Direct visualization of the dystrophin network on skeletal muscle fiber membrane.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene locus, is expressed on the muscle fiber surface. One key to further understanding of the cellular function of dystrophin would be extended knowledge about its subcellular organization. We have shown that dystrophin molecules are not uniformly distributed over the humen, rat, and mouse skeletal muscle fiber surface using three independent methods. Incubation of single-teased muscle fibers with antibodies to dystrophin revealed a network of denser transversal rings (costameres) and finer longitudinal interconnections. Double staining of longitudinal semithin cryosections for dystrophin and alpha-actinin showed spatial juxtaposition of the costameres to the Z bands. Where peripheral myonuclei precluded direct contact of dystrophin to the Z bands the organization of dystrophin was altered into lacunae harboring the myonucleus. These lacunae were surrounded by a dystrophin ring and covered by a more uniform dystrophin veil. Mechanical skinning of single-teased fibers revealed tighter mechanical connection of dystrophin to the plasma membrane than to the underlying internal domain of the muscle fiber. The entire dystrophin network remained preserved in its structure on isolated muscle sarcolemma and identical in appearance to the pattern observed on teased fibers. Therefore, connection of defined areas of plasma membrane or its constituents such as ion channels to single sarcomeres might be a potential function exerted by dystrophin alone or in conjunction with other submembrane cytoskeletal proteins.


Differential localization of Acanthamoeba myosin I isoforms.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.


Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos.

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.


Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity.

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.


Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.


Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue.

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.


Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles.

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.


Kinesin family in murine central nervous system.

In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.


Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells.

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.


Apoptosis in metanephric development.

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.


Minimal DNA sequences that control the cell lineage-specific expression of the pro alpha 2(I) collagen promoter in transgenic mice.

The pattern of expression of the pro alpha 2(I) collagen gene is highly tissue specific in adult mice and shows its strongest expression in bones, tendons, and skin. Transgenic mice were generated harboring promoter fragments of the mouse pro alpha 2(I) collagen gene linked to the Escherichia coli beta-galactosidase or firefly luciferase genes to examine the activity of these promoters during development. A region of the mouse pro alpha 2(I) collagen promoter between -2,000 and +54 exhibited a pattern of beta-galactosidase activity during embryonic development that corresponded to the expression pattern of the endogenous pro alpha 2(I) collagen gene as determined by in situ hybridization. A similar pattern of activity was also observed with much smaller promoter fragments containing either 500 or 350 bp of upstream sequence relative to the start of transcription. Embryonic regions expressing high levels of beta-galactosidase activity included the bulbus arteriosus, valves of the developing heart, sclerotomes, meninges, limb buds, connective tissue fascia between muscle fibers, osteoblasts in newly formed bones, fibroblasts in tendons, periosteum, dermis, and peritoneal membranes. The pattern of beta-galactosidase activity was similar and included within the extracellular immunohistochemical localization pattern of transforming growth factor-beta 1 (TGF-beta 1). The -315(-)-284 region of the pro alpha 2(I) collagen promoter was previously shown to mediate the stimulatory effects of TGF-beta 1 on the pro alpha 2(I) collagen promoter in DNA transfection experiments with cultured fibroblasts. A construct containing this sequence tandemly repeated 5' to a very short alpha 2(I) collagen promoter (-40(-)+54) showed preferential activity in tail and skin of 4-wk-old transgenic mice. Except for low expression of the transgene in bone, this pattern mimics the expression of the endogenous pro alpha 2(I) collagen gene. We propose the hypothesis that the tissue-specific expression of the pro alpha 2(I) collagen gene during embryogenesis is controlled by both TGF-beta 1 and cell-specific transcription factors; one of these could interact directly or indirectly with either the -315(-)-284 or the -40(-)+54 segment.


Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts.

Regulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10-25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady-state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51-80-year-old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression.


Tributyltin is a potent inducer of the heat shock response in human diploid fibroblasts.

Submicromolar concentrations of tributyltin (TBT), a commercially used organotin compound, were found to induce the expression of several stress proteins, most notably HSP89 and HSP70, in IMR-90 human diploid fibroblasts in a time- and dose-dependent manner. This induction can be demonstrated by quantitation of 1) synthesis of the heat shock proteins (HSPs), 2) relative abundance of mRNA of hsp70, and 3) transient expression of a human hsp70 promoter driven reporter gene. TBT also increased the abundance of mRNA of heme oxygenase, whereas heat shock was without effect. Analysis of protein binding to a consensus heat shock element (HSE) by electrophoretic mobility shift assay suggests that the induction of the heat shock response by TBT was attributable to activation of the heat shock transcription factor (HSTF).


Localization of p35 (annexin I, lipocortin I) in normal adult rat kidney and during recovery from ischemia.

The 35-kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca(++)-dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole-kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays.


Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.


Biphasic effect of the mitotoxin bFGF-saporin on bovine lens epithelial cell growth: effect of cell density and extracellular matrix.

We have studied the effect of a specific FGF receptor suicide antagonist on the growth of bovine epithelial cells (BEL cells) in culture. This basic fibroblast growth factor-saporin conjugate (bFGF-SAP) has a biphasic effect on bovine lens epithelial cells (BEL cells). Whereas 0.01 nM and 0.1 nM bFGF-SAP stimulate BEL cells proliferation, 1 nM and 10 nM bFGF-SAP have the predicted toxic effects on BEL cell growth. The toxicity of bFGF-SAP is observed 2 to 3 days after the initial treatment and depends on cell density. Accordingly, the sensitivity of confluent cells to bFGF-SAP is reduced compared to sparse cells. A time course analysis reveals that bFGF-SAP is effective after a short exposure to cells and that its effects are not increased with longer treatments. Cell growth on bFGF-SAP pretreated extracellular matrix (ECM) or posterior lens capsule (PLC) is also affected. Basic FGF-SAP has been shown to bind to the extracellular material, allowing a modulation of lens cells migration and survival by a single treatment in vitro. This finding raises the possibility of its use in vivo to prevent capsules invasion by lens cells after cataract surgery.


Cortical photons in early development of frog eggs: comparative study of photon emission between nuclear division and cytoplasmic fission.

In the study of cell division in the early development of frog eggs, cortical photon emission was investigated by converting small light emission from living cells into digital pulses of potentials and recording the integrals of these pulses (analog method), counting the number of pulses (photon-counting method), and counting the number of integrated pulses (improved photon-counting method). By the analog and improved photon-counting methods, changes in photon emission due to cell division could be clearly detected. The emitted light increased about 5.10(-19)W at the start of a cleavage furrow. Rapid changes in chemical reactions causing photon emission were compared during nuclear division and cytoplasmic phases. This emission occurred mainly in cytoplasmic fission, the rate being greater than in nuclear division by a factor of about 2.9. Chemical reaction rates were shown to differ according to bulk emission, thus indicating the mechanisms for the reactions to also differ.


