Comparison of the mouse L32 ribosomal protein promoter elements in mouse myoblasts, fibers, and L cells.

The sequences required for the maximal expression of the mouse L32 ribosomal protein gene and the binding of nuclear factors to L32 promoter elements were analyzed in mouse myoblasts, fibers, and L cells. Various L32 r-protein promoter sequences were linked to the chloramphenicol acetyltransferase gene (CAT), and the expression of the chimeric genes was measured transiently or after their incorporation into the genome. The sequence requirements for maximal expression of the L32 gene are very similar among the various cells and include the previously identified L32 core promoter from approximately -150 to +75. Only the promoter regions between -45 and +11 displays significant cell type specific differences. Relative to the maximal activity in each cell type, the expression of the L32-CAT gene containing the -45 to +11 region is greater in L cells than in myoblasts or fibers. This difference is correlated with the increased activity of an L cell nuclear factor(s) that binds to this fragment. In addition, our results show that deletion of sequences between -981 and -141 causes a 50-70% reduction of the expression of the L32-CAT gene in myoblasts, fibers and L cells. The transcription of all the L32-CAT genes examined decrease after myoblasts differentiate into fibers in a manner similar to the endogenous L32 gene, but we were unable to distinguish between sequences involved in controlling the expression of the L32 gene during myoblast differentiation and those sequences required for maximal promoter activity. However, gel mobility shift assays showed differences in the binding of myoblast and fiber factors to the four promoter fragments examined. The possible role of these factor binding differences in controlling L32 transcription is discussed.


Poly ADP-ribosylation: a histone shuttle mechanism in DNA excision repair.

In DNA excision repair of mammalian cells, the processing of ADP-ribose by the poly ADP-ribosylation system of chromatin is stimulated several thousand-fold. Most of this turnover is associated with the automodification reaction of the nuclear enzyme poly(ADP-ribose) polymerase and the degradation of polymerase-bound polymers by the enzyme poly(ADP-ribose) glycohydrolase. The automodification cycle catalyzes a temporary dissociation from and reassociation of histones with DNA. It is proposed that this mechanism, termed "histone shuttle", may guide specific proteins to sites of repair. In addition, histone shuttling driven by the poly ADP-ribosylation system seems to be involved in nucleosomal unfolding of chromatin in DNA excision repair.


Synchronization of hair cell regeneration in the chick cochlea following noise damage.

Pure-tone overstimulation for prolonged time leads to hair cell death in frequency-specific regions of the cochlear epithelium. Unlike mammals, birds replace missing hair cells by stimulating mitosis in an uncharacterized precursor cell. Regenerated hair cells, initially identifiable by their immature stereocilia and small surface areas, differentiate into mature cells in a manner which parallels embryonic development. In the current study, we examined whether hair cell regeneration is initiated during noise exposure or after the end of acoustic trauma. We exposed 7-to 15-day-old chicks to a 1500 Hz pure tone at 120 dB SPL (re 20 muPa) for 4, 12, and 24 hours and examined the recovering cochlear epithelium with scanning electron microscopy to determine when regenerated hair cells were first identifiable. The earliest evidence of new hair cells appeared roughly 96 hours after the onset of 4-, 12-, and 24-hour exposures. Our previous studies initially identified new hair cells 96 hours after the start of a 48-hour exposure. Therefore, hair cell regeneration follows a similar time course relative to the onset of noise exposure, regardless of the ultimate duration of exposure. Since we estimate that hair cells take at least 48 hours after their genesis to form immature stereocilia, the signal which induces hair cell precursors to re-enter the cell cycle and to divide probably has its initial effects very early during the exposure period. (A previous report of these data was given at the 1991 American Society for Cell Biology conference.)


Evolution and the meaning of metaphase.

We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.


Stimulus-dependent relocation of the microtubule organizing center in human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/- 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/- 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/- 6.27% for PL+F and 47.2% +/- 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/- 8.89% for PL+F and 39.6% +/- 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a 'pre-defined' apical location; (ii) translocated to a 'newly defined' basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.


Relocation of nucleolar proteins around chromosomes at mitosis. A study by confocal laser scanning microscopy.

The behaviour of nucleolar antigens known to associate with chromosomes at mitosis was investigated in mammalian cells (HeLa, HEp-2, PtK1, CHO) by immunofluorescence and confocal laser scanning microscopy. Serial optical sections through mitotic cells, from prophase to telophase, were used to generate three-dimensional images of the antigen distribution. Our results indicate that, at the onset of mitosis, these antigens leave the nucleoli in a highly ordered manner to form a network extending from the nucleoli towards the nuclear envelope. The migration begins at very early prophase, when the condensation of the chromosomes is not yet visible. After completion of the migration at late prophase, the labelling is found at the chromosome periphery. The antigens remain distributed as a sheath surrounding the chromosomes from prophase to telophase. Therefore, the proteins involved in the formation of this perichromosomal layer have different behaviour than those of the prenucleolar bodies. The antigens appear to interact strongly with chromosomes, since they are not lost during chromosome isolation in hypotonic buffer. Each chromosome is entirely covered from one telomere to the other, except in the centromeric region. Thus the relocation of these nucleolar proteins does not appear to be the result of a passive accumulation at the chromosome periphery, but seems rather to be due to an active targeting to specific sites. Consequently, these proteins may have a determining function in the progression of the cells through mitosis, possibly by participating in the protection and stabilization of the chromosomes.


Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts.

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.


Mammalian skeletal muscle C-protein: purification from bovine muscle, binding to titin and the characterization of a full-length human cDNA.

We report a fast method for the isolation of homogeneous C-protein from bovine skeletal muscle. In electron micrographs C-protein appears as short rods with a relatively uniform length of about 50 nm. Protein sequencing shows a single N-terminal sequence. Radio-labelled C-protein strongly decorates titin II and myosin rods but not myosin heads. Binding to titin II is retained in preparations lacking titin-associated proteins. Antibodies to bovine C-protein were used to screen a lambda gt11 cDNA library constructed from fetal human skeletal muscle. Clone HC38 is 3833 bp long and encodes a protein of 1138 amino acid residues. The start of the predicted sequence fits the N-terminal sequence of the bovine protein. All partial sequences obtained from the bovine protein (348 residues) and the sequence deduced from a partial chicken cDNA (Einheber and Fischman, 1990) can be aligned along the human sequence. The sequences of human and chicken C-proteins share 50% identity and 70% similarity. Along the repeat patterns of the human protein the fibronectin (Fn)-like domains are better conserved than the immunoglobulin (Ig)-like domains. Regions of strong divergence between chicken fast C-protein and human slow C-protein may represent differences in C-protein isoforms.


Formation of skeletal muscle in vivo from the mouse C2 cell line.

The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse. Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells. In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.


An evaluation of cell separation techniques in a model mixed cell population.

Muscle precursor cells may act not only as a means of inserting normal genes into diseased muscle fibres, in order to correct or alleviate a genetically inherited myopathy, but recent demonstrations have shown they may prove an invaluable tool for the expression of, and systemic dissemination of, non-muscle gene products. If muscle precursor cells are proved to act as such widespread vectors in terms of gene therapy, then it is imperative that methods are properly elucidated to produce large populations of pure viable myogenic cells for such purposes. In the past, many methods of cell separation have been investigated but carry with them the problems of either a lack of myogenic purity of the population or poor percentage recovery of the original cell population. In the present work we have investigated two methods for segregating myogenic from non-myogenic cells and have critically reviewed the efficiency of separation of the two techniques used. To obtain a quantitative measure of separation efficiency, segregation was carried out on a 1:1 mixture of murine C2 myogenic and murine 3T3 fibroblastic cells. To distinguish between C2 and 3T3 cells, the latter were prelabelled with the fluorescent strain carboxyfluorescein diacetate succinimyl ester (CFSE). Once incorporated into the cell, CFSE remains there, thus preventing transfer of the label to C2 cells. Both methods of separation used depend on the affinity of myogenic cells for the monoclonal antibody Mab H28, which specifically binds to the mouse neuronal cell adhesion molecule N-CAM, but differ in that one method, "panning", completes segregation by adherence of N-CAM positive cells to a dish precoated with secondary IgG antibody whereas in the other separation proceeds by the use of commercially available IgG-coated magnetic beads. Results indicate magnetic bead separation to be more efficient than panning if the beads are precoated with 0.1% gelatin.


