cells. Overexpression of exogenous HNF1 in these cells reveals that this factor is able to transactivate the albumin promoter only in variant cells where the endogenous protein is limiting. By contrast, if the HNF1-binding site is of weak affinity, overexpression of exogenous HNF1 stimulates the albumin promoter even in the HNF1-rich differentiated cells. Overexpression of exogenous LAP strongly transactivates an artificial promoter containing one LAP-binding site, but surprisingly in all the cell lines, it has little effect upon the albumin promoter. These results demonstrate that the transactivation potential of a given transcription factor depends on the degree of differentiation of the recipient cells, on the promoter structure, and on the affinity of the binding site for this factor.


Chondrons from articular cartilage. V. Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy.

The pericellular microenvironment around articular cartilage chondrocytes must play a key role in regulating the interaction between the cell and its extracellular matrix. The potential contribution of type VI collagen to this interaction was investigated in this study using isolated canine tibial chondrons embedded in agarose monolayers. The immunohistochemical distribution of an anti-type VI collagen antibody was assessed in these preparations using fluorescence, peroxidase and gold particle probes in combination with light, confocal and transmission electron microscopy. Light and confocal microscopy both showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte with reduced staining in the tail region and the interconnecting segments between adjacent chondrons. Minimal staining was recorded in the territorial and interterritorial matrices. At higher resolution, type VI collagen appeared both as microfibrils and as amorphous deposits that accumulated at the junction of intersecting capsular fibres and microfibrils. Electron microscopy also showed type VI collagen anchored to the chondrocyte membrane at the articular pole of the pericellular capsule and tethered to the radial collagen network through the tail at the basal pole of the capsule. We suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the collagens, proteoglycans and glycoproteins of the pericellular microenvironment. Secondly, specific cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. In this way type VI collagen could provide a close functional interrelationship between the chondrocyte, its pericellular microenvironment and the load bearing extracellular matrix of adult articular cartilage.


Human articular surface chondrocytes initiate alkaline phosphatase and type X collagen synthesis in suspension culture.

The type X collagen is a short chain collagen associated with calcific cartilage and/or the expression of the hypertrophic chondrocyte phenotype. In articular cartilage, type X collagen is restricted to the basal zone of calcified cartilage adjacent to the subchondral bone. However, during pathological change such as in osteoarthritis, the synthesis of type X collagen becomes more widespread but never extends to the articular surface. Using immunocytochemistry and fluorography of newly synthesised collagens, we report that surface articular chondrocytes (which occupy the uppermost 10-15% of the tissue depth) from normal human cartilage initiate de novo synthesis of both type X collagen and alkaline phosphatase when maintained in suspension culture.


Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes.

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.


Effects of the expression of mammalian annexins in yeast secretory mutants.

The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.


Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages.

The continued success of Leishmania as an intramacrophage parasite is dependent on its ability to survive within an acidic intracellular compartment, resist degradation by lysosomal hydrolases, exploit the host cell as a source of nutrients, and avoid the macrophage's antigen-presenting capabilities. All these requirements are dependent on the properties of the parasitophorous vacuole in which Leishmania resides. This study shows that the vacuole possesses membrane proteins characteristic of a lysosome, and has MHC class II molecules. The trafficking of a variety of endocytic markers supports this finding. However, a temporal study up to 14 days post-infection indicates that, as it matures, the vacuole gains mannose 6-phosphate receptor, and becomes more accessible to endocytosed ligand, suggesting that the vacuole has functionally translocated from a lysosomal to late endosomal compartment. Endocytosed material was detected in the flagellar pocket and inside the amastigote, demonstrating parasite uptake of intra-vacuolar material. Careful analysis of amastigotes suggests that they avoid antigen presentation by their host cell by limiting the release of potential antigens. These findings significantly extend our understanding of the mechanisms employed by Leishmania to ensure its survival in the macrophage.


Contact stimulation of cell migration.

Mass migrations of dense cell populations occur periodically during embryonic development. It is known that extracellular matrices, through which the cells migrate, facilitate locomotion. However, this does not explain how cells, such as neural crest, can migrate as a dense cohort of cells in essentially continuous contact with one another. We report here that unique behavioral characteristics of the migrating cells may contribute to cohesive migration. We used time-lapse video microscopy to analyze the migration of quail neural crest cells and of two crest derivatives, human melanoma cells and melanocytes. These cells migrated poorly, if at all, when isolated, but could be stimulated up to 200-fold to travel following contact with migrating cells. This phenomenon, which we have termed "contact-stimulated migration," appeared to activate and sustain migration of the mass of cells. Cells that became dissociated from the others ceased directional migration, thereby limiting aberrant cell dispersion. Fibroblasts were minimally responsive to this novel phenomenon, which may be crucial for major, mass cell migrations.


Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape.

Studies were carried out with capillary endothelial cells cultured on fibronectin (FN)-coated dishes in order to analyze the mechanism of cell and nuclear shape control by extracellular matrix (ECM). To examine the role of the cytoskeleton in shape determination independent of changes in transmembrane osmotic pressure, membranes of adherent cells were permeabilized with saponin (25 micrograms/ml) using a buffer that maintains the functional integrity of contractile microfilaments. Real-time videomicroscopic studies revealed that addition of 250 microM ATP resulted in time-dependent retraction and rounding of permeabilized cells and nuclei in a manner similar to that observed in intact living cells following detachment using trypsin-EDTA. Computerized image analysis confirmed that permeabilized cells remained essentially rigid in the absence of ATP and that retraction was stimulated in a dose-dependent manner as the concentration of ATP was raised from 10 to 250 microM. Maximal rounding occurred by 30 min with projected cell and nuclear areas being reduced by 69 and 41%, respectively. ATP-induced rounding was also accompanied by a redistribution of microfilaments resulting in formation of a dense net of F-actin surrounding retracted nuclei. Importantly, ATP-stimulated changes in cell, cytoskeletal, and nuclear form were prevented in permeabilized cells using a synthetic myosin peptide (IRICRKG) that has been previously shown to inhibit actomyosin filament sliding in muscle. In contrast, both the rate and extent of cell and nuclear rounding were increased in permeabilized cells exposed to ATP when the soluble FN peptide, GRGDSP, was used to dislodge immobilized FN from cell surface integrin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Cadherin-mediated adhesion in pancreatic islet cells is modulated by a cell surface N-acetylgalactosaminylphosphotransferase.

Rat pancreatic islet cells and RINm5F cells, an islet derived cell line, have at their cell surface an N-acetylgalactosaminylphosphotransferase (GalNAcPTase) similar to that found at the surface of chick neural retina cells and at the rat neuromuscular junction. On islet cells and RINm5F cells the GalNAcPTase is stably associated with cadherin cell-cell adhesion molecules. The effect of antibodies directed against the GalNAcPTase on homophilic, cadherin mediated adhesion was analyzed by measuring their effect on adhesion of islet and RINm5F cells to an immobilized anti-cadherin antibody. In this experimental paradigm anti-GalNAcPtase antibodies completely inhibit cadherin mediated adhesion. Furthermore, cadherin and GalNAcPTase co-distribute in islet and non-islet tissue. We conclude that pancreatic islet cell-cell adhesion is cadherin mediated and under the control of a tightly associated, cell surface GalNAcPTase.


Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.


N-cadherin and N-CAM in myoblast fusion: compared localisation and effect of blockade by peptides and antibodies.

The expression and distribution of two cell adhesion molecules, N-cadherin and N-CAM, at the surface of cultured leg muscle cells from 11-day-old chicken embryos were studied and compared. N-cadherin, which was expressed by fusing myoblasts, was down-regulated on old myotubes while N-CAM was still present. Both molecules, as viewed by confocal microscopy, appeared to have coaccumulated at the areas of contact between fusing myoblasts. However, immunogold electron microscopy did not reveal significant colocalization of N-cadherin and N-CAM, and their segregation after antibody-induced patching suggested the absence of direct interactions between N-cadherin and N-CAM. The role of the Ca2+ dependent cell adhesion molecule N-cadherin in myogenesis was investigated. Myoblast fusion was inhibited (1) with a synthetic peptide containing the H-A-V sequence and (2) with a monoclonal anti-N-cadherin antibody, demonstrating that N-cadherin-mediated cell adhesion is required for myoblast fusion. Under the same conditions no effect of anti-N-CAM antibodies was observed. Taken together these observations suggest that N-cadherin, acting independently from N-CAM, is a major cell adhesion molecule involved in embryonic myoblast fusion in vitro.