Interferon-gamma inhibits proliferation, but not commitment, of murine granulocyte-macrophage progenitors.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.


Electrochemical potentials of potassium in skeletal muscle under different metabolic states.

Intracellular potassium and membrane potential were measured simultaneously by means of double-barrelled liquid ion-exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (EK) was more negative than the simultaneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness.


In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.


Inhibition of apoptosis in human tumour cells by okadaic acid.

Gamma-radiation, tetrandrine, bistratene A, and cisplatin were all found to induce pronounced morphological changes characteristic of apoptosis and extensive DNA fragmentation in the human BM13674 cell line 8 h after treatment. Apoptosis induced in BM13674 cells by these diverse agents was markedly inhibited by 1 microM okadaic acid, a tumour promoter that inhibits protein phosphatases 1 and 2A. This compound also inhibited the appearance of apoptosis in fresh human leukaemia cells that had been exposed to gamma-radiation. The inhibition of apoptosis was confirmed using fluorescence microscopy and DNA gel electrophoresis. Dephosphorylation of a limited number of proteins was shown to be associated with apoptosis and okadaic acid prevented these dephosphorylations. Previous studies on the BM13674 cell line showed that an inhibitor of protein synthesis failed to prevent apoptosis in these cells. The present data provides further support that posttranslational modification of proteins, in particular, phosphorylation/dephosphorylation status, plays an important role in inhibition/activation of programmed cell death in different human cells after exposure to several cytotoxic agents.


Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells.

Vascular endothelial growth factor (VEGF) is a 45kDa secreted peptide that has potent mitogenic activity specific for endothelial cells in vitro and the ability to induce a strong angiogenic response in vivo. In the present study, 24 h treatment with VEGF resulted in a stimulation of expression of the metalloproteinase, interstitial collagenase, at the protein and mRNA levels 2.5-3.0-fold in human umbilical vein endothelial cells but not in human dermal fibroblasts. The dose response curve for collagenase induction was biphasic with the peak stimulatory response obtained by treatment of cells with 10-100 ng/ml (0.2-2 nM) VEGF. The dose response curve for collagenase induction overlapped with, but was not identical to, the response curve for proliferation, which showed VEGF mitogenic activity between < or = 0.1-50 ng/ml (< or = 0.002-1 nM). There was no induction seen in expression of other members of the matrix metalloproteinase family, including the 72kDa type IV collagenase, the 92kDa type V collagenase, or stromelysin. Expression of transcripts for the major metalloproteinase inhibitor, tissue inhibitor of metalloproteinases, was also unaltered by treatment with VEGF (1-200 ng/ml). These studies demonstrate that in addition to stimulating proliferation of endothelial cells, VEGF can also induce the expression of the only metalloproteinase that can initiate degradation of interstitial collagen types I-III under normal physiological conditions. Both responses are likely to contribute to the angiogenic potential of this peptide.


Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract.

We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154-160, 1984; DiMario and Strohman: Differentiation, 39:42-49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311-315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357-2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701-C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF-I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF-I, transferrin (Tf), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony-stimulating factor (M-CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M-CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities.


Effect of membrane phosphatidylethanolamine-deficiency/phosphatidylcholine-excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine.

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)-deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane-associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well-known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn-requiring rat mammary cell line 64-24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12,13-dibutyrate (PDBu) under conditions where cells have either normal or PE-deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (approximately fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE-deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP-choline pathway of PC synthesis indicated that the regulatory enzyme, CTP:phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE-deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation.


Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing.

The effect of transforming growth factor-beta 1 (TGF-beta 1) on collagen biosynthesis was investigated in confluent primary monolayer cultures of rabbit articular chondrocytes (RAC). Exposure to TGF-beta (0.1, 1, and 10 ng/ml) in serum-free medium caused a dose- and time-dependent stimulation of collagen biosynthesis associated with an increase of steady-state levels of procollagen type II mRNA. Elevation of the mRNA steady-state did not result from a stabilization of the transcript, as shown by measure of the mRNA half-life. Electrophoresis (SDS-PAGE) showed that TGF-beta stimulates the synthesis of most collagen isotypes, including type II, without qualitative change in their distribution. Moreover, pulse-chase experiments revealed that TGF-beta did not affect the processing rate of type II procollagen. TGF-beta slightly stimulated the production of prostaglandin E2 (PGE2), which could in turn exert an inhibition on collagen synthesis. However, addition of indomethacin to block prostaglandin synthesis did not further enhance the TGF-beta-induced stimulation of collagen production, suggesting that this mediator was not implicated in the effect. Moreover, TGF-beta increased steady-state levels of procollagen type II, I, and III mRNAs even in the presence of indomethacin. Despite these increased mRNA levels, only the production of type II collagen was significantly augmented, suggesting that type I procollagen mRNA was not fully translated. In addition, the TGF-beta-induced stimulation of collagen synthesis was observed whenever ascorbic acid is added or not in the culture medium. In conclusion, TGF-beta, which is present in great amount in bone and cartilage, can increase the collagen production of cultured RAC and might therefore play a role in the early events of cartilage repair, such as those observed in osteoarthritis.


Nonreceptor mediated nuclear accumulation of insulin in H35 rat hepatoma cells.

We previously demonstrated that insulin accumulated in the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time, temperature, and insulin concentration dependent, but apparently nonsaturable. This study investigated further the initial endocytotic pathways that contribute to the nuclear accumulation of insulin using trypsin treatment of the cells to prevent insulin binding to its plasma membrane receptor. Total cell-associated, intracellular, and nuclear insulin were compared in control and trypsin-treated H35 hepatoma cells. Trypsin treatment markedly decreased total cell-associated and intracellular insulin as well as the nuclear accumulation of insulin when cells were incubated with 2.8 ng/ml insulin. When the cells were incubated with 100 ng/ml insulin, trypsin treatment totally inhibited insulin binding to the plasma membrane for at least 90 min. However, intracellular accumulation of insulin was reduced by only 50% at 60 min, and trypsin treatment failed to inhibit the nuclear accumulation of insulin. Chemical extraction and Sephadex G-50 chromatography revealed nuclear associated insulin in trypsin-treated cells was identical to that in control cells incubated with either 2.8 or 100 ng/ml insulin. These results suggest that a nonreceptor mediated uptake pathway, i.e., fluid-phase endocytosis, contributed significantly to the nuclear accumulation of insulin at high insulin concentrations, but at lower insulin concentrations the receptor-mediated pathway predominated. No matter which initial endocytotic route was used to internalize insulin, the insulin apparently associated with the same nuclear matrix proteins. This association of insulin with the nuclear matrix may be involved in regulation of nuclear events such as cell growth and differentiation or gene transcription.


Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: I. Erythrocytes.