Aortic endothelial cell heterogeneity in vitro. Lack of association between morphological phenotype and collagen biosynthesis.

Previous reports dealing with the characterisation of endothelial cells derived from the same tissue have produced apparently conflicting results in fundamental cellular attributes such as matrix biosynthesis and the ability to form sprouts in vitro. One potential explanation for this discrepancy is that endothelial cells actually comprise a heterogeneous population of cells displaying a significant degree of intra-site variation in phenotype. In order to address this question, we have characterised both cloned and uncloned lines of bovine aortic endothelial cells with respect to (a) their ability to adopt both the cobblestone and sprouting cell phenotypes and (b) matrix biosynthesis by cells displaying these two phenotypes. Data are presented indicating that all of the 18 cloned and 20 uncloned cell lines examined were capable of undergoing a reversible transition between the cobblestone and sprouting cell phenotypes in response to culture conditions. In all cases, sprouting occurred spontaneously in the presence of either serum or platelet-poor plasma and did not require the addition of exogenous factors to the medium. Twelve lines of cells were examined with respect to protein biosynthesis; these lines produced different types of collagens in differing proportions. The pattern of collagen synthesis displayed by every cell line was stable and did not vary with either passage number or batch of serum. The presence of a 3-D gel of native type I collagen increased specifically the synthesis of type IV collagen by one cell line. However, in four other cell lines, even though total synthesis was increased, the type of proteins secreted by these cells was not altered.(ABSTRACT TRUNCATED AT 250 WORDS)


Changes in cell surface expression of fibronectin and fibronectin receptor during liver regeneration.

The surface expression of fibronectin and its major integrin receptor in liver, integrin alpha 5 beta 1, was studied during liver regeneration after partial hepatectomy. Using immunoblotting, plasma membranes isolated from livers at different regeneration stages were found to contain 6- to 8-fold elevated levels of fibronectin, alpha 5 and beta 1 at 12-24 h after the operation. Normal levels were gradually restored during the following 8-9 days. The membrane-associated fibronectin lacked the ED-A domain, suggesting that it consisted of plasma fibronectin. A prominent fibronectin fragment (180 kDa) was present at 12-24 h after surgery, possibly reflecting turnover of the pericellular matrix during cell division. Indirect immunohistochemical staining of liver sections revealed beta 1 and fibronectin mainly in the sinusoidal region of the hepatocyte plasma membrane. The distribution was not markedly altered during liver regeneration. The results suggest that the fibronectin-mediated contacts between the cells and the extracellular matrix increase during the pre-replicative and proliferative phases of liver regeneration. The significance of these results for the growth and for the structure of the liver during regeneration and development is discussed.


Alteration in glycosaminoglycan metabolism and surface charge on human umbilical vein endothelial cells induced by cytokines, endotoxin and neutrophils.

There is increasing evidence that the glycosaminoglycan (GAG) component of the vascular endothelium is important in regulating vascular permeability, thromboresistance and cellular interactions. We have investigated the GAG metabolism of cultured human umbilical vein endothelial cells (HUVEC) in response to a range of inflammatory stimuli. Using both chemical measurement of cellular and supernatant GAGS and 35S labelling to identify newly synthesised GAGS, interleukin 1 (IL1), tumour necrosis factor (TNF) and interferon gamma (IFN gamma) were shown to influence sulphated GAG metabolism significantly. IL1 and TNF caused a marked increase in culture supernatant GAGS and a concomitant reduction in cell-associated GAGS. This was shown histochemically to be associated with a marked reduction and redistribution of endothelial surface anionic sites. The addition of neutrophils to HUVEC pretreated with Escherichia coli endotoxin, IL1 or TNF resulted in a further reduction in both cellular GAGS and surface anionic sites. These results suggest that changes in endothelial cell GAG metabolism during inflammation may contribute to the disturbance of vascular endothelial homeostasis associated with infectious and inflammatory states.


Molecular characterisation of the gene for the 180 kDa subunit of the DNA polymerase-primase of Drosophila melanogaster.

We have cloned and sequenced the gene for the 180 kDa subunit of the a polymerase from Drosophila melanogaster. The protein shows high similarity to the 180 kDa subunits from other species. Comparative expression analysis for the transcript, protein and enzymic activity suggests that control occurs mainly at the level of transcription. In situ analyses of the RNA suggest that high levels of the transcript are synthesised in the ovaries and deposited uniformly in the egg. Immunolocalisation of the 180 kDa polypeptide in whole embryos shows that its location is mainly nuclear; however, dispersal of the protein can be seen to occur during mitotic phases of the cell cycle.


Isoprenylation of rab proteins on structurally distinct cysteine motifs.

rab proteins are low molecular weight GTP-binding proteins highly related to Ypt1p and Sec4p, which are involved in the control of secretion in yeast Saccharomyces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.


M-CSF-induced macropinocytosis increases solute endocytosis but not receptor-mediated endocytosis in mouse macrophages.

Although coated vesicles can mediate both solute and receptor-mediated endocytosis, there are other kinds of endocytic vesicles that contribute to these processes. The relative contributions of these other organelles, particularly regarding solute influx, remains unsettled. Here we describe a physiological uncoupling of solute and receptor-mediated endocytosis that occurs during growth factor-stimulated macropinocytosis. We examined how recombinant human macrophage colony-stimulating factor (rM-CSF), which rapidly stimulates solute endocytosis in murine bone marrow-derived macrophages, affected ligand internalization via receptor-mediated endocytosis. Although rM-CSF stimulated internalization and accumulation of Lucifer Yellow (LY), a probe for solute endocytosis, it had no effect on accumulation of fluorescent acetylated low-density lipoprotein (acLDL), a ligand for the macrophage scavenger receptor, or on the endocytosis of 125I-labelled diferric transferrin. Video microscopy revealed that rM-CSF immediately induced active cell ruffling and the formation of phase-bright macropinosomes. Nocodazole pretreatment of macrophages inhibited both ruffling and macropinocytosis. Macropinosomes were fluorescently labelled by incubating macrophages briefly with probes for both solute endocytosis (fluorescent dextrans) and ligand endocytosis (fluorescein-labelled transferrin or diI-labelled acLDL). Macrophages incubated for one or two minutes formed macropinosomes that were labelled predominantly with the fluorescent solute probes but with little or none of the ligand probes; the latter were localized within smaller pinosomes. When cells pulsed with the fluorescent probes were washed and chased for an additional two minutes, solute and ligand probes occasionally co-localized in macropinosomes. Nocodazole inhibited macropinocytosis with little apparent effect on endocytosis via smaller vesicles. These experiments show that macropinosome formation is dependent on microtubules and also that the macropinosomes induced by rM-CSF are solute-rich and receptor-poor. Macropinosomes differ from coated vesicles in these respects, and therefore provide a physiologically regulated mechanism for uncoupling solute and receptor-mediated endocytosis.


Chromosomes attain a metaphase position on half-spindles in the absence of an opposing spindle pole.