Molecular characterization of the secondary constriction region (qh) of human chromosome 9 with pericentric inversion.

Pericentric inversion of the secondary constriction region (qh) of human chromosome 9 is a frequent occurrence. This structural alteration is regarded as a normal familial variant, termed heteromorphism, and is inherited in a Mendelian fashion without any apparent phenotypic consequences. We characterized the qh region of chromosome 9 from five individuals using a series of molecular cytogenetic techniques. Four out of the five individuals have an additional area composed of alphoid DNA sequences on the inverted chromosome 9 while one case was found to have an apparently intact alphoid DNA sequence. Although the direct function(s) of alphoid DNA sequences remain unclear, the centromeric breakage involving these sequences in inverted chromosome 9 raises a series of questions pertaining to the monocentric, dicentric and pseudodicentric nature of pericentric inversions. Nevertheless, these findings have prompted us to suggest that the structural organization of alphoid DNA sequences of the centromeric region of chromosome 9 are apparently "breakage prone" and may be associated with a higher incidence of pericentric inversions. Furthermore, the hierarchical organization of various satellite DNA families (alpha-satellite, beta-satellite and satellite III) within the primary and secondary constriction regions of chromosomes 9 are elucidated here.


Characterization of the human involucrin promoter using a transient beta-galactosidase assay.

Involucrin, a component of the cornified cell envelope, is expressed specifically in differentiating keratinocytes of stratified squamous epithelia. To explore the regulation of involucrin expression, 3.7 kb of upstream sequences of the human involucrin gene was cloned into a plasmid containing a beta-galactosidase reporter gene and transfected into early passage keratinocytes and a variety of human cell types. The full-length construct gave maximal and tissue-specific expression. Deletion analysis showed that sequences between 900 and 2500 bp upstream of the transcriptional start site and the intron located between the transcriptional and translational start sites were required for maximal expression. Further analysis of the intron indicated that its effects on expression were independent of it being present in nascent RNA and suggested that sequences within the intron have regulatory activity. These results suggest that the involucrin intron operates in vivo to regulate expression in the epidermis.


Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau.

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.


Brain microtubule-associated proteins modulate microtubule dynamic instability in vitro. Real-time observations using video microscopy.

We used video assays to study the dynamic instability behavior of individual microtubules assembled in vitro with purified tau, purified MAP2 or a preparation of unfractionated heat-stable MAPs. Axoneme-nucleated microtubules were assembled from pure tubulin at concentrations between 4 and 9 microM in the presence of MAPs, and observed by video-differential interference contrast microscopy. Microtubules co-assembled with each MAP preparation exhibited the elongation and rapid shortening phases and the abrupt transitions (catastrophe and rescue) characteristic of dynamic instability. Each MAP preparation increased the microtubule elongation rate above that for purified tubulin alone by decreasing the tubulin subunit dissociation rate during elongation. The brain MAPs used in this study reduced the rate of microtubule rapid shortening, but allowed significant loss of polymer during the shortening phase. Purified tau and MAP2 decreased the frequency of catastrophe and increased the frequency of rescue, while the heat-stable MAPs suppressed catastrophe at all but the lowest tubulin concentrations. Thus, each of these MAPs modulates, but does not abolish, dynamic instability behavior of microtubules. We propose a model to explain how MAP2 and tau bind to the microtubule lattice at sites along protofilaments so that the MAPs promote polymerization, but do not significantly block the mechanism of rapid shortening inherent in the tubulin lattice. Rapid shortening, when it occurs, proceeds primarily by the dissociation of short fragments of protofilaments, which contain the bound MAPs.


Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation.

Serotonin, an activator of adenylate cyclase, stimulates motility in molluscan gill cilia and sperm flagella. To determine and compare potential targets of cAMP action, dynein was prepared from the lateral gill.cilia and sperm flagella of the mussel Mytilus edulis and the clam Spisula solidissima. In the flagella of both species, high-salt extraction removes about half of the ATPase activity, half of the alpha and beta heavy chains, and the outer arms. The dynein from both species sediments at 18-20 S, contains two or three intermediate chains, and three light chains. High-salt plus detergent removes most of the remaining dynein ATPase, alpha and beta heavy chains, and inner arms, also yielding a stable 18-20 S particle. In gill cilia of both species, high-salt extraction removes only 12-18% of the ATPase, up to 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and a subset of the outer arms. The dynein sediments at 18-20 S and, in Spisula, the heavy, intermediate, and light chains precisely co-sediment. High-salt plus detergent removes another 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and the remaining outer arms. The ATPase sediments mainly as a 13-14 S form showing considerable dissociation of co-sedimenting intermediate and light chains. The inner arms and at least half of the ciliary dynein ATPase activity remain unextractable, corresponding in mass mainly to an apparent beta heavy chain that is vanadate-cleavable. Cyclic AMP-dependent, calcium-independent phosphorylation takes place on specific dynein light chains in cilia but on only the dynein alpha heavy chain in flagella. Pre-activation of the flagella prevents subsequent addition of labeled phosphate. Phosphorylation has no effect on the steady-state ATPase properties. The single phosphate added to the flagellar alpha chain is located within the LUV1 vanadate photocleavage fragment. Considering the probable locus of the light chains and the site of the alpha heavy chain phosphorylation, both beyond the active site and toward the base of the molecule, these distinct phosphorylations may regulate dynein action by modulating arm flexibility or interaction.


Adsorption behaviour of lipase from Staphylococcus carnosus on a hydrophobic adsorbent.

The adsorption of proteins on a solid surface with a subsequent desorption is a well known final purification step in downstream processing. Here the adsorption behaviour of a microbial lipase on the hydrophobic Fractogel TSK butyl 650 in a crude fermentation supernatant is investigated. The measured equilibrium curves differ from fermentation to fermentation by up to +/- 65%. The adsorption capacity increases with decreasing particle diameter of the adsorbent and is influenced by the method of contacting the supernatant with the adsorbent. The rate of desorption depends largely on the adsorption conditions, which is an indication of different orientations of the adsorbed enzyme.


Study of protein-ligand binding effects by direct chromatographic on-column injection.

The direct zonal on-column injection method was applied to a high-performance liquid chromatographic study of pollutant-protein binding interactions in solution. The protein and the protein-ligand complex are excluded on the basis of the size from the diol support, and the free ligand penetrates into the pores and is more retained. The pattern of the ligand elution profile depends on the protein-ligand dissociation constant. This effect was quantitatively analysed by developing a numerical simulation algorithm in which the column is divided into slices of given thickness. The column length, flow-rate and shape of the injection signal are given as input parameters. A global dispersion coefficient accounts for peak broadening. A rapid equilibrium is assumed with the hypothesis that a monovalent ligand interacts with a single binding site on the protein. The interaction of bovine serum albumin with pentachlorophenol was studied, and an apparent dissociation constant for the protein-ligand complex was determined by fitting the theoretical profile to the experimental one. The effect of the acetonitrile content in the solvent was studied. An important decrease of the dissociation constant is observed that affects the chromatographic elution pattern.


Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods.

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughout. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 microns). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-microns preparative column, led to the conclusion that the overloaded 20 microns column gave a higher throughput than the 40-microns column.


Fast separation of macrophages by retention on cross-linked amylose and release by enzymatic amylolysis of the chromatographic material.

Macrophages from mice peritoneal exudate were isolated on basis of specific adherence on epichlorohydrin cross-linked amylose (CLA), a chromatographic gel presenting a high susceptibility to advanced amylolysis with alpha-amylase. The cell suspension, containing predominantly macrophages and lymphocytes, was applied onto the column and incubated for 30 min at 37 degrees C for the adherence of macrophages. After this interval the non-adherent cells were eluted with buffered medium and the CLA support was incubated in the column with an alpha-amylase-buffered solution liquefying the matrix and releasing, in situ, the adherent cell population containing 90% macrophages with a viability higher than 90%.


Quantitative separation of Trichoderma lipid classes on a bonded phase column.

Bond Elut aminopropyl columns were used to purify the different lipid classes of Trichoderma harzianum and Trichoderma viride. This methodology permitted good separation of the fungal lipid classes in less time than traditional techniques. The incorporation of [1 (14)C]linoleic acid into neutral lipids, free fatty acids and phospholipids was quantified for both strains. The fatty acid profile of the different lipid classes of these fungal strains was determined as a function of culture time.


Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.


Rational design of purification processes for recombinant proteins.