Chick embryos rendered calcium (Ca) deficient by shell-less (SL) culture develop hypertension and tachycardia. Since hypocalcemia is accompanied by hypernatremia systemically but not by lower cellular Ca (Koide and Tuan, 1989), we speculate that cellular Ca handling may be altered in the SL embryo, perhaps involving Na transport. Using erythrocytes (RBC) from day-14 SL and normal (NL) embryos as the experimental cell, cellular Ca handling was studied under varying extracellular osmotic and ionic conditions by analyzing 45Ca uptake and cell volume regulation. Two agents, p-chloromercuriphenylsulfonate (PCM), and inosine/iodoacetamide (INI) were used to treat the RBCs to modify plasma membrane ion permeability and to deplete cellular ATP, respectively. Other cellular functions and activities related to Ca homeostasis, including ATP content and Ca(2+)-ATPase activity, were also analyzed. These analyses showed: (1) in NaCl, Ca uptake was similar in NL and SL cells, except after INI treatment, which resulted in slower Ca uptake by the SL cells, (2) in choline and sucrose, Ca uptake by SL RBCs was higher, (3) Ca uptake by RBCs of both embryos changed depending on the osmotic agent (Na < K < or = choline < sucrose), (4) Ca(2+)-ATPase activity was higher in SL RBC, although there was no change in the size or charge of the enzyme, and (5) in any osmotic agent, cellular Na was significantly lower, whereas cellular K was higher, in SL RBC. Based on these results, three features of RBC Ca handling were apparent: (1) Na-Ca exchange was functional and was more active in SL RBCs, (2) Ca uptake was dependent on the total ionic electrochemical gradient but not on bulk H2O movement, and (3) Ca pumping out capacity was directly correlated with Ca(2+)-ATPase activity. Elevated Ca uptake in sucrose-treated SL RBC is therefore indicative of its greater ion permeability. Taken together, these findings indicate that cellular Ca handling of the RBCs of SL chick embryos is characterized by a more active Na-Ca exchange system, greater ion permeability, and higher Ca pumping out capacity, thereby suggesting an up-regulated Ca handling function in the SL RBCs. The abnormal cellular Ca handling may be a direct result of the systemic Ca deficiency of the SL chick embryo and may be functionally related to its hypertension and tachycardia.


Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes.

We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca(2+)-deficient, shell-less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher alpha-adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long-term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura-2 via transient membrane damage with glass beads. Compared to Fura-2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i-dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura-2 loaded ventricular myocytes were washed in Ca(2+)-free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3.5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01-1 microM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by beta-blockade (1 microM propranolol). In contrast, in SL cells, NE remained effective after beta-blockade, and combined alpha-blockade (1 microM prazosin) and beta-blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency.


Application of triple quadrupole tandem mass spectrometry to the analysis of pyridine-containing derivatives of long-chain acids and alcohols.

An improved derivatization procedure for the preparation of nicotinate and 3-picolinyl esters from mixtures of fatty alcohols and acids has been developed. The derivatives can be analysed by capillary gas chromatography on an SE-54 type column, which affords separation of the acid and alcohol derivatives with the same carbon chain. Detection with tandem mass spectrometric techniques on a triple quadrupole instrument is feasible, and yields informative spectra devoid of coeluting interferences.


Simultaneous analysis of azidothymidine and its mono-, di- and triphosphate derivatives in biological fluids, tissue and cultured cells by a rapid high-performance liquid chromatographic method.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro in the human MT-4 lymphocyte cell line did not require a prior extraction, and AZT and its phosphorylated derivatives could be accurately analysed in one HPLC run. However, plasma, bile, liver homogenate and urine samples could not be injected directly into the chromatograph. Therefore, a solid-phase extraction procedure was developed, using azidodideoxyinosine as internal standard. The extractions of the compounds of interest from all but urine samples were reproducible, with recoveries between 65% (AZT triphosphate from plasma) and 100% (AZT from plasma).


High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.


High-performance liquid chromatographic determination of pamaquine, primaquine and carboxy primaquine in calf plasma using electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection is described for quantification of pamaquine, primaquine and carboxy primaquine in calf plasma. After the proteins had been precipitated with acetonitrile, the drugs were separated on a 5-microns C18-modified polymer gel column with an isocratic mobile phase. The detection limit was 0.01 microgram/ml in plasma for all three compounds. The applicability of the method in pharmacokinetic studies was demonstrated by determining the plasma concentrations of the three substances in calves administered a single dose of pamaquine or primaquine.


Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with carbohydrate and alcohol intake based on 24-h dietary recall.

Quantitative gas chromatographic estimates of the major lipid classes and molecular species in fasting plasma were correlated with total carbohydrate, starch, fibre, sucrose and alcohol intake based on 24-h dietary recall. Spearman coefficients (rs) and tests of significance (P) were obtained for groups of 775 males and 471 females aged 20-59 years from a Toronto-McMaster Lipid Research Clinics Population Study. The most significant correlations varying from rs 0.1 to 0.2 and P 0.001 to 0.0005 (n = 400-773) were between increased intake of alcohol and increased ratios of C50/C54 triacylglycerols, C34/C36 phosphatidylcholines and phosphatidylcholine/free cholesterol (PC/FC) of plasma. Increase in total dietary carbohydrate, starch and fibre correlated with decreasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios (rs = -0.1-0.2; P less than 0.002-0.04; n = 400-773). In contrast, consumption of high levels of alcohol was associated with increasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios. A high intake of alcohol (50-150 ml per day) distinguished itself from other simple carbohydrate-induced lipid profiles by its marked effect on increased C50/C52 triacylglycerol and PC/FC ratio.


Direct-gradient high-performance liquid chromatographic analysis and preliminary pharmacokinetics of flumequine and flumequine acyl glucuronide in humans: effect of probenecid.

A gradient high-performance liquid chromatographic analysis for the direct measurement of flumequine, with its acyl glucuronide, in plasma and urine of humans has been developed. In order to prevent hydrolysis and isomerization of flumequine acyl glucuronide, the samples were acidified by the oral intake of four 1.2-g amounts of ammonium chloride per day. In contrast to the acyl glucuronides of non-steroidal anti-inflammatory drugs, flumequine and its acyl glucuronide were stable in urine of pH 5.0-8.0. Flumequine acyl glucuronide is unstable at pH 1.5. In acidic urine (pH 5-6), almost no flumequine is excreted unchanged (1%): it is excreted chiefly as acyl glucuronide (84.2%). Probenecid co-medication reduces the renal excretion rate of flumequine acyl glucuronide from 662 to 447 micrograms/min (p = 0.00080), but not the percentage of glucuronidation.


Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation.

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 microns in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle-wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.


Determination of methacrylonitrile in serum using gas chromatography and flame ionization detection.

A gas chromatographic method is described for the quantitation of methacrylonitrile in serum. Methacrylonitrile was extracted from rat serum with diethyl ether and then quantified using a gas chromatograph equipped with a flame ionization detector and a 60-m megabore column coated with polyethylene glycol polymer. The recoveries obtained following a one-step extraction with diethyl ether varied from 60% at 3.2 micrograms/ml to 70% at 80 micrograms/ml. The coefficient of variation for the analysis ranged from 2.5% at 400 micrograms/ml to 15.0% at 3.2 micrograms/ml.