To examine the relative roles of chromosomes, spindle poles and microtubules in the formation of the metaphase spindle and metakinesis, I have experimentally placed an extra centrosome-free pronucleus close to a forming bipolar spindle in a living cell. The chromosomes from the extra nucleus induce the formation of an extra half-spindle from one pole of the otherwise normal bipolar spindle with chromosomes positioned at the putative metaphase plate. I conclude that chromosomes determine the location of half-spindles by sustaining a higher than normal density of microtubules. These results are surprising for two reasons: first, because previous in vivo experiments in tissue culture cells show that mono-oriented chromosomes with functional attachments to spindle microtubules do not support half-spindle formation but oscillate unstably or move to one spindle pole. Additionally, the generally accepted view is that chromosomes attain a metastable condition at the metaphase plate as a result of a balance between forces directed to opposite spindle poles. However, our observation that chromosomes on extra half-spindles attain a metastable position in the absence of an opposing spindle pole, suggests that Ostergren's model does not account for metakinesis in sea urchin embryos.


A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites.

Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrin-radixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.


Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.

Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably resulted from local filament assembly and put forward the hypothesis that microfilament reorganization in two centres drives the morphological polarization of PMNs.


Tubulin in bovine retinal rod outer segments.

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this nonaxonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes. This fraction remains membrane-associated after extensive low- or high-salt washing, requiring detergents or protein denaturants for release from ROS membranes. Unlike ROS soluble tubulin it associates tightly with liposomes upon detergent solubilization and reconstitution. The ROS membrane-associated tubulin is highly enriched in isolated ROS plasma membrane fractions compared to the total outer segment membrane pool and can be localized to the plasma membrane but not to disks by immunofluorescent staining, suggesting a possible role in the structure or electrophysiology of the rod outer segment plasma membrane.


Association of myosin with the connecting cilium of rod photoreceptors.

The cilium of a vertebrate photoreceptor cell connects the phototransductive outer segment of the cell to the inner segment. Previous studies have shown that, within the connecting cilium, there is a small cluster of actin filaments, which play a critical role in the formation of new disk membranes. Here, we have detected a polypeptide in rat rod outer segments that is recognized by myosin heavy chain antibodies and was found to possess other characteristics of conventional non-muscle myosin heavy chain: it comigrates in SDS-PAGE with non-muscle myosin heavy chain; it associates with the cytoskeleton of rod outer segments in an ATP-sensitive manner; and it binds to purified actin filaments in the absence of ATP. Myosin ATPase activity was also detected in isolated rod outer segments. Electron immunomicroscopy revealed that myosin is present in the small actin-containing domain within the connecting cilium at the site of disk membrane morphogenesis. These results pose the possibility that an actin-myosin contractile mechanism functions in the formation of new photoreceptor disk membranes.


Radial movement of lysosomes along microtubules in permeabilized macrophages.

In murine bone marrow-derived macrophages, lysosomes often form tubulovesicular compartments, whose extended distribution in the cytoplasm depends on the integrity of cytoplasmic microtubules. When macrophages with fluorescently labeled lysosomes were plated onto coverslips opsonized with IgG, they engaged that surface in a phagocytic response (frustrated phagocytosis). The tubular lysosomal compartment of these cells collected in a central, perinuclear region, despite the continued presence of a radiating array of cytoplasmic microtubules. Using methods developed in the study of melanophores, we permeabilized macrophages engaged in frustrated phagocytosis, then re-activated lysosome extension along microtubules. Permeabilization was selective for plasma membranes, in that high molecular weight probes such as trypan blue or IgG could enter cells, while fluorescent probes previously loaded into lysosomes via endocytosis remained contained therein. Addition of 2 mM ATP, GTP or UTP to these permeabilized cell models produced centrifugal extension of tubular lysosomes. Selective depletion of ATP, using Escherichia coli glycerol kinase, inhibited ATP-dependent extension but not that which occurred with GTP or UTP, indicating that the mechanism of radial movement can use any of these three nucleotide triphosphates. Extension was independent of pH between 6.8 and 7.4, and was inhibited by AMP-PNP and by GMP-PNP. Depolymerization of cytoplasmic microtubules with nocodazole prevented subsequent ATP-inducible lysosome extension, whereas preincubation of cells with cytochalasin D did not inhibit the response. These results are consistent with the in vitro mechanochemical properties of kinesin (Cohn et al., 1989), and support earlier evidence, obtained in living cells, that kinesin is the mechanochemical motor of lysosome extension along microtubules in macrophages.


Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.


Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier.

Primary culture of rat brain endothelial cells is described, based on the method of C. C. W. Hughes and P. L. Lantos. The cells have been characterised using morphological and immunocytochemical techniques, and systematic studies undertaken to determine the optimal culture medium and conditions required to grow the cells at high purity on a variety of substrata. The endothelial cells have a spindle-shaped morphology, and proliferate as plaques from small clusters of cells associated with capillary fragments in the starting material. Tight junction-like cell:cell appositions are seen at the electron-microscopic level. The cells show characteristic staining for antigens recognized by antibodies against von Willebrand factor (Factor VIII-related antigen), angiotensin-converting enzyme (ACE), the transferrin receptor (Ox-26), actin and vimentin. They also show binding of the lectin from Ulex europaeus (UEA I). Potential contaminating cells include smooth muscle, fibroblasts, pericytes and meningeal cells. Contaminants can be kept to < ca. 5% by careful removal of large vessels and meninges during dissection, by brief treatment with Ca(2+)- and Mg(2+)-free saline, by growth in medium supplemented with plasma-derived serum treated for removal of platelet-derived growth factor (PDGF), and by occasional use of medium in which D-valine is substituted for L-valine. Cells attach well to collagen-coated plastic, less well to glass. Cells can be grown on transparent collagen filters (ICN, Cellagen and Costar, Transwell-Col), and on microcarrier beads (Pharmacia, Cytodex-3). The culture has proved to be a useful preparation for studies of cellular physiology, pharmacology and biochemistry of the brain endothelium, and represents a first step in producing an in vitro model of the rat blood-brain barrier.


Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture.

The extracellular matrix may be considered as an insoluble local mediator which plays an important role in regulating cell function. Communication between the cell and its matrix occurs via the integrins, a family of transmembrane proteins composed of non-covalently linked alpha and beta subunits. The aim of this study was to establish which integrins are present on human bone cells in situ and in culture, using cryostat sections of undecalcified human bone, osteoclastoma tissue and cultured human osteoblasts. Integrin subunit expression was identified indirectly using alkaline phosphatase anti-alkaline phosphatase conjugates and FITC-labelled secondary antibodies. Subunits expressed by cultured human osteoblast-like cells were then quantified by FACS analysis. Staining patterns observed in situ show that osteoblasts and osteoclasts possess different integrin subunits. Osteoblasts primarily express alpha 1, alpha 3 and beta 1 and weakly express alpha 2. Osteoclasts express alpha 2, alpha V, beta 1 and beta 3. Subunits alpha 4, alpha 5, alpha 6, alpha L, alpha M and beta 2 were not expressed by either of these cell types. Expression of beta 1 by all cells of the osteoblastic lineage was constitutive, but alpha 1 and alpha 3 subunits were expressed by osteoblasts actively synthesizing bone and some of the osteoblast lining cells. All integrin subunits identified on osteoblasts in situ were maintained on culture but there was an increased expression of alpha 2 and alpha V subunits were weakly positive. Expression of alpha 2, alpha 3, alpha V and beta 1 subunits was independent of cell density but expression of alpha 1 was much greater in confluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)


Cell guidance by micropatterned adhesiveness in vitro.