This paper discusses the elements important for rational design of purification processes for recombinant proteins. Main issues involved in selection of operations and process design are reviewed with particular emphasis on the challenges posed by recombinant proteins. This includes thermodynamic characterization of target protein and main contaminants, use of correlations and of expert knowledge for the development of an expert system for optimization and design (selection) of separation and purification (chromatographic) processes. The main deficiency in accurate information for rational process selection is in that required for high-resolution chromatographic processes. The authors show that a database with detailed information on properties of the main contaminants present in the fermentation streams of usual recombinant protein sources can be integrated to an expert system with an open architecture. This will allow more precise selection of unit operations for the design of protein purification processes.


Immunoaffinity chromatography in biorecovery: an application of recombinant DNA technology to generic adsorption processes.

The constant region of human kappa light chain (Ck) was linked to Escherichia coli beta-galactosidase, using standard molecular cloning techniques. The binding of Ck-beta-galactosidase fusions to a number of different murine monoclonal antibodies, specific for Ck, was improved by the insertion of spacers between Ck and beta-galactosidase: a cleavable linker was then introduced. Over-expressed Ck-beta-galactosidase fusion protein was purified using monoclonal antibodies immobilised on Sepharose 4B. Elution conditions were found that maintained beta-galactosidase activity so purified enzyme could be released on breaking the cleavable linker. A number of practical problems associated with maintaining stable fusion proteins and immunoaffinity column performance were identified.


Detection of fungal carbohydrate antigens by high-performance immunoaffinity chromatography using a protein A column with covalently linked immunoglobulin G.

Fungal carbohydrate antigens were analysed by high-performance immunoaffinity chromatography (HPIAC) with immunoglobulin G (IgG) antibodies raised against extracellular polysaccharides of Mucor racemosus. The protein A-IgG complex was covalently bound with dimethyl pimelimidate, which enabled the use of strong acidic buffers to release the tightly bound antigens from the column. Prior to pulsed-amperometric detection, an anion-micromembrane suppressor was used to raise the pH of the effluent to above 12 without dilution. The HPIAC system provides a sophisticated method for the rapid and sensitive detection of antigenic oligomeric carbohydrates in biological samples and is proposed as an alternative to quantitative enzyme-linked immunosorbent assay techniques.


Purification of glycogen phosphorylase isozymes by metal-affinity chromatography.

Mammalian phosphorylase isozymes from muscle, brain and liver were expressed in Escherichia coli and purified from the crude bacterial cell extracts in one step using a copper-loaded, metal-affinity matrix. Good chromatographic behavior, enzyme activity and protein stability were maintained by judicious choice of pH and buffer which contained 250 mM sodium chloride and 25 mM beta-glycerophosphate at pH 7.0. Small amounts of beta-mercaptoethanol and EDTA in the buffers further stabilized the enzymes, but stripped some of the metal from the column which, nonetheless, retained good chromatographic characteristics. Owing to the presence of multiple surface histidine residues in the phosphorylase dimers, good enzyme purities (90-98%) and recoveries (>90%) were routinely obtained from crude bacterial lysates after two passes through the copper column. Of the various metal ions which were investigated, Cu2+ gave the best chromatographic results. Imidazole gradients at constant pH were used to selectively desorb the phosphorylase from the metal column whose capacity for phosphorylase binding in the presence of bacterial proteins exceeded 30 mg enzyme per milliliter of matrix.


Purification of S-oxynitrilase from Sorghum bicolor by immobilized metal ion affinity chromatography on different carrier materials.

The purification of the hydroxynitrile lyase (EC 4.1.2.11, S-oxynitrilase) from Sorghum bicolor is compared using different strategies. A new procedure is presented, which exploits the affinity of S-oxynitrilase towards metal ions as a key step in purification. The metal ions are immobilized by chelators on different carrier materials, e.g. Sepharose beads, microporous membranes or poly(ethylene glycol). A systematic examination demonstrates the excellent potential of immobilized metal affinity chromatography as a preparative separation method.


Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.


Modified on-column interface for coupled high-performance liquid chromatography-gas chromatography and its application to the determination of levamisole in milk.

A modified on-column interface is reported for the coupling of high-performance liquid chromatography with gas chromatography, incorporating an adapted, commercially available multidimensional gas chromatography switching system. Novel features include cryogenic analyte focusing, total solvent exclusion from the analytical column and independent carrier gas supplies to the analytical GC column and uncoated pre-column. The instrumentation was used for the determination of the veterinary anthelmintic drug levamisole in milk with analyte detection by both flame ionisation and nitrogen-phosphorus detectors. Detection limits for the assay were 2.2 micrograms l-1 and 0.4 micrograms l-1 by flame ionisation and nitrogen-phosphorus detectors, respectively. The assay was applied to a survey of fourteen milk samples from different dairy outlets.


O-(2,3,4,5,6-pentafluorophenyl)methylhydroxylamine hydrochloride: a versatile reagent for the determination of carbonyl-containing compounds.

A review on the use of O-(2,3,4,5,6-pentafluorophenyl)methylhydroxylamine hydrochloride (PFBHA) for the determination of carbonyl-containing compounds is presented. PFBHA has been used in the determination of such diverse compounds as thromboxane B2, prostaglandins, amygdalin and a variety of other aldehydes, ketones and acids. PFBHA has been used for the determination of these compounds found in water, blood, urine, air and even clothing. The review covers literature referenced in Chemical Abstracts from 1975, when PFBHA was first synthesized, through March 1992.


Characterization of the influence of displacing salts on retention in gradient elution ion-exchange chromatography of proteins and peptides.

It has been shown earlier that the choice of displacing salt has a large effect on the retention in ion-exchange chromatography of proteins and peptides. The influence of different displacing salts cannot be predicted or quantitatively explained, owing to the current lack of an adequate theoretical framework. In this work a general characterization is made by using a considerable number of proteins and peptides and all displacing salts found feasible. Principal component analysis is used to interpret the large amount of data that is generated. The results of the analysis indicate that most of the retention variations are due to non-specific effects and can be explained by changes in the apparent gradient slope, i.e., the increase in elution strength per unit volume, and the elution strength of the starting buffer. This differs from the interpretation given earlier, where the selectivity changes were attributed to specific effects of the salts. However, as it is impossible to test all existing proteins and peptides, specific effects are still possible, but they might be less common than previously considered.


Immobilized metal ion affinity chromatography of synthetic peptides. Binding via the alpha-amino group.

Peptides synthesized by the solid-phase method can be efficiently purified in a single immobilized metal affinity chromatography step based on interaction with the alpha-amino group if, after coupling of each amino acid residue, unreacted amino groups are irreversibly blocked by acetylation and if no strongly metal-binding amino acids (His, Trp, Cys) are present in the sequence. A difference in basicity for alpha- and epsilon-amino functions of ca. 2 pH units is sufficiently large to allow selective binding of peptides to immobilized metal ions via the unprotonated alpha-amino group. The binding is pH-dependent: on Cu(2+)- and Ni(2+)-loaded supports most peptides are maximally retarded at pH values around 7.5 and 8.5, respectively. The decreased binding strength at lower pH values is due to protonation of the alpha-amino function, whereas the reduced affinity at higher pH is caused by metal ion transfer from the matrix to the peptide. The metal ion is captured in a multidentate chelate where, in addition to the alpha-amino group, up to three adjacent deprotonated amide nitrogens are coordinated to the metal. If the pH is raised further, additional metal ions may be bound in biuret-like structures. Immobilized Ni2+, owing to its higher selectivity and affinity, is the preferred chromatographic support if slightly basic conditions can be tolerated.


Purification, subunit structure and inhibitor profile of cathepsin A.

Cathepsin A (EC 3.4.16.1), a lysosomal carboxypeptidase, has been purified 1374-fold from pig kidney. Purification steps included concanavalin A-Sepharose and phenyl-Sepharose chromatography and chromatofocusing. The specific activity (16.9 U/mg) of the purified enzyme was significantly higher than previously reported values. The enzyme preparation appeared homogeneous when analyzed by non-denaturing polyacrylamide gel electrophoresis and was free of detectable protease contamination. The molecular mass (M(r) = 97,000), isoelectric point (5.0), and sensitivity to inhibitors were consistent with reported properties of cathepsin A. However, the previously reported three-peptide chain structure was not observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol demonstrated that the enzyme is composed of two M(r) 47,000 subunits, each of which dissociate in the presence of 2-mercaptoethanol into two polypeptide chains of 19,000 and 31,000.


Purification and determination of the binding site of lactate dehydrogenase from chicken breast muscle on immobilized ferric ions.