Improved high-performance liquid chromatographic method for the determination of ethylmorphine and its metabolites in microsomal incubations and cell culture media.

Ethylmorphine N-demethylation is used as a marker pathway in studies of rat cytochrome P450 3A and 2C11 biotransformations. At present, microsomal activities are generally measured by a colorimetric determination of the formed formaldehyde. In the present study, a high-performance liquid chromatographic method of separating and quantifying both the N-demethylated (norethylmorphine) and the O-de-ethylated (morphine) metabolites is described. Either samples are extracted with ethyl acetate or proteins are precipitated with zinc sulphate-barium hydroxide. Separation is achieved on a CN reversed-phase column, using a mobile phase of phosphate buffer (pH 4.5)-acetonitrile (90:10, v/v). At a flow-rate of 1.5 ml/min, the analysis time is 30 min. The limit of detection (ultraviolet, 210 nm) for ethylmorphine and its metabolites is 0.5 micrograms/ml.


Reversed-phase high-performance liquid chromatographic separation of the stereoisomers of labetalol via derivatization with chiral and non-chiral isothiocyanate reagents.

The antihypertensive agent labetalol is a mixture of two racemates. We report reversed-phase high-performance liquid chromatographic (HPLC) methodology for the separation of the four stereoisomers of labetalol via derivatization with the chiral reagent (4S-cis)-2,2-dimethyl-5-isothiocyanato-4-phenyl-1,3-dioxane. The derivatives were separated on octadecylsilane columns with a methanol-ammonium phosphate buffer mixture as mobile phase. Separations of the diastereomeric forms of labetalol were achieved with the non-chiral derivatizing reagents benzyl isothiocyanate and 1-naphthalenemethyl isothiocyanate. In all cases the derivatives of the R,S/S,R forms eluted before those of the R,R/S,S forms. Isothiocyanates may have general utility in stereoisomer separations of amines by HPLC.


Determination of nalbuphine using high-performance liquid chromatography coupled to photodiode-array detection and gas chromatography coupled to mass spectrometry.

A procedure involving capillary column gas chromatography coupled to mass spectrometry and a method involving liquid chromatography coupled to a diode-array detector have been developed for the analysis of nalbuphine. The extraction step is the same for both techniques and involves extraction under alkaline conditions in chloroform-2-propanol-n-heptane (50:17:33, v/v/v) with levallorphan as the internal standard. After purification by acidic extraction and back alkaline extraction, drugs are derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane for gas chromatography-mass spectrometry and directly injected for high-performance liquid chromatography-diode-array detection. The limits of detection are 2.0 and 25.0 ng/mg, respectively.


Improved high-performance liquid chromatographic procedure for the determination of lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns.

A high-performance liquid chromatographic (HPLC) method for the determination of the ionophore coccidiostat lasalocid in poultry muscle and eggs was developed. The drug was extracted from tissue with acetonitrile. The extract was partitioned between saturated salt and carbon tetrachloride and the organic layer evaporated to dryness. Clean-up was by solid-phase extraction on a silica column. HPLC analysis was carried out on either a polymeric PLRP-S or a porous graphitic carbon Hypercarb column with a basic mobile phase and fluorescence detection with excitation at 310 nm and emission at 420-430 nm. Average recoveries from poultry muscle at the 0.002, 0.010 and 0.050 mg kg-1 levels were 65.7, 72.0 and 77.9%, respectively. Average recoveries from egg at the 0.010 and 0.100 mg kg-1 levels were 76.2 and 76.4%, respectively.


High-performance liquid chromatography-thermospray mass spectrometry of hydroperoxy polyunsaturated fatty acid acetyl derivatives.

A method for the analysis of hydroperoxy polyunsaturated fatty acids was developed. The hydroperoxy groups were acetylated by acetic anhydride, and the mixture was partially purified on a Sep-Pak C18 cartridge and analysed by high-performance liquid chromatography with thermospray mass spectrometry. Generally, the base ion, [M+H - n(60)]+ or [M+H - n(60) - n(H2O)]+, is produced through elimination of acetic acid or water (n = number of hydroperoxy groups). The detection limit for these derivatives was ca. 1 pmol at concentrations of hydroperoxy polyenoic acids prior to derivatization. Using this method, many hydroxy and hydroperoxy polyunsaturated fatty acid derivatives could be detected simultaneously within 30 min on a selected-ion monitoring detection chromatogram without a gradient system. The assay was successfully applied to hydroxy and hydroperoxy polyunsaturated fatty acids from an incubation mixture of rat brain homogenate to which polyunsaturated fatty acids had been added.


Determination of carnosine and other biogenic imidazoles in equine plasma by isocratic reversed-phase ion-pair high-performance liquid chromatography.

The isocratic reversed-phase ion-pair high-performance liquid chromatographic technique presented provides a sensitive, rapid and reproducible analytical method for the selective determination of carnosine and other biogenic imidazoles in equine plasma. Plasma was deproteinized with 5-sulphosalicylic acid and the compounds of interest were isolated by sorbent extraction on Bond Elut PRS cartridges. Recoveries were 97-105% and the lowest limits of detection were 58.3-80.1 nM. All compounds of interest were well resolved within a maximum retention time of 9.2 min. The mean equine plasma carnosine level determined by this method was 11.31 microM. Comparative determinations were made in canine and human plasma. Carnosine was not detected in human plasma. Concentrations of imidazole in canine plasma are reported here for the first time.


Measurement of total plasma and cerebrospinal fluid homocysteine by fluorescence following high-performance liquid chromatography and precolumn derivatization with o-phthaldialdehyde.

Precolumn derivatization of amino acids with o-phthaldialdehyde followed by high-performance liquid chromatographic separation and fluorescence detection is used in many clinical and experimental laboratories for the measurement of primary amino acids. This technique was adapted for the measurement of homocysteine in plasma and cerebrospinal fluid (CSF) following alkylation of the free sulphydryl group with iodoacetate. The minimum detection limits are less than 1 microM in plasma and 80 nM in CSF. Within-day and between-day coefficients of variation for plasma and for CSF are less than 10%. Values for normal plasma homocysteine range from 6.04 to 16.2 microM and for CSF from 0.28 to 0.66 microM.


Monitoring of an experimental red blood cell pathology with gravitational field-flow fractionation.