Tracks of adhesiveness are believed to be involved in guiding morphogenetic cell migrations. Here, electronics microfabrication technology was used to manufacture patterns of alternating tracks of adhesive and non-adhesive substratum (untreated fused quartz and adjacent parallel tracks of hydrophobic treatment) of varying period (4, 6, 12, 24 and 50 microns). These experimental substrata were used to model, in vitro, possible differentially adhesive guidance cues. The effect of such patterned substrata was assessed using fibroblastic BHK cells and epithelial MDCK cells. Cells were oriented and elongated by these surfaces. Their responsiveness was dependent on cell type, cell-cell interactions, and the geometry of the patterns. Alignment of BHK cells increased with increasing pattern period. Single MDCK cells aligned to all pattern periods, their elongation being period-dependent, whereas colonies were mainly unaffected. These in vitro data have important implications, which are discussed with regard to in vivo guidance cues. The ability of cells to bridge over non-adhesive regions will influence the effectiveness of linear cues, and will be important for guideposting. The geometry of patterns of differential adhesion is here shown to be an important factor in determining the precision with which local guidance of cells may be controlled.


The role of the cytoskeleton and intercellular junctions in the transcellular membrane protein polarity of bovine aortic endothelial cells in vitro.

This project examines the transcellular membrane protein polarity of bovine aortic endothelial cell (BAEC) monolayers in vitro with respect to the roles that intercellular junctions (as defined by comparing confluent and subconfluent monolayers) and the submembranous cytoskeleton play in controlling this phenomenon. Plasma membrane (PM) proteins obtained from apical (AP) and basolateral (BL) PM domains of confluent BAEC monolayers were isolated using the cationic colloidal silica technique and resolved by two-dimensional gel electrophoresis (2-D PAGE). To facilitate the identification of domain-specific PM proteins, an isoelectric point/molecular weight database of the proteins from AP and BL PM domains was constructed. Domain-specific PM proteins were assessed for their interaction with the cytoskeleton by determining whether they co-isolated with a Triton X-100 detergent-resistant cytoskeletal/extracellular matrix fraction. The maintenance of polarized PM protein segregation by intercellular junctional complexes was determined by comparing AP and BL protein patterns of confluent monolayers with patterns generated by subconfluent monolayers, which lack such junctional structures. Proteins isolated from AP and BL PM domains from both confluent states were immunoblotted with antibodies to angiotensin-converting enzyme (ACE) and collagen receptors (CR). ACE was restricted exclusively to the AP PM domain in the subconfluent condition, even though no apparent cytoskeletal interaction was observed. CRs, found to interact with the cytoskeleton in either confluence state, were predominantly segregated to the BL PM domain regardless of the presence or absence of cell-cell contact. Membrane proteins found by 2-D PAGE to be asymmetrically distributed in the absence of intercellular junctions were assessed for cytoskeletal interaction by their inability to be extracted by Triton X-100 from monolayers in the subconfluent state. Computer cross-referencing of 2-D PAGE peak lists and immunodetection generated from the above fractionation protocols identifies a set of four proteins associated with the cytoskeleton that remain segregated in the proper domain, and five proteins associated with the cytoskeleton that become equally distributed between AP and BL PM domains in the absence of intercellular junctions. Additionally, six proteins not associated with the cytoskeleton remain asymmetrically distributed to the AP domain in the subconfluent state. The data suggest that BAEC monolayers have unknown mechanisms, apart from intercellular junctions expressed at confluency or cytoskeletal binding, for maintaining transcellular PM protein polarity.


Dimeric transcription factor families: it takes two to tango but who decides on partners and the venue?

Dimeric transcription factors that bind to DNA are often grouped into families on the basis of dimerization and DNA-binding specificities. cDNA cloning studies have established that members of the same family have structurally related dimerisation and DNA-binding domains but diverge in other regions that are important for transcriptional activation. These features lead to the straightforward suggestion that although all members of a family bind to similar DNA elements, individual members exhibit distinct transcriptional effector functions. This simple view is now supported by experimental evidence from those systems that have proved amenable to study. There are however some largely unaddressed questions that concern the mechanisms that allow family members to go about their business without interference from their highly related siblings. Here I will discuss some insights from studies of the bZIP class of transcription factors.


Recent advances in the treatment of otitis media.

Otitis media (OM) continues to be a major cause of morbidity in infants and children. Antibiotics are presently the mainstay of treatment. A variety of agents including vaccinations against the most common causative organisms, prednisone, non-steroidal anti-inflammatory agents, intravenous immune globulin, antihistamines, decongestants, and topical antibiotics have been investigated. This review will address the recent developments in the treatment and prevention of OM.


Progress in self treatment for psoriasis vulgaris.

Chronic plaque psoriasis is by far the most frequent form of the disease and is usually amenable to home treatment. The therapeutic armamentarium for self-treatment of psoriasis has, until recently, been limited to emollients, tar, dithranol and topical corticosteroids. Although limited progress has been made in improving formulations and treatment regimes for these compounds, they still have significant drawbacks in terms of either unwanted effects or cosmetic acceptability. Topical vitamin D analogues offer a new, effective, convenient and safe option for self-treatment of psoriasis. Most research has been performed on calcipotriol. This has compared well to beta-methasone valerate and short-contact dithranol in controlled studies. Skin irritation is frequently noticed by patients, but rarely requires treatment to be discontinued. The mechanism of action appears most likely to be a direct regulation of keratinocyte proliferation and differentiation. Calcipotriol has relatively little effect on calcium metabolism and appears to be safe when used according to established guidelines but hypercalcaemia may develop if excessive quantities are used.


Sorption studies of dipotassium clorazepate salt (Tranxène) and midazolam hydrochloride (Hypnovel) in polyvinyl chloride and glass infusion containers.

The compatibilities of dipotassium clorazepate and midazolam hydrochloride with polyvinyl chloride (PVC) bags have been studied and compared with those observed with glass flasks. At room temperature, dipotassium clorazepate showed chemical instability which was independent of the container. At 4 degrees C, dipotassium clorazepate showed no decrease in concentration over 12 h, both in 5% dextrose solution and 0.9% sodium chloride solutions. When stored for longer periods of up to 72 h, a slight decrease not exceeding 5% was observed. The compatibility of midazolam with the PVC bags was found to be good at room temperature. It was slightly better in 5% dextrose solution than in 0.9% sodium chloride solution. High sorption of the drug was observed at pH 7.0 when the primary ring structure is maintained. The results make it possible for the hospital pharmacist to prepare dipotassium chlorazepate salt and midazolam admixtures up to 3 days before administration, provided the former are stocked in a refrigerator and the latter are prepared in dextrose isotonic solutions.


Pilot study to compare the bronchodilator and metabolic responses to terbutaline infusion in seven asthmatic subjects--are they correlated?

We compared the changes in peak expiratory flow rate (PEFR), plasma potassium and plasma glucose during and after an infusion of the beta 2-agonist, terbutaline, in seven asthmatic subjects. Our aim was to establish whether these metabolic parameters could be correlated with PEFR. The percentage changes in PEFR were significantly correlated with the percentage changes in glucose (r = 0.60, P less than 0.001) and potassium (r = -0.64, P less than 0.001). Values for glucose and potassium changes were also correlated significantly with each other (r = 0.86, P less than 0.001). This suggests that in asthmatic subjects on beta 2-agonist treatment, plasma potassium could be used as a surrogate marker for beta 2 activity at bronchi. It also implies that the most effective beta 2-agonist bronchodilators will produce the greatest fall in plasma potassium. Further studies with a larger number of patients are needed to confirm this.


Determination of dissociation constants of some pharmaceutical compounds using derivative spectrophotometry.

Derivative spectrophotometry is applied to the determination of the dissociation constants (pKa) of some weakly dissociating compounds. The pKa value(s) can be calculated by measuring the amplitude of derivative maxima (D) at the selected wavelengths for the dissociated, undissociated and partially dissociated forms of the compound. A graphical technique, based on plotting the derivative spectrophotometric titration curves and interpolating pKa at D1/2 is also presented. Both methods are applied to pharmaceutical compounds with single and double dissociation constants. The results are in good agreement with the reported pKa values of the investigated compounds.


A survey of prescription label preferences among community pharmacy patrons.