Lactate dehydrogenase from chicken breast muscle was purified to homogeneity in one step by immobilized metal ion affinity chromatography. The purified enzyme was used to localize the binding site to immobilized Fe(III) ions. After cyanogen bromide degradation and digestion with trypsin, small enzyme fragments capable of binding to immobilized Fe(III) ions were obtained. It is proposed that several histidyl groups are involved in the binding.


Supercritical fluid chromatography of Fusarium mycotoxins.

Capillary- and packed-column supercritical fluid chromatography has been used for the separation of Fusarium mycotoxins of various structure types such as the trichothecenes including deoxynivalenol and its acetylated derivatives and T-2 toxin, as well as butenolide, culmorin, sambucinol and zearalenone. The effect of modifier concentration and column temperature and pressure was also studied. Retention indices based on alkylphenones were determined for these mycotoxins on two of the capillary columns employed.


Determination of propionate in bread using capillary zone electrophoresis.

A method for the determination of propionate in bread is described. The propionate was extracted from the bread with a repeated extraction procedure and measured using capillary zone electrophoresis in the indirect UV mode applying a background electrolyte of 0.005 M Tris adjusted at pH 4.6 by adding benzoic acid. Using laboratory-baked bread containing known amounts of sodium propionate, recoveries of ca. 95% could be established, validating the method.


Determination of oxytetracycline in marine shellfish (Crassostrea gigas, Ruditapes philippinarum and Scrobicularia plana) by high-performance liquid chromatography using solid-phase extraction.

A reversed-phase high-performance liquid chromatographic method was developed to detect oxytetracycline (OTC) in three species of marine shellfish (Crassostrea gigas, Ruditapes philippinarum and Scrobicularia plana). Shellfish tissues were analysed after solid-phase extraction by using a mobile phase containing acetonitrile and 0.02 M orthophosphoric acid solution. The linearity and precision of the method were checked over the concentration range 0.100-1.500 micrograms/g. The limits of detection and determination of OTC were 0.040 and 0.100 microgram/g, respectively. The recovery of OTC from spiked shellfish tissues was 79.8%. The OTC concentration in oyster (Crassostrea gigas) spiked at 0.500 microgram/g and stored at -20 degrees C was stable for 6 weeks. The method was applied to a 21-day experimental study performed with oysters.


"Paralogs", sorbent families for protein separations.

Novel peptide-based (paralog) sorbents are evaluated with respect to performance, reproducibility and reusability in a 96-well test plate screening format, and to utility in protein separations. The results demonstrate that this approach to constructing sorbents provides a new and generally applicable set of tools for separating proteins.


Determination of fatty acids in fish oil dietary supplements by capillary chromatography with laser-induced fluorescence detection.

The 4-bromomethyl-7-methoxycoumarin derivatives of 14 saturated and unsaturated fatty acids, including the omega-3 fatty acids, were separated by reversed-phase liquid chromatography and detected by laser-induced fluorescence. Baseline resolution was obtained by using a high-efficiency packed capillary column with 240,000 theoretical plates, together with a systematic optimization of the mobile phase composition. The retention indices of the fatty acid derivatives correlated well with a predictive empirical model, showing accuracy better than 0.46% relative error and reproducibility better than +/- 0.1% relative standard deviation. The physiologically important fatty acids with 12-22 carbon atoms and 0-6 double bonds were determined at the femtomole level in fish oil dietary supplements by using this methodology.


The autistic label and its potentially detrimental effect on the child's treatment.

This study asks whether adults teach a child labeled "autistic" differently from a child labeled "normal". Twenty undergraduate college students served as student-teachers. Results showed that the student-teachers gave significantly more praise and reward marbles for incorrect responses, as well as significantly less verbal correction, when the child was labeled "autistic". The inference is that such treatment could make it more difficult for the autistic child to acquire new skills.


A methodological critique of the current status of eye movement desensitization (EMD).

Eye Movement Desensitization (EMD) has been recently advocated as a rapid treatment for the elimination of traumatic memories responsible for the maintenance of a number of anxiety disorders and their clinical correlates. Despite a limited conceptual framework, EMD has attracted considerable interest among clinicians and researchers. The popularity and interest generated by EMD will likely result in wide usage. We present a methodological critique of it with reference to assessment, treatment outcome, and treatment process. We also provide guidelines for judging the methodological adequacy of research on EMD and suggest intensive research to assess effectiveness, treatment components, and comparisons with other procedures.


Eye movement desensitization: a critique of the evidence.

The scientific evidence supporting the efficacy of eye movement desensitization (EMD), a novel intervention for traumatic memories and related conditions, is reviewed. The sparse research conducted in this area has serious methodological flaws, precluding definite conclusions regarding the effectiveness of the procedure. Clinicians are cautioned against uncritically accepting the clinical efficacy of EMD.


A meta-analysis of treatments for panic disorder with agoraphobia: imipramine, alprazolam, and in vivo exposure.

The most common pharmacological treatments for panic disorder with agoraphobia (PDA) include the use of imipramine and alprazolam while the most common behavior therapy is the use of graded in vivo exposure. Several studies have found these treatments to be superior to placebo in the treatment of PDA, but it has not been clear if there are differences among these three treatments. It has also not been clear for what aspects of PDA each treatment is the most effective. The purpose of this study was to conduct a meta-analysis of the results of relevant treatment outcome studies on a number of dependent variables (e.g., panic attack severity, dysphoria, avoidance behavior). Few studies satisfied the minimum criteria for inclusion and the final data pool consisted of 34 treatment studies. Imipramine was found to be generally ineffective for most variables. Alprazolam was significantly effective for panic and anxiety variables in PDA, while exposure was significantly effective for phobia variables. Exposure had the most consistently strong effect sizes.


Booster sessions and long-term effects of behavioral family therapy on adolescent substance use and school performance.

This study investigated the long-term impact on adolescent substance use and academic failure of 6 months of booster sessions following the termination of behavioral family therapy. Subjects were four 14-16-year-old white and Hispanic males and females whose parents or teachers had referred them for 5-12 months of behavioral family therapy. The first subject received no booster sessions; after the typical decrease in substance use and academic problems at the end of treatment, his problems recurred and subsequently worsened during follow-up. The other three subjects responded similarly to behavioral family therapy, but this was followed by 6 months of booster sessions, leading to a second decrease in substance use and academic problems. Improvement was maintained throughout follow-up. These results suggest that booster sessions can prevent the recurrences of adolescent problem behavior that often follow treatment termination.


Treatment integrity of relaxation training for tension headaches.

Treatment integrity procedures, generally lacking in previous research, were employed for evaluation of relaxation training for tension headaches. Treatment integrity is the extent to which the therapist implements the relaxation procedure as described, and the degree to which the patients comply with the therapist's instructions. Objective compliance with the home practice of relaxation training was assessed using a microcomputer-based method which required the patient to squeeze a hand control when instructed to tense a muscle. A single-case replication design with three tension headache patients was used. The dependent variables were taken from patients' self-reported daily headache data. Results indicate that: (a) the therapist accurately adhered to the relaxation training protocol; (b) headache frequency decreased in all patients from baseline to 1-year follow-up (improvements ranged from 72.7% to 98.2%); and (c) improvement was greater with higher compliance.


Somatic symptoms in social phobia: a treatment method based on rational emotive therapy and paradoxical interventions.

Social phobia is often accompanied by somatic symptoms such as trembling, blushing, and sweating. In cases where these symptoms are predominant and, rather than the social situation, represent the feared stimulus, their unpredictable occurrence may reduce the effectiveness of an otherwise successful treatment, such as exposure in vivo. In this study, a paradoxical treatment approach, combined with rational emotive therapy, is employed with three social phobic patients with fears of blushing, trembling, and sweating, respectively. Results indicate that a paradoxical treatment may be an effective strategy in reducing somatic symptoms in social phobia.


Development and validation of a behavioral observation measure for the syndrome of psychosis, intermittent hyponatremia, and polydipsia.

A behavioral observation scale (Virginia Polydipsia Scale; VPS) for monitoring drinking patterns was developed and its reliability tested during 25 hours of tandem ratings among six patients with the syndrome of psychosis, intermittent hyponatremia, and polydipsia (PIPS). These ratings were compared to those collected from a control group of six psychiatric inpatients who were similarly observed for 25 hours. The scale was subsequently used to assess day-long drinking in a single PIPS patient. Results demonstrated that the VPS can be reliably administered by trained raters and that it clearly differentiates the drinking patterns of PIPS patients from controls. In addition, our findings highlight associations among drinking behaviors, psychiatric functioning and low serum sodium concentration. On balance, these results support using observational measures of drinking behaviors in future studies of PIPS patients.