Gravitational field-flow fractionation is a simple method suitable for the separation of micrometre-size particles, for example the red blood cells (RBC). The variation in the composition of the RBC population in a rabbit during an experimental reversible anaemia induced by phenylhydrazine was studied. Blood samples taken at different stages of the anemia showed differences in the retention and shape of the elution profiles. Microscopic observations of original RBC samples and fractions collected at the outlet of the fractionation channel make a description of the RBC population possible. The three species observed were the normal RBC, the newly produced reticulocytes, and the blood cells containing Heinz bodies (intracellular haemoglobin precipitates). A decrease of the normal RBC from 96 to 1% was observed over five days. The production of reticulocytes in bone marrow is stimulated by the anaemia and increases in percentage after the second day of the anaemia (from 1 to 16%). RBC with Heinz bodies, which appear on the third day, were also studied. Granulometric studies were performed on the RBC sampled from the rabbit each day as well as on some fractions eluted by field-flow fractionation. Reinjection procedures of some cell subpopulations of known size distribution were also performed. The relaxation process of these cells was then studied to approximate their density properties. It was observed that RBC of different density but of the same average size were selectively eluted, as were cells of equivalent density but of different size. Injection of the cells at different stop-flow times enabled the study of the relaxation process on the elution profiles. The results, compared with systematic microscopic observation and size analysis, permit the description of modifications in the RBC composition as well as the purification of subpopulations at each stage of the anaemia. The correlation observed between the fractionation profiles and the progress or the regression of the anaemia opens a new field in the analytical monitoring of this type of pathology.


Quantitation by gas chromatography with selected-ion monitoring mass spectrometry of "natural" diazepam, N-desmethyldiazepam and oxazepam in normal human serum.

During the past five years, the literature has tended to prove the occurrence of "natural benzodiazepines" in tissues and biological fluids of non-medicated humans. Several have been identified but very few papers deal with their quantitation in biological material. We present here a method for the specific and sensitive measurement of serum levels of diazepam, N-desmethyldiazepam and oxazepam by gas chromatography with selected-ion monitoring mass spectrometry in twenty human volunteers without medication. Diazepam was found over the whole population, in the range 7.3-32.0 pg/ml, identical in males and females. The other two were present in only some individuals (1.0-7.6 pg/ml for N-desmethyldiazepam and 2.0-13.0 pg/ml for oxazepam). The origin (endogenous, dietary or microbial) of these substances is still to be elucidated.


Electron-impact and chemical ionization detection of nicotine and cotinine by gas chromatography-mass spectrometry in rat plasma and brain.

Nicotine and its metabolite, cotinine, were measured in rat plasma and brain by gas chromatography-mass spectrometry. Both agents were extracted from plasma and brain, separated on a capillary column, and quantified by single-ion monitoring. The major fragment ions of nicotine and cotinine at m/z 84 and m/z 98, respectively, were monitored by electron-impact ionization detection and the protonated molecular ions at m/z 163 and m/z 177, respectively, were monitored by chemical ionization detection. Both compounds were quantified using deuterium-labeled nicotine and cotinine, respectively, as internal standards.


Simultaneous determination of fluoxetine and norfluoxetine enantiomers in biological samples by gas chromatography with electron-capture detection.

An electron-capture gas chromatographic procedure was developed for the simultaneous analysis of the enantiomers of fluoxetine and norfluoxetine. The assay involves basic extraction of these enantiomers from the biological samples, followed by their conversion to diastereoisomers using the chiral derivatizing reagent (S)-(-)-N-trifluoroacetylprolyl chloride. The method was utilized to detect and measure the quantity of these enantiomers in plasma and urine of patients and in liver and brain tissue of rats treated with (R,S)-fluoxetine.


Rapid and sensitive anion-exchange high-performance liquid chromatographic determination of radiolabeled inositol phosphates and inositol trisphosphate isomers in cellular systems.

A rapid and sensitive high-performance liquid chromatographic method for the determination of multiple inositol phosphates and inositol trisphosphate isomers was developed. The separation of inositol phosphates was optimized by controlling the ionic strength with stepped gradient programs and the pH of mobile phase. Six inositol phosphates were determined within 22 min or the six compounds plus an inositol trisphosphate isomer within 24 min using a single anion-exchange column containing the quaternary ammonium functional group. This technique was successfully applied to the determination of inositol phosphatide turnover by AlF4-stimulation in a small amount (5.10(5)-1.10(6) cells) of cultured retinal capillary pericytes. Because of its efficiency, accuracy and applicability to the separation of inositol phosphates from biological samples, this method may be useful in signal transduction studies in cellular systems.


Rapid fluorimetric assay for the detection of the peptidyl alpha-amidating enzyme intermediate using high-performance liquid chromatography.

Peptidylglycine alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to their corresponding amidated peptides via a stable alpha-hydroxyglycine intermediate. Using a new rapid fluorimetric reversed-phase high-performance liquid chromatographic assay, we have demonstrated that the substrate and product of the amidation reaction, as well as both stereoisomers of the alpha-hydroxyglycine intermediate, can be separated and detected in quantities as low as 1 pmol. The method is highly reproducible and requires less than 11 min for separation and quantification.


Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.


Sensitive method for the determination of organophosphorus pesticides in fruits and surface waters by high-performance liquid chromatography with ultraviolet detection.

A sensitive method for the high-performance liquid chromatographic determination of five organophosphorus pesticides (paraoxon, methyl-parathion, ethyl-parathion, guthion and fenitrothion) in fruits and tap and river water samples is described. For the determination of pesticides in fruits a simple and rapid sample preparation procedure was developed that allowed pesticides to be determined at 50-100 micrograms/kg levels with recoveries ranging from 83 to 118% and relative standard deviations below 6%. The determination of pesticide residues in surface water samples was also successfully accomplished. Concentrations at sub-ppb levels can be measured by using a solid-phase concentration step, the recoveries being over 80%. In analyses of both fruits and surface waters, the sensitivity levels achieved were 2-10 times lower than legal limits admitted in the European Economic Community.


Studies on the methanolysis of small amounts of purified phospholipids for gas chromatographic analysis of fatty acid methyl esters.

The methanolysis of small amounts of purified phosphoglycerides and sphingomyelin was studied and a quantitative comparison of five methods for the methanolysis of standard phosphoglycerides was made. These methods were based on methanolysis with boron trifluoride-methanol, methanolic sodium methoxide (at ambient temperature and with heating) and methanolic sulphuric acid. A further method was based on saponification with methanolic sodium hydroxide and subsequent esterification with boron trifluoride-methanol. Under the experimental conditions, only the sodium methoxide-catalysed method at ambient temperature gave complete methanolysis of phosphoglycerides. For methanolysis of sphingomyelin, boron trifluoride-methanol, methanolic sulphuric acid and methanolic hydrochloric acid were used. It was found that complete methanolysis of sphingomyelin takes 15 h at 90 degrees C. Based on these results, procedures for the methanolysis of phosphoglycerides and sphingomyelin separated by high-performance liquid chromatography are presented.


Coculture of embryos on Vero cells and transfer of blastocysts in humans.