Fifty-five randomly selected community pharmacy patrons were surveyed regarding their preferences between four different prescription labels. The variables displayed on the four labels were: (i) laser printed, (ii) dot-matrix printed, (iii) glossy surface, and (iv) matt surface. The study population was stratified by age and gender. Statistical analysis of the results indicated a clear preference by all groups for the laser printed labels (P less than 0.001), and the only other statistically significant finding was the preference for the matt surface (P less than 0.05) of females between the ages of 16 and 39. The implications for these differences on label readability are discussed, and recommendations are offered for the use of improved laser technology for computer-generated prescription labels.


Development of a stable oral liquid dosage form of spironolactone.

A clear, stable, oral liquid dosage form of spironolactone has been developed. Solubility profiles of spironolactone were obtained in several co-solvent blends. Using this data, a co-solvent blend containing polyethylene glycol 400 (30% v/v), propylene glycol (10% v/v), glycerin (10% v/v) and ethyl alcohol (10% v/v) was used to solubilize spironolactone at a concentration of 2 mg/ml. The final formulation contained sweetening agents (sucrose, saccharin sodium), flavours (cherry, sweet), a desensitizing agent (menthol), a dye (FD&C Red #40) and a preservative (benzoic acid) to incorporate the desired organoleptic and preservative properties. A phosphate buffer was used to maintain a pH value of 4.5 (pH of maximum stability as reported earlier) to minimize hydrolysis. The final dosage form was stable for at least 93 days at 40 degrees C (loss of potency less than 4%). According to FDA guidelines, a tentative expiration date of 2 years at 25 degrees C is justifiable.


Influence of age and co-medication on steady-state phenobarbital serum level-dose ratios in Japanese paediatric patients.

The effects of age and co-medication on steady-state phenobarbital (PB) level-dose (L/D) ratios were evaluated retrospectively in 539 paediatric patients. The PB L/D ratio increased significantly with age up to 15 years. Associated anti-epileptic therapy affected the PB L/D ratio, which was significantly increased in patients on polypharmacy as compared to patients on monopharmacy. Therefore, routine monitoring of PB serum levels is extremely useful, particularly in the paediatric age group, and in patients who require associated anti-epileptic medication.


[Value an immediate histological study of the lymph nodes at the time of surgical treatment of cancer of the uterine cervix].

From 1978 to 1989, 500 external iliac lymph node dissections were carried out at the Institut Curie during surgery for cancers of the uterine cervix. The percentage of error of the histological study of the lymph node was 2.8%, including 13 false-negative findings and 1 false-positive. In 1.8% of cases (9), there were doubts as to lymph node invasion, and no confirmation was possible. The most frequent cause of error was the failure to recognize partial invasion of a lymph node. Fibrosis and necrosis secondary to preoperative radiation therapy make the intraoperative histological study more difficult.


[Linitis plastica of the proximal part of the duodeno-jejunal angle. Apropos of a case].

The authors report the first case to date of primary linitis plastica localised to the proximal part of the small bowel. A 44-year-old man with a one-month history of vague abdominal symptoms was treated for a proximal jejunum perforation due to a linitis plastica and died 2 months later.


[Intestinal ascariasis. A rare cause of obstruction of the small intestine].

Ascariasis is a benign and common disease in tropical areas. Due to its endemicity and high prevalence, serious abdominal complications requiring urgent and versatile surgical therapy do occur. An observation of intestinal obstruction by ascaris bundle, diagnosed during operation, is reported. The postoperative course was uneventful.


[Gunshot wounds of the ureter].

Lesions of the ureter caused by penetrating wounds of the abdomen rare a are complication of abdominal gunshot wounds, of which the reported incidence is 2 to 17%. The preoperative diagnosis is difficult and unrecognized initially in every third case. There are no early clinical signs of ureteral lesions. There is often no hematuria. Only IVP can make the preoperative diagnosis possible. As a matter of fact, the diagnosis will often be established by the surgical exploration made during the laparotomy required by the extent of the associated lesions. The ureteral blast contusion is an important cause of secondary necrosis with fistula. The treatment is mainly based on ureteroureterostomy after debridement. The transanasmotic drainage of urine is controversial. Mortality results from associated abdominal lesions and morbidity consists in a risk of secondary nephrectomy.


[Volvulus of the sigmoid colon. Emergency resection and anastomosis].

Eleven cases of sigmoid volvulus in patients aged 76 years in average, without visible signs of necrosis on endoscopy, are reported. All patients were treated within 6 to 48 hours by colic resection and immediate restoration of continuity with a mechanical anastomosis. Morbidity was low, including one case of evisceration and one of pneumonia, and there was no mortality after three months. Early resection seems to prevent the risks of necrosis and recurrence, and to have a good prognosis for survival.


[Cockett's syndrome caused by an ectopic kidney. Apropos of two cases].

Two original cases of Cockett's syndrome caused by an ectopic and/or malformed kidney are reported. Intravenous pyelography and computed tomography allow establishing the diagnosis. The choice of the therapy depends on the patency of the left common iliac vein.


[Intestinal invagination in adults. Apropos of a case of idiopathic ileocolic invagination].

Intestinal intussusception is a rare condition in adults, in whom it is caused by an organic lesion in 60% of all cases. On the basis of one case of ileocolic intussusception in a 64-year-old patient, the author reviews the literature and sums up the clinicopathological data, as well as the elements of the clinical diagnosis and, most importantly, of the radiological diagnosis for which enema with hydrosoluble contrast media is the method of choice. The treatment mainly consists in intestinal resection with anastomosis.


[Idiopathic segmental infarction of the greater omentum. Apropos of a new case].

Idiopathic segmental infarction of the greater omentum is an unusual cause of acute abdomen. The preoperative diagnosis is difficult. Before laparotomy the diagnosis is hardly ever considered; the authors report a personal case and describe etiologic hypotheses and the clinical picture of this disease.


[Arthroscopy of the shoulder].

Arthroscopy of the shoulder is currently gaining importance because of the diagnostic aid it can provide to understand the pathology of this complex joint, and also because of the technical possibilities it offers for treatment. From a diagnostic point of view, it has allowed, for example, a better understanding of the anatomical lesions observed in instability, or a better analysis of partial rotator cuff lesions. The therapeutic possibilities are the reason for the current enthusiasm for this endoscopic technique: removal of a foreign body, articular washing, synovectomy, ligament reinsertion of abrasion are now accessible with some training. The two indications of shoulder arthroscopy that are most resorted to are the endoscopic treatment of some cases of shoulder instability with reinsertion of disinserted elements at the anterior capsular-ligamentous level, and anterosuperior decompression of the rotator cuff when there is a conflict between this tendinous area and the acromiocoracoid osteoligamentous dome.


[Iliac eventration after posterior iliac bone removal. An original technique of reconstruction].

A tricortical bone graft removal from the iliac crest causes a parietal defect that may be complicated by iliac or iliolumbar eventration, according to the extent of the bone loss. On the basis of their experience with a particularly characteristic clinical case, the authors describe an original reconstruction technique. The surgical techniques aimed at parietal restoration described in the literature include either the use of foreign materials or of those provided by the patient (skin, fascia lata, etc.), or the use of the surrounding anatomical elements to reconstruct a solid wall. Most of these procedures may be difficult to implement if the loss of substance is extensive, or in a posterior site. Thus, in order to prevent possible recurrence in case of major bone defects, we propose to combine the cure of the eventration with a restoration of bone continuity with a cold-conserved allograft and the reconstruction of the musculoaponeurotic curtain by the translation of the lumbosacral fascia. This procedure allowed obtaining a very satisfactory result, which was stable after 18 months both as regards parietal reconstruction and as regards pain, owing to the restoration of balance in the paraspinal muscular support.


[Technique of percutaneous drainage under ultrasound guidance for acute cholecystitis].