An alternative biofeedback-based behavior modification treatment for a case of chronic constipation.

A 31-year old female with chronic constipation was referred for treatment of a paradoxical contraction of the external anal sphincter (EAS) muscle during straining to defecate. The patient was dependent on laxative use and the digital removal of feces. The present program emphasized biofeedback training of the EAS utilizing the inexpensive, noninvasive procedure of disposable surface electrodes placed on the anal verge. Outcome of treatment resulted in an 89% decrease in laxatives used, complete termination of the digital removal of feces, and a notable decrease in EAS tension. The patient was able to maintain these gains at an 8 month follow-up.


Behavioral analysis and treatment of reflexive vomiting associated with visceral sensations: a case study of interoceptive conditioning?

Behavior analytic methods are applied to the conceptualization and treatment of a complex case of interoceptive classical conditioning. The subject exhibited a gagging and vomiting response to bladder and/or similar visceral sensations. A two-factor learning model of the problem led to a successful exposure and response prevention intervention, which was based on single-case experimental methodology. We discuss the possible relevance of this case in the context of the growing attention to interoceptive conditioning in behavioral problems, and comment upon the importance of behavior analytic principles and procedures to the behavioral clinician.


Opiate antagonists and eating behavior in humans: a review.

Animal studies have demonstrated a robust role for the endogenous opioid system in the control of food intake. In humans, selective opioid antagonists such as naloxone, naltrexone, and nalmefene have been shown in some studies to reduce total food intake by up to 30% and to alter food preferences in short-term experimental trials in normal-weight subjects, as well as in obese and bulimic patients. The value of naloxone and naltrexone in the long-term treatment of eating disordered patients, however, must be considered very limited. The published treatment studies do not justify the routine use of naloxone and naltrexone in patients with Prader-Willi syndrome, obesity, bulimia nervosa, or anorexia nervosa because of their unprofitable risk/benefit ratios, although further work, particularly focused on some of the newer antagonists, should be undertaken.


Pharmacotherapy and pharmacodynamics in the management of bacterial infection.

Minimum inhibitory concentration testing is the most common standard used to evaluate antibacterial activity of antimicrobials against specific pathogens. The consideration of pharmacodynamic factors in conjunction with these tests can improve the management of bacterial infections. Further, the incorporation of MIC values into pharmacodynamic ratios may provide clinically useful tools for selecting optimal antibiotic selection, determining proper dosing strategies, and predicting therapeutic outcomes. Physiologic consequences of infection and antibiotic treatment, such as endotoxin release and initiation of the septic cascade, also must be considered when choosing appropriate anti-infective therapy. The introduction of adjuvant immunotherapy, along with improvement, validation, and implementation of pharmacodynamic predictors of antibiotic efficacy, undoubtedly will provide the medical community with an effective arsenal to further reduce the morbidity and mortality rates associated with bacterial infections.


Effects of meals and meal composition on the bioavailability of fenretinide.

The effects of meals and meal composition on the bioavailability of fenretinide, N-(4-hydroxyphenyl) retinamide, a synthetic retinoid undergoing clinical trials, were examined in two separate studies using an open, randomized, crossover design. In the first study, 13 healthy male volunteers received 300-mg doses of fenretinide (1) while fasting and (2) after a high-fat breakfast. In a subsequent study, 15 subjects received 300 mg fenretinide after each of three different test meals (high-fat, high-protein, and high-carbohydrate) separated by a 1-week washout period. Plasma specimens obtained over a 72-hour period after each treatment were assayed by high-pressure liquid chromatography to characterize the effects of a meal and meal composition on the bioavailability of fenretinide. Results from the initial study demonstrated a significant increase in the bioavailability of fenretinide after a high-fat meal. In the follow-up study, the bioavailability of fenretinide, as assessed by total area under the plasma concentration curve, was three times greater after the high-fat meal than after the high-carbohydrate meal. This supported the findings of the first study. Although to a lesser extent, the high-protein meal also produced a greater area under the curve than the high-carbohydrate meal. These combined findings demonstrate that the bioavailability of fenretinide is markedly influenced not only by administration with meals but also by the specific composition of such meals.


The influence of diltiazem versus cimetidine on propranolol metabolism.

The present study was undertaken to examine whether the inhibitory effect of diltiazem on the metabolism of propranolol differs from that of cimetidine. Six healthy male volunteers received a single oral dose of 20 mg propranolol with pretreatment with placebo, 60 mg diltiazem, or 400 mg cimetidine three times daily for 4 days. Diltiazem and cimetidine increased the area under the concentration (AUC) of propranolol and its glucuronide. Cimetidine also increased the urinary excretion of propranolol glucuronide. There were no significant differences in the AUC of 4-hydroxypropranolol (4OHPPL) and its conjugates or the urinary excretion of conjugated 4OHPPL. Diltiazem increased the AUC of naphthoxylactic acid (NLA) and the urinary excretion to NLA. After cimetidine pretreatment, there was the trend toward a decrease in the partial metabolic clearance to 4OHPPL and a significant decrease in that of NLA. These results suggest that diltiazem and cimetidine inhibit the oxidation pathways of propranolol in different manners. Cimetidine might inhibit both oxidative pathways to 4OHPPL and NLA, whereas diltiazem might not inhibit the pathway to NLA.


Pharmacokinetics of sotalol enantiomers in humans.

The chiral beta-blocker, sotalol (STL), is marketed as a racemic mixture. Although both STL enantiomers have equal Class III antiarrhythmic activity, beta-blocking activity has been ascribed mainly to the R-enantiomer. The pharmacokinetics of STL enantiomers were studied in young (mean age 32 +/- 3 years), healthy male volunteers after oral administration of 160 mg. Subsequent plasma and urine samples were collected over 24 hours, and STL enantiomer concentrations were determined using a stereospecific high-performance liquid chromatography assay. There were no significant differences between pharmacokinetic parameters of enantiomers. The area under the time-concentration curves (mean +/- standard deviation [SD]) were 6.95 +/- 0.85 and 6.76 +/- 1.2 (mg/L)hour for S- and R-STL, respectively. Maximal plasma concentrations of S- and R-STL were 615 +/- 167 and 619 +/- 164 ng/mL, respectively, which were obtained on average, 3.13 +/- 0.60 hours after dosing. The mean residence time (mean +/- SD) was 13.2 +/- 1.2 and 12.9 +/- 1.8 hours for S- and R-STL, respectively. Respective renal clearance values for S- and R-STL were 8.98 +/- 1.5 and 9.46 +/- 2.3 L/hour, and were approximately 1.5 times greater than creatinine clearance. Renal clearance constituted approximately 76% of the oral clearance. Although stereoselective disposition of STL was absent after racemate administration, these results should not be extrapolated to patients with significantly altered physiology, or to the pharmacokinetics of S-STL after administration of pure-S-STL.


The effect of food or sucralfate on the bioavailability of S(+) and R(-) enantiomers of ibuprofen.

This randomized, multiple cross-over pharmacokinetic study was undertaken to determine if food or sucralfate alter the bioavailability of the active S(+) enantiomer of ibuprofen. Eleven healthy adult male volunteers were given three single 600-mg doses of ibuprofen (separated by 1 week) administered either in a fasting state, after a standardized breakfast, or with sucralfate 1 g. The main outcome measures were area under the concentration (AUC), maximum peak plasma concentration (Cmax), and time to reach peak concentration (tmax) for total, S(+), and R(-) enantiomer serum ibuprofen levels, drawn up to 10 hours after dosing. The AUC for R(-) ibuprofen was significantly lower than S(+) ibuprofen in all three treatment groups. The treatments had no different effects on AUC for S(+), R(+), or total ibuprofen. There was no difference in the ratio of S(+):R(-) enantiomers across different treatment groups, but the intersubject variability was significant (P < .05). The S(+) ibuprofen Cmax was greater than the R(-) ibuprofen Cmax for all treatment groups (P < .05). Sucralfate reduced the peak concentration of both S(+) and R(-) enantiomers when compared with fasting (P < .05). There was a slight but nonsignificant increase in the mean time to achieve peak concentration of both S(+) and R(-) enantiomers. Neither food nor sucralfate has a significant effect on ibuprofen enantiomer pharmacokinetics, but interindividual variability contributes significantly to the variability of enantiomer bioavailability.


The effect of antacid and ranitidine on droxicam pharmacokinetics.