Preliminary results are presented concerning the first clinical application of cocultures of human embryos. In the experimental group, the embryos were cultured and transferred on day 5 post-insemination. Blastocyst formation was not dependent upon the stimulation regimen. Long term or ultrashort stimulation of the ovaries after gonadotrophin releasing hormone analogue gave 55-60% blastocyst formation. Serum was not necessary to obtain blastocysts. When the embryos were cocultured, we observed an increase in the implantation rates per embryo in the pregnant patients. However, a real increase in the pregnancy rate per transfer was observed in a population of patients who had had repeated failures of embryo transfer. This observation is discussed as possibly bypassing an effect of uterine motility, but the overall beneficial effect has to be assessed in a double blind randomized study. It is probable that improvements will not be observed for all the indications for in vitro fertilization.


Preimplantation diagnosis.

Although healthy babies have been born after preimplantation diagnosis for sex determination, this technique is still in an experimental phase. To date, removal by micromanipulation of one or two blastomeres from an eight-cell embryo seems an acceptable procedure. Since a reasonable number of biopsied embryos will implant and develop in utero, the methodology for the analysis of the blastomeres at the chromosomal, gene product or DNA level, needs to be improved before further clinical application. In the meanwhile, more information about early embryo development will become available.


Transvaginal gamete intra-fallopian transfer and embryo intra-fallopian transfer.

This study describes the conduct and results of a recently developed technique for transvaginal catheterization of the Fallopian tube in order to transfer gametes or early embryos. Transvaginal gamete intra-Fallopian transfer (TV-GIFT) was performed in 46 patients after stimulation with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) and transvaginal oocyte retrieval. This resulted in 11 (23.9%) pregnancies. Eight patients delivered healthy children, including one set of twins. Two patients had abortions at 8 and 11 weeks of gestation and one had an ectopic pregnancy. In a first series of 11 women, oocytes were fertilized in vitro and a maximum of three embryos at the 2- to 8-cell stages were transferred into one tube. Three of the 11 cycles with tubal embryo-stage transfer' (TV-TEST) resulted in clinical pregnancy.


Indications for in-vitro fertilization and results.

During the last few years, many healthy children have been born after in vitro fertilization and embryo transfer, fulfilling the wish for a child for many couples who had often nearly given up hope. Because of in vitro fertilization and other related methods developed subsequently (gamete intra-Fallopian transfer, intratubal embryo transfer), progress in the diagnosis and treatment of involuntary childlessness took place and has helped many patients. Furthermore, by dealing with these methods, new knowledge about ovarian function, fertilization and early embryonic development could be gained. This has also led to better treatment of childless couples. Despite the broad diagnostic and therapeutic possibilities of reproductive medicine, its limits must also be taken into account. These limitations have been recognized early by physicians and scientists dealing with this subject and also by society. Guidelines and laws have been drawn up which now give a clear framework.


The male IVF patient--psychosomatic considerations.

In order to study psychosomatic considerations of the male IVF patient, three issues were investigated. First, the personality traits of sterile males (n = 180) were recorded. Male IVF patients were found to have largely the same personality traits as the general population although they tended to have more dominant and retentive traits. Second, in examining the specific stress experiences during IVF, the individual treatment steps were ranked as being similarly stressful by both partners. Males felt the waiting periods (fertilization? pregnancy?) to be clearly more unpleasant than the physical aspects of treatment (e.g. masturbation). Third, an individual comparison of sperm parameters taken during the diagnostic phase with those measured during the IVF attempt, showed that density and morphology were significantly different. Some practical recommendations to reduce the stress that men experience during IVF include his presence during the treatment steps that the female must undergo, as well as his participation in a self-help discussion group.


Fallopian tube sperm perfusion: first clinical experience.

We have developed an insemination method using a large (4 ml) volume of the inseminate. This method incorporates ovarian stimulation, isolation of an optimal number of motile spermatozoa and, finally, Fallopian tube sperm perfusion (FSP). In a clinical study, 139 couples with various causes of infertility, had a total of 239 treatment cycles. The pregnancy rate per treatment in groups with endometriosis, ovulation disorders, tubal impairment, combined male and female factors and subnormal sperm quality were low, ranging from 2.7% to 7.7%. In patients with unexplained infertility, the pregnancy rate per treatment was 26.9% and for this group, the pregnancy rate in the first treatment cycle was 37.3%. In the cervical hostility group, two pregnancies occurred after five treatments. FSP seems to be a favourable treatment for couples with cervical hostility or unexplained infertility. About half of the women in these groups conceived after three treatment cycles. FSP is easier to perform and is less expensive than other methods of assisted procreation, such as in vitro fertilization and gamete intra-Fallopian transfer.


Genetic research in embryology.

Based on findings on the molecular mechanisms of differentiation in drosophila, developmental genes have been identified in mammals similar to those in drosophila. Moreover, sequence similarities between drosophila developmental genes and mammalian growth factors provide evidence for common ancestors of such genes and suggest a complex link between growth and differentiation. As numerous oncogenes display their action as growth factors and are expressed during embryogenesis, differentiation and oncogenesis seem to be two sides of the same coin. Therefore, the aim of molecular embryology is twofold; elucidating differentiation processes and disclosing oncogenesis by studying the physiological function of oncogenes during development. Recent progress in molecular genetic technology now allows gene function to be studied by modification or disruption of genes. Models of clinical diseases have already evolved from such work and it is anticipated that progress in molecular embryology will further stimulate work on the diagnosis and therapy of genetic diseases as well as clinical oncology.


The use of gonadotrophin releasing-hormone analogues (GnRHa), in in-vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary.

Several lines of investigation suggest a direct modulatory role for gonadotrophin-releasing hormone (GnRH) on granulosa cell functions. Also, GnRH and its analogues (GnRHa) have been implicated in the resumption of meiosis, both in vivo and in vitro. Despite the presence of specific receptors for GnRH on human granulosa and luteal cells, very little is known about the possible effects of this hormone on the ovary. The use of GnRHa for long periods of time in patients undergoing in vitro fertilization (IVF) may influence granulosa cell function and/or oocyte maturation. We describe our clinical and experimental data in which we have searched for evidence of a direct action of GnRHa on the ovary. We have found that the retrieval of higher numbers of oocytes in women treated with GnRHa is correlated with oocytes of lower quality, manifested by a decreased fertilization and implantation rate. This impairment seems to be the consequence of oocyte immaturity, as ascertained by cytogenetic analysis of unfertilized oocytes in which an increase in diploidy, as well as prematurely condensed sperm chromosomes of the G1 phase, was observed in women with an excessive response to the stimulating drugs. Follicular atresia was not increased in women treated with GnRHa. Thus, there was no evidence for a direct effect of GnRHa on the human oocyte. Rather, the observations reflect the harmful effect of pushing follicles in early stages of development using this stimulation protocol. We have also searched for possible effects of GnRHa on granulosa-luteal cells obtained at ovum collection. In vitro culture of these cells has shown that the steroidogenic pathway is affected.(ABSTRACT TRUNCATED AT 250 WORDS)


Is there an indication for embryo reduction?

