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Biomedical subjects

L W Haynes

Publications and source records attributed to L W Haynes.

At least 37 records · Page 2Linked to original sources
PubMed1993

Localization and activity of transglutaminase, a retinoid-inducible protein, in developing rat spinal cord.

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.

Animals↗
PubMed1993

Cellular transglutaminases in neural development.

Enzymes of the transglutaminase family catalyze the Ca(2+)-dependent covalent cross-linking of peptide-bound glutamine residues of proteins and glycoproteins to the epsilon-amino group of lysine residues to create inter- or intramolecular isopeptide bonds. Transglutaminases can also covalently link a variety of primary amines to peptide-bound glutamine residues giving rise to two possibilities; firstly, where the primary amine has two or more amino groups, further catalysis can result in the formation of cross-linked bridges between glutamine residues, and secondly, where the primary amine is a monoamine, glutamine residues are rendered inert to further modification. The products are therefore in the main, homo- or heterodimers, or extensive, metabolically-stable multimeric complexes or matrices. Ca(2+)-dependent transglutaminase activity is present in the mammalian peripheral and central nervous systems and transglutaminase-catalyzed cross-linking of endogenous substrates has been demonstrated in neurons of Aplysia and the mammalian brain. Transglutaminase activity increases in the brain during development, principally owing to the increasing preponderance of glial cell activity. In a few regions including the cerebellar cortex, activity is also high in early development. Cellular transglutaminases occur widely in differentiating cells and tissues in mammals, with more than one transglutaminase frequently associated with a single cell type. The primary protein sequences of three cellular transglutaminases have been fully determined in different species, together with that of a mammalian protein homologue (band 4.2) which shares extensive sequence homologies with transglutaminases, but lacks the active site cysteine residue. The upstream sequences of two mammalian cellular transglutaminase genes (C and K) contain numerous regulatory sites, and an invertebrate transglutaminase, annulin, is spatially regulated within homeodomains. Multiple molecular forms of transglutaminase C and possibly other cellular transglutaminases exist in mammalian brain. The emerging picture is one of a family of cytosolic and membrane-bound proteins central to several regulatory pathways whose functions is to stabilize the cellular and intercellular superstructure in growing organisms. The targeted formation of glu-lys isopeptide bonds between proteins is central to this function. Cytoskeletal proteins, membrane-associated receptors, enzymes in signal transduction pathways and extracellular glycoproteins are candidate substrates as are polyamines, but few cellular proteins have been identified as components of naturally-occurring covalently-bonded matrices. Transglutaminases participate in the programme of neuronal differentiation in some but not all classes of neurone. Both neuronal and non-neuronal expression of transglutaminases may be important for guidance of migrating neurons or growth cones and sustainment of cell shape and coordinates during development.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Activity and distribution of tissue transglutaminase in association with nerve-muscle synapses.

We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or in coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of epsilon-(gamma-glutamyl)lysine isopeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and particulate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5-15 microM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.

Animals↗
PubMed1992

Low-voltage-activated calcium channels in human retinoblastoma cells.

Retinoblastoma cells represent pluripotent neural-progenitor cells which, if induced to differentiate, express many features of mature human retinal neurons. Ca channel currents were recorded from isolated, undifferentiated human retinoblastoma Y79 cells in a bath solution containing 20 mM BaCl2 using whole-cell patch-clamp pipettes containing CsCl. The transient, macroscopic currents inactivated with a time constant of about 20 ms at -20 mV and had other properties similar to low-voltage-activated calcium channels described in other cell types: Activation curves fit by the Boltzmann relation had a midpoint of -32 mV and a slope factor of 6.8 mV (-80 mV holding potential) and inactivation curves had a midpoint of -40 mV and a slope factor of 3.7 mV. Non-stationary fluctuation analysis of the currents performed over a 2 kHz bandwidth indicated that each channel contributed 0.44 pA to the macroscopic transient current at -20 mV, suggesting a unitary conductance of about 7 pS.

Calcium Channels↗
PubMed1992

Eosinophil chemotactic peptide sequences in rat alpha-CGRP. Activation of a novel trophic action by neutral endopeptidase 24.11.

Rat alpha- and alpha-CGRP are substrates for endopeptidase 24.11 in vitro. Cleavage of both peptides occurs at several points, including an unusual substrate recognition site to the amino side of ala36. In alpha-CGRP this resulted in the early formation of val32-gly-ser-glu35, a sequence previously reported to be a component of the eosinophil chemotactic factor of anaphylaxis (ECF-A). The biological activity of this peptide fragment was confirmed by bioassay. Chemotactic activity in other hydrolysis fragments of both alpha- and beta-CGRP was observed. Both alpha- and beta-CGRP could thus serve as precursors to different eosinophil chemotactic peptide fragments. A novel function of endopeptidase 24.11 may be to modify rather than to terminate the biological activity of CGRP peptides.

Amino Acid Sequence↗
PubMed1992

Endopeptidase-24.11 cleaves a chemotactic factor from alpha-calcitonin gene-related peptide.