Droxicam is a nonsteroidal anti-inflammatory drug that is a pro-drug of piroxicam. The influence of concomitant administration of antacid or ranitidine on droxicam pharmacokinetics has been investigated. On three separate phases, 15 healthy volunteers received a single oral 20-mg dose of droxicam either alone, with antacid (400 mg aluminum hydroxide + 400 mg magnesium hydroxide, three times/day), or with ranitidine (300 mg, two times/day) for 6 days. Piroxicam, the active substance from droxicam, was quantified by high-performance liquid chromatography. The pharmacokinetic parameters for droxicam given alone were: maximum peak plasma concentration (Cmax) = 1.53 +/- .21 micrograms/mL (mean +/- SD), time to peak concentration (Tmax) = 7.5 +/- 2.1 hr, t1/2a = 1.38 +/- .82 hour, t1/2el = 53.3 +/- 11.9 hr, Cl/F = 2.98 +/- .71 mL/min, volume of distribution (Vd/F) = 13.2 +/- 1.8 L and area under the curve (AUC) = 117.6 +/- 26.8 micrograms/hour/mL. The subject effect was significant for all the pharmacokinetic parameters except for the absorption half-life (P < .05). Concomitant antacid or ranitidine administration had no significant effect on any of the droxicam pharmacokinetic parameters. The results of this study suggest that antacid or ranitidine do not significantly alter the oral absorption or pharmacokinetic disposition of single-dose droxicam.


Comparative effects of verapamil and volume overload on atrial natriuretic factors and the renin-angiotensin aldosterone-vasopressin system.

The authors compared the effects of verapamil (120 mg three times daily for 3 days) with those of acute volume expansion with normal saline on the plasma levels of atrial natriuretic factors (ANF), renin (PRA), angiotensin II (AII), aldosterone (ALD), and arginine-vasopressin (AVP) in healthy subjects. A randomized, double-blind, placebo-controlled study of crossover design was employed, where each individual received two acute volume overloads 1 week apart, one during placebo and the other during treatment with verapamil. Verapamil reduced blood pressure (BP) and increased the plasma levels of ANF, PRA, AII, ALD, and AVP. Strong positive correlations were observed between PRA, AII, ALD, and AVP, but not with ANF. Acute volume expansion (1500 mL saline in 15 minutes, in supine legs-up position) similarly to verapamil increased ANF levels; however, opposite to verapamil, it reduced PRA-AII-ALD, did not modify AVP levels, and increased BP. The mechanisms of these changes are discussed. In verapamil-treated subjects, volume expansion produced an additional increase in ANF and inhibited the PRA-AII-ALD axis, suggesting that in young healthy individuals, verapamil does not interfere with the reflex compensatory hormonal mechanisms activated under circumstances of acute volume-salt overload, with rapid expansion of the central vascular compartment. Our study indicates that verapamil and volume expansion represent two different stimuli for ANF secretion associated with opposite changes in the PRA-AII-ALD axis. In addition, verapamil can be used as a tool to study and understand the simultaneous increases in ANF and in PRA, AII, and AVP, characteristics of congestive heart failure.


Pyrazinoylguanidine: antihypertensive, hypocholesterolemic, and renin effects.

In a single-blind, placebo-controlled study of 12 subjects diagnosed as having mild to moderate hypertension and hypercholesterolemia, pyrazinoylguanidine (PZG) in a dose of 600 mg twice daily for 4 weeks reduced systolic blood pressure and heart rate. Pyrazinoylguanidine also reduced diastolic pressures, but to a lesser extent. Pyrazinoylguanidine reduced total serum cholesterol and low-density lipoprotein (LDL). Regression analysis indicated a dose-dependent reduction of both total cholesterol and LDL by PZG, i.e., the higher the presenting serum concentration, the greater the reduction by PZG. The extent of the reductions produced by PZG in elevated cholesterols and LDLs was highly correlated (r = .949). Normal high-density lipoprotein levels were unchanged by PZG. Pyrazinoylguanidine increased 24-hour urine volume and urinary excretion of sodium. Serum Na+, K+, or Cl- concentrations were unaltered. Means for plasma aldosterone and renin activities tended to decrease, but these trends did not attain statistical significance. Pyrazinoylguanidine was well tolerated. An activity profile that includes antihypertensive effects as well as reduction in hypercholesterolemia without major impact on serum renin or electrolyte balance makes PZG an attractive candidate for the management of hypertension.


A study in normal human volunteers to compare the rate and extent of levothyroxine absorption from Synthroid and Levoxine.

Numerous branded and generic formulations of levothyroxine (LT4) sodium tablets are currently available. Results from previous studies attempting to examine the comparative bioavailability of these formulations are difficult to interpret because of subject heterogeneity, single time-point blood sampling, varying degrees of hypothyroidism, and other factors. This study was devised to compare the rate and extent of absorption of LT4 from different LT4 sodium tablet formulations, in a simple model using a single-dose two-way single-blind, randomized cross-over design in 30 normal, healthy, nonpregnant, female subjects. This design controlled for many factors that limited previous LT4 bioavailability studies. Subjects were given a single 600 micrograms dose of LT4 as either Synthroid (Boots Pharmaceuticals, Inc., Lincolnshire, IL) tablets (formulation A) or Levoxine tablets (Daniels Pharmaceuticals, St. Petersburg, FL; formulation B). Measurements of baseline-corrected total T4 serum concentrations determined at multiple time points demonstrated statistically significant differences between the two formulations at the 1.00, 3.00, 5.00, and 18.00 hour sampling times. Statistically significant differences for area under the curve (AUC) (0 to 48 hours) (formulation A, 159.9 +/- 9.4 micrograms-hour/dL; formulation B, 193.4 +/- 10.1 micrograms-hour/dL) and maximum peak plasma concentration (Cmax) (formulation A, 5.91 +/- .34; formulation B, 7.12 +/- .32) also were demonstrated. Furthermore, the ratio of the baseline-corrected total T4 concentrations (B/A x 100) were 120.9% for AUC and 120.5% for Cmax. These data demonstrate that the administration of Synthroid and Levoxine result in a significantly different rate and extent of absorption of LT4, and therefore these two formulations cannot be considered bioequivalent.(ABSTRACT TRUNCATED AT 250 WORDS)


Bioavailability studies of drugs with nonlinear pharmacokinetics: I. Tracer dose AUC varies directly with serum concentration.

The authors show that for a drug cleared by one enzyme the area under the serum concentration-time curve from time 0 to infinity (AUC0-INF) of a test dose can be expressed as AUC0-INF = TD x F x (Km + C)/Vmax where TD is test dose size, F is fraction absorbed, C is drug serum concentration at the time of the study, and Km and Vmax are the Michaelis constant and maximum velocity of the enzyme. This equation predicts the AUC0-INF produced by a given tracer dose of drug will vary directly with C in drugs with nonlinear pharmacokinetic properties (i.e., drugs whose value for C approaches or exceeds Km) if C is held constant by administration of tracer and maintenance doses of drug. The AUC0-INF produced by intravenous tracer doses of 150 mg of 13C15N2-sodium phenytoin was determined in 15 subjects at 30 different values of C. AUC0-INF showed a high degree of direct linear correlation with C (AUC0-INF (ug x hr/mL) = 35.4 + 8.1 x C (ug/mL), r = 0.885, P < .0001). Consequences of this observation for relative bioavailability studies of drugs with nonlinear pharmacokinetic properties are discussed.


Single-dose antibiotic therapy: what has the past taught us?

The proper dosage schedule of antibiotics has generally been determined empirically, due to the difficulty of clinical trials. Initially, the dosage was chosen to allow high sustained levels greater than MIC in the blood. Antibiotics (beta lactams, tetracyclins, macrolides) were given at high doses three to six times daily, whatever their kinetic properties. The data obtained by Eagle3 with beta lactams in animal models of streptococcal and treponemal infections outlined the importance of interval between doses on the in vivo efficacy. They also showed that increasing the dose of penicillin had a positive effect on the bactericidal activity only through the persistence of effective levels (greater than MIC) at the site of infection. Further illustrations were given through experimental and clinical studies with beta lactams or other compounds on different types of infections: LRTIs, UTIs, meningitis, and endocarditis. The importance of both dynamic (i.e., pattern of bactericidal effect) and kinetic (elimination half-life) parameters was thus further identified. Information on toxicity with some compounds with a narrow therapeutic index, such as aminoglycosides, indicated that increasing the dose to enhance efficacy had some limitations. This led to numerous studies on the relations between concentration and toxicity, stating that nephro- or ototoxicity were not directly related to peak level in serum. Experimental studies showed that OD administration of aminoglycosides was both more efficient and less toxic than the multiple-dose regimen of the same daily amount. Economic considerations progressively justified attempts to both reduce the dose and the work load related to multiple administrations.(ABSTRACT TRUNCATED AT 250 WORDS)


Can fluoroquinolones be considered once-daily therapy?