The selective reduction of embryos in multiple pregnancies poses numerous medical, technical, ethical and psycho-social problems. In a retrospective study, we analysed nine hundred and twenty-two pregnancies obtained using medically assisted procreation between May 1982 and May 1990. Among 922 successful pregnancies, 372 were singleton, 102 were twin and 13 were triplet. Data from this analysis and from a French multicentre study of 262 embryo reduction procedures demonstrated the value of an embryo quality score for minimizing the risk of multiple pregnancy and the existence of extremely infrequent, ethically acceptable indications for embryo reduction. These indications included ultrasound-proven malformations of one fetus, multiple pregnancies in patients with extensive uterine scarring, and multiple pregnancy despite the appropriate use of preventative measures which can be expected to make this technique unnecessary in the future.


Transcervical access and intra-luminal imaging of the fallopian tube in the non-anaesthetized patient; preliminary results using a new technique for fallopian access.

A study was performed to evaluate a new transcervical, Fallopian tube access system for use in falloposcopy procedures without the need for conventional hysteroscopy or uterine distension. Visualization was accomplished by the use of a 0.5 mm falloposcope which was delivered to the Fallopian tube by a new linear everting catheter. The linear everting catheter allows access to the Fallopian tube by means of a flexible, tubular rolling membrane which can safely negotiate the tortuous anatomy from the ostium to the ampulla. Concurrent laparoscopy was performed solely for supervision purposes and not for tubal manipulations in 13 Fallopian tube access procedures in 10 patients. The ostia were visualized in 12 cases and the Fallopian tube was accessed in all 12 without complications. As a precursor for future gamete and embryo transfer procedures, intra-tubal insemination was performed by visualizing the ostia, accessing the Fallopian tubes, and obtaining successful isthmic-ampullary, intra-luminal images.


Treatment of male infertility by gamete micromanipulation.

Over the past decade, in vitro fertilization (IVF) has become a routine and acceptable tool in the treatment of infertility. However, major limitations in solving certain infertility problems still remain. Male infertility is one area in which only a small fraction of patients have benefited from IVF. Union of male and female gametes, either in vivo or in vitro, requires sperm penetration through the cumulus oophorus and the zona pellucida. Failure of fertilization despite the increased number of spermatozoa introduced into the oocyte's vicinity by IVF, has been shown to be directly related to abnormalities in sperm cell morphology and motility. The improved technology for micromanipulation of gametes has made it possible to circumvent the oocyte barriers to sperm penetration, thereby greatly reducing the number of normal sperm cells needed to achieve fertilization. This paper reviews the three major micromanipulative strategies which have been developed over the past five years, patient selection for the different procedures and the methods of sperm preparation to improve the yield of the applied technique.


Cryopreservation of rabbit zygotes.

Rabbit embryos at the pronuclear stage (n = 480) were frozen using a mixture of propanediol and sucrose. The survival rate was 71% according to their morphological features shortly after thawing. The viability rate after 5 days of culture was 10% according to the number of embryos that reached the blastocyst stage, while the rate of formation of blastocysts from freshly collected and cultured pronuclear embryos was higher (59%). The implantation rate was higher (24%) if the frozen/thawed zygotes were directly transferred to foster mothers after 2-4 hours of culture, whereas with zygotes cultured for 24 hours (4 to 8-cell stages) the implantation rate was poor (4%). Both rates were low compared with the implantation rate (52%) achieved with freshly collected and directly transferred pronucleate embryos.


Indications for oocyte donation.

Donated oocytes were transferred on 92 occasions to 87 women without gonadal function and 5 with functional ovaries. Twenty-three pregnancies were established (25% pregnancy rate), 9 after transfer of fresh embryos in 30 synchronous donor and recipient cycles and 14 after transfers of frozen-thawed embryos in 62 asynchronous donor and recipient cycles. Twenty-two pregnancies were obtained in agonadal patients (25.3% pregnancy rate) and 1 in a gonadal woman (20% pregnancy rate). Pregnant women were younger than those who did not become pregnant, but the difference was not significant. The pregnancy rate was higher when intra-Fallopian transfer was performed (46%) as compared with intrauterine transfer (21.5%) and when micronized progesterone was given intravaginally (pregnancy rate 30.3%) as compared to intramuscularly injected natural progesterone in oil (pregnancy rate 22%). Twenty healthy infants have been born including one set of twins; four pregnancies miscarried.


Fertilization abnormalities in human in-vitro fertilization.

Fertilization abnormalities (premature chromosome condensation of spermatozoa (PCC), triploidy, haploidy) were analysed in order to determine their origin. PCC occurs in 9% of unfertilized oocytes and seems to be the consequence of a failure of oocyte activation, leading to the continuing presence of cytoplasmic chromosome-condensing factors, causing the sperm nucleus to undergo chromosome condensation prematurely. This anomaly appears to be related to incomplete nuclear and/or cytoplasmic maturation. Triploid zygotes (6.5% of fertilized oocytes) display an original type of division: half of them divide into 3 and 6 cells, whereas at the same time diploid zygotes divide into 2 and then 4 cells. A cytological study, using both antitubulin antibodies and Hoechst dye, allowed us to demonstrate that they divide into 3 cells by means of a tripolar spindle. Triploidy seems to be correlated with four of 16 clinical or biological parameters examined: semen origin (fresh or frozen), type of stimulation treatment, number of oocytes recovered and embryo morphology. Haploid eggs (1.6% of inseminated oocytes) result from parthenogenetic activation. A correlation was found between a high number of recovered oocytes and triploid zygotes, and the occurrence of oocyte activation. These data show that increasing follicular recruitment decreases the overall oocyte quality and maturity leading to an overall 9% with impaired fertilization.


Chlorinated hydrocarbon content of fetal and maternal body tissues and fluids in full term pregnant women: a comparison of Germany versus Tanzania.

Chlorinated hydrocarbons are distributed worldwide and due to their lipophilic properties and chemical stability they accumulate in the foodchain. The concentrations of 19 different chlorinated hydrocarbons (hexachlorohexane (HCH), DDT and various metabolites and nine different polychlorinated phenyl (PCB) congeners were detected in various body tissues and fluids (maternal and fetal serum, adipose tissue, placenta, amniotic fluid) of full term pregnant women from Germany and Tanzania. Great variation of total toxin burden and toxin distribution within the different body compartments was found. This was in part due to local differences of exposure to some of the chlorinated hydrocarbons. Comparing samples from Germany and Tanzania, typical distribution patterns reflected the specific economic situation of the two countries with a high burden of insecticides (DDT and Dieldrin) in the agricultural country and high levels of constituents of industrial products (hexachlorobenzene (HCB) and PCBs) in Germany. Different chlorinated hydrocarbons seem to show different distribution patterns in body tissues, probably due to their chemical structure, the lipid content of the compartment and the overall toxin burden of the individual. A 10 to 100 fold accumulation of chlorinated hydrocarbons was observed in maternal adipose tissue compared with the other compartments. The concentrations of certain toxins in fetal cord serum and placenta were higher than in maternal serum.