Gallbladder percutaneous drainage under ultrasound guidance could offer an interesting option compared to surgery in multi-systemic disease patients with acute cholecystitis and who are considered as high-risk patients towards anesthesia and surgery. The authors describe this technics which is an easy, quick and eventually repetitive procedure with no major complications.


Accuracy in establishing identity in edentulous individuals by means of intraoral radiographs.

The aim of this study was to evaluate the degree of accuracy in establishing identity in edentulous individuals by using occlusal "ante- and postmortem" radiographs of the maxilla. Twelve examiners with the following background; six dental radiologists, four other specialists in dentistry and two dental students were asked to combine the radiographs. The results showed that six of the 12 observers were able to correctly establish identity for all 20 cases. Five of these observers were dental radiologists and one of them was a prosthodontist. The other specialists made 3-8 identifications. The senior and the junior dental students made two and four incorrect identifications. The findings from this study emphasise the importance of having more than one forensic specialist signing the identification form. The results also indicate that examiners well trained in oral radiology are able to determine a positive identity even in cases where the teeth are not present.


Dental identification after the Dash 7 aircraft accident at Torghatten, Northern Norway, May 6th, 1988.

The accident occurred when a Dash 7 aircraft hit a steep mountain side and fell approximately 70 meters. A fire broke out immediately and all 33 passengers and three crew members perished. Most of the victims were found in or near the wreckage and burnt by the fire. The identifications were carried out by the Norwegian Identification Commission which included four dentists. One dentist, together with a pathologist, co-operated with the police at the site of the accident. All 36 victims were identified and in 32 cases (89%) dental identity was established. This success was mainly due to systematic radiographic recordings of the victims and a comparison with ante-mortem radiographs. The Norwegian computer programme for identification was successfully tested during this investigation.


International co-operation in a dental identification.

A case of international identification of an unknown male, where the teeth were essential in establishing identity is described. A noteworthy aspect of the procedure was the teamwork between the police and other authorities, and the forensic odontologist and medical examiners in Albania and Sweden. In Albania a forensic medical examiner handled the dental examination, since forensic odontology is not yet acknowledged. The importance of having dental and medical ante-mortem registers for missing persons in every country is stressed. This is best administered in co-operation with the police authorities.


Exhumation following incorrect identification. A case report.

The exhumation of two victims of a light aircraft accident was performed in February 1991 at Oudtshoorn, Cape. The exhumation was ordered by the magistrate on request of the parents of one of the victims who suspected that their son had been wrongly identified at the postmortem examination. At the pre-burial autopsy all the victims were positively identified, but the identity of two of the victims was subsequently reconsidered and reversed because one of them wore a silver bracelet. Burial then took place. The exhumation was ordered one month after the accident at which stage the bodies were in an advanced state of decomposition. Oral autopsies were performed on each of the victims, the jaws were radiographed and postmortem dental records drawn up. Dental records were obtained from the dentists who treated them and the information from these records was used to draw up ante-mortem dental records. A comparison between the postmortem and ante-mortem records showed that the victims were incorrectly identified and reburial in the correct graves was subsquently carried out. This case again emphasies the importance of dental identification in cases of severe burning, multilation or decomposition in order to avoid mistaken identity and incorrect burial in the future.


Prevention of sudden cardiac death.

Since the vast majority of these deaths are triggered by arrhythmias, the first step in prevention is to understand the risk factors for malignant rhythms. The second is to identify patients who may benefit from preventive intervention. Finally, who should get drugs, who automatic cardioverter-defibrillator implantation. The determinants of that choice remain to be elucidated.


Management of HIV infection: a summary of summaries (I).

Often the hardest task in managing HIV-infected patients is keeping up-to-date on the latest in HIV care. Presented here is a summary of 32 current, accessible articles for the primary care physician.


Coronary heart disease in women: a 'new' problem.

Recent reports of "sex bias" in the management of heart disease serves to focus on a more fundamental problem. Too little is known about the biology of heart disease in women or the reasons for striking differences in women's risks, prognosis, and treatment outcome--medical or surgical. Studies more sharply focused on these issues could improve treatment for half of the patient population.


The acquisition of Italian morphology: implications for models of language development [see comment].

This study explores the spontaneous acquisition of Italian inflectional morphology by three children (age range I;4-3;O). Longitudinal, free speech samples are examined, focusing on the development of the morphological paradigms of Italian verbs, pronouns and articles. Data analysis is conducted using criteria appropriate to allow reliable cross-linguistic comparisons with data from English. By this means we evaluate the plausibility of a nativist, parameter-setting account of language development in Italian and English, as recently proposed for these two languages. Results show that the general developmental patterns observed in Italian are not significantly different from those found in English. These findings are not consistent with current interpretations of parameter-setting accounts of language development. Alternative explanatory models are discussed.


Comprehension and production of gesture in combination with speech in one-word speakers.

This study explores the role that gesture plays in the earliest stages of language learning. We describe how one-word speakers use gesture in combination with speech in their spontaneous communications, and interpret gesture presented in combination with speech in an experimental situation. Forty one-word speakers (ages 1;2.22 to 2;4.6) were videotaped in a free-play session which provided data on the child's spontaneous gesture and speech production. The children were also given a comprehension task in which the presence and absence of gesture were systematically varied in relation to speech. We found that (1) all of the children spontaneously produced gestures in combination with speech, and (2) all of the children were able to understand gesture when it was presented in combination with speech, not only when the gesture was redundant with speech but also when the gesture substituted for speech. These data suggest that, even at this young age, gesture naturally forms an integrated system with speech in both production and comprehension.


Discourse and the acquisition of eat.

English has several classes of transitive verbs which can optionally appear without an undergoer. Despite their similar syntactic subcategorization, there are at least three different semantic subclasses that allow undergoer omission. Information sources based on surface structure, for example, syntactic bootstrapping, cannot inform the child of the semantic representation of these verbs. The focus of this paper is the acquisition of a single English verb, eat. The transcripts of 40 children, who were audiotaped monthly from 1;0 to 3;0, showed that eat was the first member of this verb class to be acquired. Some 1276 eat sentences were analysed for the presence of overt undergoer arguments across levels of cumulative verb lexicon (CVL) size, and two discourse conditions: (1) UNDERGOER ACCESSIBLE and (2) OPEN (to undergoer omission). Results indicate that undergoer omission became associated with discourse conditions when CVL size rose above 75 types, at MLU approximately 2.4 and age approximately 2;3. This suggests that two-year-old children are sensitive to a relationship between undergoer omission and discourse context.


Phonological characteristics of words young children try to say.

To examine the possible persistence of phonological selectional constraints on young children's lexical choices, the words attempted in the conversational speech of a longitudinal sample of 12 normally-developing preschoolers from age 2;0 to 5;0 were scored for syllabic length, presence of consonant clusters, and distribution of constituent phonemes. Except at the youngest ages, few developmental changes in target word characteristics were seen, and the observed differences were largely accounted for by syntactic, lexical, and pragmatic factors. The results suggest that selectional constraints persist only briefly in the course of language acquisition.


Mental terms in mothers' and children's speech: similarities and relationships.

In this study, mental terms in mothers' and their children's speech at two and three years of age were studied in order to examine the relationships between maternal and child use. Nineteen mother and child dyads were videotaped for one hour on each of two days when the children were 2;0 and again for two one-hour sessions on separate days when they were 3;0, and mental terms were noted. The utterances in which mental terms were used were coded for function. Results supported the existing picture of children's mental term use. Few terms appeared at 2;0, but many were used at 3;0 with think and know predominating. Mental terms occurred more commonly in utterances used to regulate the interaction between the participants than in utterances referring to mental states. Children's mental term use mirrored that of their mothers. Further, mothers' use of mental terms for particular functions when their children were 2;0 predicted their children's use at 3;0. While allowing no conclusions about causation, our findings suggest that the development of mental state language, and thus presumably a theory of mind, is fostered by the linguistic environment. Specifically, it is argued that the tendency of mothers to focus their children's attention on mental processes by talking about them and, more importantly, by using utterance types which conceivably direct the children to reflect on their own mental states, is associated with children's use of mental terms.