Fluoroquinolone antimicrobial agents inhibit most Enterobacteriaceae at extremely low concentrations, less than or equal to 0.5 microgram/mL. The half-lives of the agents range from 4 to 18 hours. Most of the available fluoroquinolones can be administered once daily to treat urinary tract and diarrheal infections. Newer agents with long half-lives that inhibit gram positive organisms at lower concentrations than the older fluoroquinolones, less than or equal to 1 microgram/mL, and have a long post-antibiotic effect have the potential to be used once daily as treatment of respiratory, skin-structure and selected bone infections as well. Careful clinical studies are needed to establish the efficacy of once daily use of fluoroquinolones, to determine that clinical efficacy is equivalent to multiple doses, and that once-daily dosing does not select more resistant bacteria. Single-dose therapy with quinolones would be an improvement in cost and patient compliance.


What preclinical data are needed to justify once-daily therapy?

Before applying in clinical practice once-daily dosing of antimicrobials, one must take into consideration several factors that may influence the pharmacodynamic interaction between antimicrobials and microbes at the site of infection. The ideal agent should demonstrate rapid concentration-dependent killing activity and a post-antibiotic effect that could allow for a clinically significant delay with levels below the minimal inhibitory concentration before regrowth of the microorganism. The pharmacokinetic properties of the antibiotic should allow for a good therapeutic ratio (concentration/MIC) at the site of infection. To evaluate the importance of dosage schedule on outcome, investigators have to use animal models where peak levels, half-life, area under the curve, time above MIC in interstitial fluid or infected tissues, and other pharmacodynamic properties can be evaluated simultaneously. The pharmacodynamics of several antibiotics administered at different dosing interval is compared using an animal model of infected fibrin clots. In this model, once-daily therapy resulted in better killing than other modes of administration. Aminoglycosides and quinolones may be better suited for once-daily therapy than beta-lactams unless these latter agents have a long half-life.


Cost effectiveness of once-daily oral antimicrobial therapy.

Once daily dosing of oral antimicrobials achieves significantly better patient compliance than three or four times daily dosing, and limited data suggest that this is associated with greater efficacy. Comparison of once daily and twice daily dosing is less consistent, and most studies show only marginally better compliance with once daily dosing versus twice daily dosing. Detection of urinary antimicrobial activity provides a simple method for checking compliance with oral antimicrobials and deserves wider study. Counts of residual tablets have been shown to overestimate compliance. Intravenous formulations are always more expensive than equivalent oral formulations, and preparation and administration of intravenous drugs adds significant additional costs. Moreover, intravenous regimens are complex, and a number of studies have shown that serious errors occur in both preparation and administration of intravenous drugs. There is increasing evidence that serious infections can be adequately treated with oral drugs, and the excellent bioavailability of quinolones makes them particularly attractive for these indications. Clinicians require a method for checking absorption by patients with severe infection, and the Serum Bactericidal Test may provide a practical method for monitoring a wide variety of drugs.


Introduction of a composite parameter to the pharmacokinetics of venlafaxine and its active O-desmethyl metabolite.

Venlafaxine is a structurally novel, nontricyclic compound that is being evaluated for the treatment of various depressive disorders. A randomized three-period crossover study was conducted to obtain pharmacokinetic and dose proportionality data on the drug and its active metabolite, O-desmethylvenlafaxine. Eighteen healthy young men received single doses of venlafaxine 25, 75, and 150 mg followed by 3 days of administration every 8 hours (q8h). Steady-state elimination half-life was 3 to 4 hours for venlafaxine and 10 hours for O-desmethylvenlafaxine; both were independent of dose. Venlafaxine had a high oral-dose clearance, ranging from 0.58 to 2.63 L/hr/kg across doses with the lowest mean clearance, 0.98 L/hr/kg, at the highest dose. The apparent clearance of O-desmethylvenlafaxine was lower than venlafaxine, ranging from 0.21 to 0.66 L/hr/kg, and the lowest mean clearance, 0.33 L/hr/kg, occurred at the lowest dose. The area under the metabolite curve was two to three times greater than that for venlafaxine. Each compound had linear dose proportionality up to 75 mg q8h. A composite parameter incorporating venlafaxine plus O-desmethylvenlafaxine was introduced (i.e., AUC [area under the curve] + activity factor.AUCm), which extended linearity to 150 mg q8h. In summary, venlafaxine is a high-clearance drug that forms a metabolite with almost equal activity and demonstrates linear dose-proportionality.


Pharmacokinetic interactions of cefprozil with food, propantheline, metoclopramide, and probenecid in healthy volunteers.

Cefprozil, a new oral cephalosporin antibiotic, is composed of cis and trans isomers in an approximate 90:10 ratio. The objectives of this study were: (1) to assess the effects of alterations in gastrointestinal motility by metoclopramide and propantheline on the pharmacokinetics of cis and trans isomers of cefprozil, and to compare them with the effects of food on the pharmacokinetics of cefprozil; (2) to assess the effects of inhibition of renal tubular secretion by probenecid on the pharmacokinetics of cefprozil isomers. In this four-way crossover study, 15 healthy male volunteers received a 1000-mg dose of cefprozil after fasting, pretreatment with metoclopramide or propantheline, after breakfast, or after probenecid in an incomplete, balanced block design. There was a 1-week washout period between each treatment. Blood and urine samples collected over a 24-hour period were assayed for the cis and trans isomers. The concentrations of the trans isomers were generally 1/10 of the cis isomer. The means and variances of the pharmacokinetic parameters of the cis and trans isomers of cefprozil were similar in fasting subjects and were affected in a parallel manner by food, metoclopramide, propantheline, and probenecid. The pharmacokinetics of the cis isomer under the fasting condition were as follows: maximum peak plasma concentration (Cmax), 14.0 +/- 2.7 micrograms/mL; median time to reach Cmax (tmax), 1.5 (range, 1.0-3.5) hours; half-life (t1/2), 1.24 +/- 0.27 hours; area under the concentration (AUC0-infinity), 47.3 +/- 7.7 micrograms.hour/mL; mean residence time after oral administration (MRTpo), 2.9 +/- 0.4 hours; CLR, 219 +/- 60 mL/minute; and Xu% (percent cumulative urinary excretion in 0-24 hours), 68.1 +/- 12.5.(ABSTRACT TRUNCATED AT 250 WORDS)


Language samples using three story elicitation tasks and maturation effects.

The spoken language skills of 20 elementary students with verified learning disabilities were explored using a story format and discourse analysis procedure. The purpose was to compare the performance of LD students on three types of story tasks (story generation from a picture, story generation from memory, and story generation based on a game) over a one-year period. Results indicated statistically significant main effects for approximately half (9/17) of the discourse variables by task and one main effect for time. Overall, stories from memory were longer and characterized by more as well as different types of errors than were stories from pictures or stories based on games. Implications for use of stories and type of story collection task in language sampling are discussed.


Lectin-binding profiles for normal skin appendages and their tumors.

A histochemical investigation of lectin-binding sites was carried out on formalin-fixed paraffin-embedded sections of 60 skin appendage tumors and adjacent normal skin appendages, using four different biotinylated lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), and Ulex europaeus agglutinin-1 (UEA-1), and avidin-biotin-horseradish peroxidase labeling. In the secretory segments of eccrine sweat glands, the superficial dark cells showed strong cytoplasmic staining with UEA-1, whereas DBA and SBA strongly stained the plasma membranes of basal clear cells. The acinar cells of apocrine sweat glands revealed sporadic apical membrane staining with all four lectins. In some cases, the luminal membranes of sweat gland ducts showed apical membranous staining with all four lectins. In the hair follicles, the inner root sheath was positive for all four lectins, and the outer root sheath was stained by PNA. The sebaceous ducts, as well as the outer root sheath at the level of sebaceous duct insertion, were also labeled by all four lectins. Sebaceous lobules showed cytoplasmic and membrane staining of mature sebocytes with PNA and SBA. Although sweat gland tumors revealed differences in lectin binding when compared to their corresponding normal tissues, the lectin-binding pattern of pilosebaceous tumors was analogous to the pilosebaceous apparatus.