Peripheral inflammatory root resorption. A review of the literature with case reports.

External resorptions associated with inflammation in marginal tissues are many times misdiagnosed and confused with caries and internal resorptions. A lack of uniformity in nomenclature has added to the confusion. This paper reviews the literature and presents cases to illustrate a rational approach to diagnosis and treatment. A new name is suggested to better reflect the features of this type of root resorption: peripheral inflammatory root resorption.


Granulocyte elastase in gingival crevicular fluid. A possible discriminator between gingivitis and periodontitis.

The granulocyte elastase activity and the immuno-reactive (antigenic) granulocyte elastase of gingival crevicular fluid (GCF) were studied in 16 periodontitis patients and in 10 gingivitis patients. The elastase activity was measured with a low molecular weight substrate specific for granulocyte elastase. The antigenic elastase was determined with specific antibodies against granulocyte elastase. Intracrevicular sampling of GCF with paper strips for 30 s seemed to provide representative values of elastase. The elastase activity correlated with probing depth and attachment loss and appeared to be a measure of the degree of tissue destruction. Antigenic elastase represents the number of granulocytes in GCF and should thus be related to the degree of inflammation. The periodontitis patients and the gingivitis patients both had a similar degree of inflammation as measured by antigenic elastase per microliter GCF and gingival index. The elastase activity per microliter GCF, however, was higher in the periodontitis group. Elevated granulocyte elastase activity in GCF seems to be independent of inflammation and could thus be an indicator of patients at risk for periodontitis.


Reproducibility of an electronic probe in relative attachment level measurements.

4 probing designs have been employed to investigate the reproducibility of the Florida Probe. 3 groups (each composed of 10 subjects) were selected for the study: healthy adults, gingivitis subjects, and periodontitis subjects. The 4 probing designs were as follows: (a) the probe tip was left in the sulcus between successive probings; (b) the probe tip was removed from the gingival margin between probings but the next probing followed immediately; (c) successive whole-mouth probings were interrupted by a 5-min interval and a mouthrinse; (d) there was a 4-week interval between each probing. 3 measurements were taken for each design. The main purpose of this study was to identify variance components in the attachment level variation. The maximum probing error standard deviation was found to be around 0.3 mm, which is considerably smaller than that found in most previous studies. The errors associated with the periodontal condition and probing effect were also estimated. The variance components obtained here can be used for determining the sample size in controlled clinical studies.


Evaluation of a new periodontal curet. An in vitro study.

Removal of plaque and calculus from subgingival root surfaces is a fundamental aspect of periodontal therapy. A new type of periodontal curet (Gracey Curvette Sub-0) has been designed to better adapt to the root surfaces that may be encountered in periodontal pockets of incisor teeth. The new curet (test) was compared to a Gracey curet 1/2 (conventional) in their ability to remove deposits from the subgingival root surfaces of incisor teeth in an in vitro model. A dentiform model mounted on a dental chair was used with artificial gingiva, 6 mm pocket depths and subgingival root surfaces covered with black enamel paint. 2 groups of 12 hygienists used, on a timed basis, either the test or conventional curet on 4 of the incisor teeth and then switched to the alternate instrument for the remaining four incisors. The extent of surface material removal was determined using a computerized video routine. The test curet removed significantly (p less than 0.001) more of the surface material from all subgingival incisor root surfaces than the conventional curet (60.7% versus 46.3%). Both instruments were most effective at labial surfaces. The test curet was least effective at proximal surfaces and the conventional curet least effective at lingual surfaces. The greatest statistical difference between instruments was apparent at lingual surfaces of maxillary incisors and the least at mesial surfaces of mandibular incisors.


An imaging routine for assessing the efficacy of instruments used for scaling and root planing.

Deposits of dental plaque or calculus are typically quantified using planimetric techniques. However, error is introduced into a quantitative analysis of plaque deposits using these methods, since they require a significant amount of human intervention. The purpose of this study is to describe and validate a computerized imaging routine which has the potential to objectively identify material on dental roots and measure the area covered by these deposits. Dentiform teeth with simulated plaque were videorecorded. A computer routine was developed based on a flood-fill algorithm which analyzed images of the dentiform teeth and determined the amount of simulated plaque on their root surfaces. Results showed that the dentiform teeth and their simulated plaque patterns are duplicated by the imaging routine in a rapid and reliable fashion. The system shows a high degree of accuracy with an average error factor of only 0.58%. As well, the system enables precise reproducibility with an average error factor of only 0.71%.


Comparative in vitro studies of sonic, ultrasonic and reciprocating scaling instruments.

Flat root surface areas of formalin-stored mandibular incisors with plaque and calculus were scaled by sonic (PHATELUS SONIC SCALER, SONIC FLEX 2000, TITAN-S SONIC SCALER) or ultrasonic instruments (HYGIENIST ULTRASONIC SCALER, CAVITRON) or by a new reciprocating scaling insert for the EVA/PROFIN system. The test areas were photographed by SEM and coded micrographs were independently graded by three examiners using the RCI (Remaining Calculus Index) and the RLTSI (Roughness Loss of Tooth Substance Index). The findings revealed that the sonic scalers as a group removed calculus more completely but also left significantly more roughness and loss of tooth substance than the other instruments tested. No difference was seen between the two ultrasonic scalers. The reciprocating insert gave results similar to those of the ultrasonic except for the scaling time which was significantly longer for the new "cleansing principle".


A comparison of natural product, triclosan and chlorhexidine mouthrinses on 4-day plaque regrowth.

There is a continuing search for ingredients to enhance the chemical plaque inhibitory action of oral hygiene products. Sanguinarine, other natural extracts and triclosan have already been used in products. The aim of this study was to evaluate a number of triclosan and natural product rinses for effects on plaque regrowth. In particular, the influence of other rinse components were assessed, notably sodium lauryl sulphate (SLS) and zinc. The study was a randomised, blind, 9-cell cross-over design to measure the effects of each rinse on 4-day plaque regrowth from a zero baseline. 15 volunteers rinsed 2x daily with each product and plaque was recorded by area and score. The 0.2% chlorhexidine rinse (positive control) was significantly more effective, and the saline rinse (negative control) significantly less effective, than other rinses. Sanguinarine alone was little different from saline and the addition of zinc made a modest improvement in activity. The 3 triclosan/SLS rinses were more effective than the sanguinarine rinses but similar to their minus triclosan control rinse. A natural product/SLS experimental rinse was second to chlorhexidine and, in many analyses, significantly better than all other rinses, but caused some oral erosions. The results indicate that the plaque inhibitory properties of basic ingredients such as SLS may be difficult to enhance or surpass. However, the possible range of recipes, particularly using natural ingredients, provides scope for research and development in the field of oral hygiene products.