Can bilingual two-year-olds code-switch?

Sociolinguists have investigated language mixing as code-switching in the speech of bilingual children three years old and older. Language mixing by bilingual two-year-olds, however, has generally been interpreted in the child language literature as a sign of the child's lack of language differentiation. The present study applies perspectives from sociolinguistics to investigate the language mixing of a bilingual two-year-old acquiring Norwegian and English simultaneously in Norway. Monthly recordings of the child's spontaneous speech in interactions with her parents were made from the age of 2;0 to 2;7. An investigation into the formal aspects of the child's mixing and the context of the mixing reveals that she does differentiate her language use in contextually sensitive ways, hence that she can code-switch. This investigation stresses the need to examine more carefully the roles of dominance and context in the language mixing of young bilingual children.


The development of children's knowledge structures: events, slots, and taxonomies.

Seventeen preschool (age range 2; 10-3;6), 26 kindergarten (age range 5;5-6;7), and 26 fourth-grade (age range 9;5-10;5) children's knowledge structures were examined with a word association task and a match-to-sample picture task to determine whether or not children used slot-filler categories as a mediating structure between event-based and taxonomic knowledge structures, as proposed by Nelson (1985, 1986). In general, preschool children were able to provide event-based, but not slot-filler or taxonomic, relations; kindergarten children were able to provide event-based or slot-filler relations, but very few taxonomic relations; and fourth-grade children were able to provide all three relations. These findings support Nelson's hypothesis that taxonomic knowledge structures are derived from event-based knowledge structures with the aid of slot-filler categories.


Interpreting and responding to spoken language: children's recognition and use of a speaker's goal.

This study had two purposes. The first was to examine age differences in the extent to which children infer and use a speaker's interpersonal goal to understand speech acts. To this end, the subjects responded to Do you have...? requests in a role-playing task. The goal behind such a request is implicit in the speaker's choice of noun phrase. We sought to determine whether children, like adults, use the noun phrase to infer the speaker's goal and thereby decide whether the request is a yes-no question or a directive. The second purpose was to examine age differences in the extent to which children select responses that carry implications appropriate to the speaker's interpersonal goal. To do this, Do you have...? requests containing general category labels were addressed to the children when they possessed all the category exemplars and when they possessed only a few exemplars. A simple yes implies that the listener has nearly all exemplars and, therefore, is inappropriate for the speaker's goal in the latter situation. The subjects were six-, seven-, nine-, and eleven-year-olds and adults, with 12 subjects per age. Only the eleven-year-olds and the adults used the speaker's noun phrase and considered the implications of yes.


Morphosyntactic development in Italian and its relevance to parameter-setting models: comments on the paper by Pizzuto & Caselli.

Pizzuto & Caselli (1992) claim that data from the morphosyntactic development of Italian-speaking children are inconsistent with nativist, parameter-setting models of language development. In the present Note it is argued that much of the data which Pizzuto & Caselli adduce is irrelevant to the specific hypotheses they are evaluating and that those data which are relevant fully support parameter-setting and linguistic-theoretic models, contrary to their claims.


The Human Communication Research Centre dialogue database.

The HCRC dialogue database consists of over 700 transcribed and coded dialogues from pairs of speakers aged from seven to fourteen. The speakers are recorded while tackling co-operative problem-solving tasks and the same pairs of speakers are recorded over two years tackling 10 different versions of our two tasks. In addition there are over 200 dialogues recorded between pairs of undergraduate speakers engaged on versions of the same tasks. Access to the database, and to its accompanying custom-built search software, is available electronically over the JANET system by contacting liz@psy.glasgow.ac.uk, from whom further information about the database and a user's guide to the database can be obtained.


A comparison of twins and singletons with child psychiatric disorders: an Item Sheet study.

An important assumption underlying the twin method of comparing MZ and DZ twins is that results from twin samples can be generalized to the larger singleton population. This study uses Item Sheet data on child psychiatric patients to compare twins and singletons on demographic, diagnostic and symptomatic variables. Although little difference was found between the two groups, conduct disorders were significantly over-represented in the twin population. The significance of this finding is discussed. This study lends overall support for the use of the twin method in child psychiatric samples.


Behavioural and cognitive characteristics of conduct disordered-hyperactive boys from age 6 to 11: a multiple informant perspective.

Three subgroups of kindergarten boys (stable conduct disordered-hyperactive (SCDH), stable conduct disordered (SCD), stable non-deviant control (SC)) were defined and followed up three, four and five years later in primary school. Using assessments from multiple informants (teachers, mothers, peers, self), the results showed that SCDH boys were more hyperactive (i.e. overactive/inattentive) and displayed a greater diversity of conduct problems in school and home at follow-up compared to the other groups. These results and the fact that during early adolescence SCDH boys tend to develop a worse prognosis than SCD boys suggest that they should be distinguished.


A motor presetting study in hyperactive, learning disabled and control children.

Motor presetting was investigated in hyperactive children, learning disabled children and normal controls. The reaction time of the hyperactive group was more sensitive to increases in interstimulus interval (event rate) than was that of the learning disabled and the controls. This finding indicates that hyperactive children have difficulty with motor presetting.


Observations of hyperactive behaviour during vigilance.

A sustained attention deficit implies a deficit originating in the course of a task. A series of studies previously indicated that task inefficiency in hyperactive children is independent of task duration. The present study shows that differences in body activity between hyperactive and control children are also independent of task duration. However, the groups did differ in visual behaviour, i.e. hyperactive children did look away from the task more frequently as time proceeds. This visual behaviour did not interfere with task accuracy. It is argued that the results indicate that hyperactive children may have prevented a further deterioration of task efficiency over time through self stimulation.


The performance of hyperactive and control preschoolers on a new computerized measure of visual vigilance: the Preschool Vigilance Task.

This study examines the ability of a new visual vigilance task, the Preschool Vigilance Task (PVT), to differentiate between hyperactive and control preschoolers. The PVT was designed to be an objective measure of attention specifically for use with the preschool-age population. Hyperactive and control preschoolers were tested on the PVT and the Continuous Performance Task (CPT). Results indicated that both were useful in differentiating between hyperactive and control preschoolers. Implications for the utility of the PVT and future research directions are discussed.


School-age children's perceptions of coping with distress: strategies for emotion regulation and the moderation of adjustment.

An inventory was developed to assess children's perceptions of what they did to feel better in stressful situations, and to examine the role of these perceptions in moderating the relationship between stress and outcome. A total of 345 6-12 year old children from diverse backgrounds were interviewed. Children's perceptions of what they do to relieve distress could be organized into six factors including the use of social support, distraction/avoidance, and other categories of coping which show continuity with those identified for infants and toddlers. Several types of coping served as compensatory moderators of the relationship between stress and self-worth.


Formal thought disorder in pediatric complex partial seizure disorder.

We compared the formal thought disorder ratings of 27 children with complex partial seizure disorder, 31 schizophrenic children and 58 normal children. The epileptic children with fullscale IQ scores below 100 had significantly more formal thought disorder than normal children with a similar IQ. The severity of their formal thought disorder was related to the age of seizure onset, seizure control and a diagnosis of schizophrenia spectrum disorder. The schizophrenic children had thought disorder irrespective of IQ scores. The cognitive correlates of their formal thought disorder scores differed from those of the epileptic children. Possible anatomical substrates of thought disorder in childhood complex partial seizure disorder and schizophrenia are discussed.