Distant cutaneous metastasis of pleural malignant mesothelioma.

We report a facial tumor that was proven to be a metastatic mesothelioma. The diagnosis was not established pre-mortem. The patient died shortly after the facial biopsy, and an autopsy revealed a large pleural-based mass which had the gross appearance typical of a mesothelioma. Electron microscopic examination of tissue from the pleural tumor was diagnostic for mesothelioma. The patient had extensive visceral metastatic disease. Inclusion of this entity in the differential diagnosis of certain cutaneous tumors is important, in part because this lesion may be confused with angiosarcoma, particularly when it occurs in the skin of the face or head in older patients.


Acoustic patterns of an adolescent with multiple articulation errors.

This is a case study on an adolescent who demonstrated multiple articulation errors. In particular, an acoustic analysis was performed on the same features as those investigated by Kent and Forner (1980) in children with normal articulation and by Glasson (1984) in speech- and language-disordered children. The results of the analysis were compared to the data obtained by Kent and Forner and Glasson. Our subject demonstrated longer phrase durations and more variability than any of the groups with whom he was compared. In addition, he displayed an interaction between syntactic and articulatory performance.


A fluent language disorder following antepartum left-hemisphere brain injury.

This article describes the development of language in a left-handed girl with a left middle cerebral artery infarction. Seven language samples of parent-child interaction, obtained when she was between 36 and 60 months of age, were transcribed and analyzed using the Child Language Data Exchange System. At 36 months of age, only 42 (20%) of the child's 214 utterances contained words; the other 80% were composed of jargon or interactional markers such as "uh-huh" and "uhn-uhn." Jargon incorporated familiar intonational contours and prosodic features to convey emotional states and communicative functions. Between 36 and 45 months of age, her jargon became differentiated into increasing approximations of English sentences. Simultaneously, her use of words and word combinations increased. By 54 months of age, no jargon was heard. The pattern of development observed in this child can be described as a transient jargon or fluent aphasia. It may have resulted from initial reliance on an uninjured right hemisphere. However, given the similarity between this pattern and the expressive or gestalt style of learning seen in some normal children, the pattern may also be related to other variables including characteristics of the parental input.


Transient bullous dermolysis of the newborn. Retention of anchoring fibril- and basal lamina-like structures in keratinocytes and evidence of collagenolysis.

In a freshly induced blister from transient bullous dermolysis of the newborn, we found in epidermal basal cells structures similar to anchoring fibrils and lamina densa, often in combination. In the upper dermis, collagenolysis and phagocytosis of degenerated collagen fragments were observed. It is concluded that the stellate inclusions previously reported in this disease are prematurely assembled anchoring fibrils and lamina densa. A disturbance of delivery mechanism of precursor materials for anchoring fibrils and lamina densa may cause the deficiency of these structures which are essential for the integrity of dermo-epidermal adhesion. The presence of many normal anchoring fibrils and lamina densa in perilesional skin and their prompt degeneration after rupture of the inclusions previously led us to conclude that lytic enzymes are involved in the formation of blisters in this disease. The new finding of collagen phagocytosis in the lesional dermis seemed to support this contention.


Another look at semantic relational categories and language impairment.

Earlier comparative studies of language-impaired and normal children involved semantic-relational analyses of broad categories (e.g., action, locative action, and so on) in which utterance types were not differentiated. In this study, locative action utterances were differentiated by the types of locative words used singly and in combination. The types of utterances used by a language-impaired child were tracked between 1 year 6 months and 3 years and compared with those of three age- and MLU-matched normal children. The results suggested that differences in the semantic properties of language-impaired and normal children's utterances may go undetected unless a fine-grained analysis is performed on the types of expressions used within a global relational category. The potential value of extending semantic-relational analyses by exploring word use in syntactic contexts is addressed.


Two alternative program models for serving speech-disordered preschoolers: a second year follow-up.

This study investigates the appropriate roles of parents and speech-language pathologists in early intervention by longitudinally comparing the costs and effects of two programs for speech-disordered preschoolers: a home parent training program and a clinic-based, low parent involvement program. Results from follow-up testing one year after the intervention ended corroborated the results immediately following intervention. Specifically, the home parent training group performed at least as well as the clinic-based group on measures of speech and language functioning as well as on a measure of general development. On one variable measuring personal/social skills and one measuring adaptive behavior, the home parent training group performed significantly better than the other group. Results of the cost analysis indicated that, excluding the value of parent time, there was no meaningful difference in program costs. The implications of this study are that parents can be given significant responsibilities in early intervention and that program administrators have the viable option of training parents to provide the primary early intervention services. Findings support the need for therapists to be trained to work with parents as well as with the child.


HIV and AIDS among adolescents in the United States: increasing risk in the 1990s.

Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) are growing problems among U.S. adolescents. By examining recent data on AIDS surveillance and HIV seroprevalence, surveys on teenagers' knowledge, beliefs, and behaviors related to HIV/AIDS, key treatment issues, and barriers to prevention, this manuscript reviews the problem and proposes possible ways of combating it. African American youth have the highest rates of AIDS and white youth the lowest. However, the largest number of AIDS cases overall has been recorded in white males, reflecting relatively high case rates in boys with hemophilia and in young male homosexuals. Predominant HIV risk factors for adolescents are unprotected sex and/or sharing injection drug equipment with an infected partner. Relatively high rates of HIV infection in adolescent females may indicate their greater physiological vulnerability than adult females to sexually transmitted diseases (STDs). Data from HIV seroprevalence studies suggest a substantially increased heterosexual epidemic in the 1990s, especially in large east coast cities and southeastern rural areas where drug use and/or STDs are highly prevalent. More comprehensive prevention and treatment services are needed to prevent ongoing expansion of HIV infection and AIDS in the adolescent age group.


Sexuality and AIDS: attitudes and behaviors of adolescents in east and west Berlin.

The study which was conducted in 1990 in Berlin, Germany, compares two samples of 542 adolescents from West Berlin and 340 from East Berlin. The present investigation shows that the general trend toward sexual liberality among adolescents is continuing and that necessary processes of adolescent sexual development appear not to be influenced by the threat of AIDS. Though the level of knowledge about the threat of AIDS is, on the whole, satisfactory, this does not in itself guarantee determined and consistent AIDS-preventive behavior. Fewer than one-third of sexually-active adolescents, and even fewer than one-fourth of those in the most sexually-active group, can be described as consistent users of condoms. The attitudes of adolescents in regard to contraceptive characteristics only partly suggest a preference for condoms. When adolescents are asked directly about condoms, they additionally emphasize their effect of reducing feeling and pleasure. Adolescents with experience of sexual intercourse rate condoms more negatively than do those without such experience. However, AIDS-education efforts can make use of the fact that consistent condom-users expressed clearly more positive attitudes to condoms than did others. Although the comparison between East and West Berlin adolescents yielded differences in certain details, these differences are not of significance to AIDS-education programs which seek to increase AIDS-preventive behavior.


The AIDS awareness and sexual behaviour of young people in the South-west of England.

A programme of surveys was undertaken in collaboration with District Health Authorities to assess the AIDS awareness and sexual behaviour of young people, in order to help the development and implementation of HIV/AIDS risk-reduction strategies. Given that the patterns of findings were strongly replicated in the different districts, the data is presented in terms of a combined data set comprising 2955 respondents aged 16-24. The surveys indicate fairly reasonable levels of knowledge about HIV/AIDS but very low personally perceived vulnerability to infection. The sexual culture is one of fairly early ages of first intercourse, moderate levels of partner change and low (one third) levels of condom use. The findings are discussed in terms of their implications for HIV/AIDS risk-reduction strategies.


The social context of adolescent sexuality: safe sex implications.

This study was an examination of the sexual worlds of 153 adolescents aged 15 to 18 years through the content analysis of interviews on the topics of love, romance, relationships between the sexes, sexual values and sexual behaviors. The aim was to develop more detailed descriptions of the dimension of adolescent sexuality and relate these dimensions to sexual risk, that is, the tendency to engage in unprotected intercourse, an activity which increases vulnerability to AIDS and other sexually transmitted diseases. To this end, seven themes were isolated from the interview scripts, these being permissiveness, double standards, belief about sexual control (the Id Factor), romance, regrets about permissiveness, sexual aggression, and questioning. Measures of four of these themes were constructed, and sex and sub-group differences explored, as were the relationships between themes and sexual risk. The implications of different "pathways" to sexual risk-taking were discussed.