Stable isotope dilution mass spectrometry for the simultaneous determination of cortisol, cortisone, prednisolone and prednisone in plasma.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of cortisol, cortisone, prednisolone and prednisone in human plasma is described. [1,1,19,19,19-2H5]Cortisol, [1,1,19,19,19-2H5]cortisone, [1,19,19,19-2H4]prednisolone and [1,19,19,19-2H4]prednisone were used as internal standards. Formation of the bismethylenedioxy-3-heptafluoro-n-butyryl (BMD-HFB) derivatives made possible the separation of the four corticosteroids with good gas chromatographic behaviour. The new double derivatization has been demonstrated to be of value for sensitive and selective quantification by this technique. Detection was performed by monitoring the molecular ion (M+) of the BMD-HFB derivatives for cortisone and prednisolone, the [M - 18]+ ion for cortisol, and the [M - 30]+ ion for prednisone. The method requires no complex corrections for contributions and provides good accuracy and precision.


Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate.

A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.


Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.


Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization.

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicyclic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde-3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.


Characterization of thyroxine-albumin binding using high-performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin.

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.


Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites.

A sensitive, specific capillary gas chromatographic-electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs greater than 76% and for GTN and the GDNs greater than 95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1.3-GDN maximum concentrations (Cmax) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the Cmax ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN Cmax ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.


Chiral separation of barbiturates and hydantoins by reversed-phase high-performance liquid chromatography using a 25 or 50 mm short ODS cartridge column via beta-cyclodextrin inclusion complexes.

A high-performance liquid chromatographic method on a 25 or 50 mm short ODS cartridge column has been developed for the resolution of the enantiomers of some optically active barbiturates and hydantoins in human serum. beta-Cyclodextrin was used in the mobile phase. This method also seems to be an easy and effective way to test whether beta-cyclodextrin would be a useful chiral discriminator for a particular racemate.


High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.


High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver.

A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection is described for determining the production of the major N-dealkylated metabolite of amiodarone in rat liver microsomes. The principal advantages of this method are its simple sample preparation (protein precipitation by acetonitrile), low detection limit for N-desethylamiodarone (0.05 mumol/l) and relatively short analysis time (16 min). Its analytical applicability is demonstrated by the comparison of the kinetic parameters (maximum velocity and Michaelis-Menten constant) between Sprague-Dawley and Dark-Agouti rats.


Determination of the pyridinium metabolite derived from haloperidol in brain tissue, plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method for the determination of the pyridinium metabolite (HPP+) derived from the antipsychotic drug haloperidol (HP) in brain tissue, plasma and urine using high-performance liquid chromatography with fluorescence detection is described. The HPP+ present in biological samples was extracted using a Sep-Pak C18 cartridge. Recoveries of HPP+ ranged from 78 to 90%. Final separation and quantitative estimations of HPP+ were achieved on a C18 reversed-phase column employing a mobile phase of acetonitrile-30 mM ammonium acetate (40:60, v/v) containing 10 mM triethylamine and adjusted to pH 3 with trifluoroacetic acid. The fluorescence detection utilized an excitation wavelength of 304 nm and an emission wavelength of 374 nm. Standard curves were linear in the range of 2.5-100 ng/ml for brain tissue homogenate and plasma samples and 10-500 ng/ml for urine samples. The detection limit of HPP+ was about 1 ng/ml in all biological samples. The concentrations of HPP+ in brain tissue, plasma and urine from HP-treated rats were determined using this method.


On-line solid-phase extraction and high-performance liquid chromatographic determination of nortriptyline and amitriptyline in serum.

An isocratic reversed-phase high-performance liquid chromatographic method with on-line solid-phase extraction for the simultaneous determination of amitriptyline and nortriptyline in serum has been developed. A 250-microliters serum sample is injected directly onto a commercially available CN cartridge and, after a washing step, the retained solutes are backflushed onto a bonded-phase CN column using a column-switching technique and a mobile phase composed of acetonitrile (26%) and 0.05 M phosphate buffer with diethylamine. Serum is diluted with 0.1 M sodium lauryl sulphate and centrifuged before the injection. Detection at 210 nm ensures sufficient sensitivity. The recovery is almost quantitative and the relative standard deviation ranges from 2.8 to 8.0% for concentrations of 200-40 ng/ml. Being rapid and simple, the method is convenient for routine use.


Simultaneous determination of omeprazole and its metabolites in plasma and urine by reversed-phase high-performance liquid chromatography with an alkaline-resistant polymer-coated C18 column.

Omeprazole (OPZ) is a proton pump inhibitor in gastric parietal cells. A reversed-phase high-performance liquid chromatographic method was developed that enables concentrations of OPZ and its major metabolites, omeprazole sulphone (OPZ-SFN) and hydroxy-omeprazole (H-OPZ), to be determined simultaneously in plasma and that of H-OPZ in urine. To prevent decomposition of OPZ, all the processes (extraction, injection and elution) were carried out under alkaline conditions. Recoveries of the analytes and internal standard were greater than 93.1%. The intra- and inter-assay coefficients of variation were less than 9.1 and 6.4% for plasma samples and less than 2.9 and 3.9% for urine samples, respectively. The minimum determinable concentration (relative standard deviation 10-15%) was 10 ng/ml for all analytes in plasma and H-OPZ in urine samples. The clinical applicability of this assay method was evaluated by determining plasma concentration-and urinary excretion-time courses of the respective analyte(s) in four healthy volunteers after an oral dose of 20 mg of OPZ. The present assay is considered to be simple, precise and accurate and suitable for the study of the kinetic disposition and metabolism of OPZ, which is an extensively metabolized drug in the human liver.


High-performance liquid chromatographic determination of N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methionine, the active plasma metabolite of a prodrug atriopeptidase inhibitor (SCH 42495), using a thiol selective (Au/Hg) amperometric detector.

A high-performance liquid chromatographic assay for the determination of N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methionine (SCH 42354; II), the active metabolite of the atriopeptidase inhibitor prodrug, N-[2(S)-(acetylthiomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methi onine ethyl ester (SCH 42495; I), in human plasma was validated for use in clinical pharmacokinetic studies. Plasma (200 microliters) was processed by protein precipitation with acetone containing the internal standard, N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L- ethionine (III). Compound II was recovered (ca. 90%) in the supernatant after centrifugation and prepared for injection by the addition of 0.15 M monochloroacetic acid containing 0.2 mM EDTA. Separation of II and III was accomplished on commercially available reversed-phase C8 columns designed for the separation of basic compounds. Both compounds were detected using amperometric detection (+0.125 V versus Ag/AgCl) on a thin-layer Au/Hg amalgam electrode. The lower limit of quantitation was 10 ng/ml, where the inter-assay precision (coefficient of variation) was +/- 11.4% and the inter-assay accuracy (bias) was +1.0%. No endogenous interferences were observed in the extracts obtained from drug-free plasma. The detector response (using either peak area or height ratios of II to III) was linear from 0.01 to 1.0 micrograms/ml. Compound II was stable in plasma supplemented with EDTA and sodium hydrogensulfite for at least 3 months when stored frozen at -78 degrees C; no significant decomposition of II was observed following three freeze-thaw cycles. The feasibility of this liquid chromatographic assay with electrochemical detection was demonstrated with plasma samples from hypertensive subjects administered 100 mg of compound I.


Determination of cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid in the urine of a patient with cystathioninuria using column liquid chromatography-mass spectrometry.

A method for the measurement of cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid in the urine of a patient with cystathioninuria has been developed, using column liquid chromatography-mass spectrometry. Cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid were determined by scanning the [M + H]+ ions of each compound. The recoveries were 80-92.4% for cystathionine and 80-100% for perhydro-1,4-thiazepine-3,5-dicarboxylic acid after ion-exchange treatment. The results agreed well with those obtained using an amino acid analyser. The concentrations found for cystathionine and perhydro-1,4-thiazepine-3,5-dicarboxylic acid were 1.289 +/- 0.099 mg/ml and 0.310 +/- 0.0067 mg/ml, respectively.