The sequence of rat alpha-calcitonin gene-related peptide (CGRP-alpha) contains the tetrapeptide eosinophil granulocyte chemotactic factor Val32-Gly-Ser-Glu35. Peptide fragments formed following hydrolysis of rat CGRP-alpha in vitro by endopeptidase-24.11 were identified. The tetrapeptide fragment was generated following cleavage at a substrate recognition site unusual for this enzyme (-Glu-Ala-). Chemotactic activity of rat CGRP-alpha was increased following hydrolysis. Furthermore, rat CGRP-beta, which lacks the tetrapeptide sequence and is completely devoid of chemotactic activity, displayed low but measurable activity after hydrolysis. Val-Gly-Ser-Glu was identified as the principle fragment with chemotactic activity in rat CGRP-alpha. The results show that the chemotactic activity of the neuropeptide rat CGRP-alpha towards eosinophil polymorphonuclear leukocytes is increased following its hydrolysis in vitro by endopeptidase 24.11 through the formation of a previously identified eosinophil chemotactic tetrapeptide.

Amino Acid Sequence↗
PubMed1992

Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

Animals↗
PubMed1990

Localisation of acetylcholinesterase in rat myotubes in the presence of beta-endorphin and beta-endorphin-(1-27).

In rat embryo skeletal myotubes, acetylcholinesterase is present, as multiple forms, and can be detected in deposits at the cell surface. Myotubes cultured in the presence of beta-endorphin, exhibit an increased predominance of the globular (precursor) forms of the enzyme, which are largely restricted to intracellular sites associated with nuclei. In the presence of beta-endorphin-(1-27), the relative proportions of the different forms of acetylcholinesterase is similar to that seen in the controls, but the enzyme is intracellular and has a characteristic focal localisation in the myotube.

Acetylcholinesterase↗
PubMed1990

Immunocytochemical detection of beta-endorphin in mouse spinal motoneurons cocultured with astrocytes from spinal cord and forebrain.

Beta-endorphin was detected by immunocytochemistry in motoneurons from mouse embryo spinal cord enriched by differential sedimentation and plated onto cultures of embryonic astrocytes from appropriate (cord) and inappropriate (cerebral cortex) sources. Growth of motoneurons on astrocytes for 5 d in vitro improved cell differentiation and suppressed beta-endorphin immunoreactivity. Immunoreactivity was suppressed to a greater degree in motoneurons plated onto cortical rather than spinal cord astrocytes. Some small, dense spinal cord cells also exhibited beta-endorphin immunoreactivity, but staining was unaffected by cultivation on astrocytes. Cell contact and/or hormonal factors deriving from glia may contribute to the regulation of beta-endorphin, a trophic neuropeptide conditionally expressed in normal and pathologic motoneurons.

Animals↗
PubMed1990

Glycyl-L-glutamine stimulates the accumulation of A12 acetylcholinesterase but not of nicotinic acetylcholine receptors in quail embryonic myotubes by a cyclic AMP-independent mechanism.

Myotubes prepared from the Japanese quail embryo at 9 days gestation were cultivated in the presence of glycyl-L-glutamine (Gly-Gln, beta-endorphin C-terminal dipeptide) or glycyl-glutamic acid (Gly-Glu), and changes in the activity of acetylcholinesterase (AChE) molecular forms and binding of 125I-alpha-bungarotoxin (alpha BGT) to cell surface nicotinic acetylcholine receptors were measured. The A12 oligomer was the major form of AChE in the cultures. The activity of all molecular forms of the enzyme was increased in the presence of Gly-Gln, but Gly-Glu did not alter AChE activity. In cells infected with the temperature-sensitive mutant, La31C, of Rous sarcoma virus (ts-RSV) and transferred to the nonpermissive temperature, the A12 form of AChE was absent, but its activity could be induced following exposure of the cells to Gly-Gln. When cells treated in this way were incubated in the presence of collagenase, there was a small but significant loss of A12 AChE activity, indicating that Gly-Gln stimulated the activity of a pool of this oligomer which was mainly but not entirely intracellular. Neither Gly-Gln nor Gly-Glu influenced 125I-alpha BGT binding after exposure of the cells to the peptides for any duration. Neither Gly-Gln nor Gly-Glu influenced the accumulation of cyclic AMP in the cultures. beta-Endorphin is one of a family of peptides that coexist transiently with acetylcholine in lower motoneurones of vertebrates in the perinatal period. This report provides evidence for the selective trophic activity of one of its derivatives toward the postsynaptic cholinergic system in avian muscle cells.

Acetylcholinesterase↗
PubMed1990

Single-channel measurement from the cyclic GMP-activated conductance of catfish retinal cones.

1. A patch of plasma membrane was excised, in the inside-out configuration, from the outer segment tip of a catfish cone and recorded electrically with a patch pipette. A solution of 118 mM-NaCl was present on both sides of the membrane. 2. With the solution outside the pipette containing a low concentration (typically several micromoles per litre) of cyclic GMP and the membrane potential held at a non-zero level, brief steps of current indicative of the openings of single ion channels could be detected. There was no sign of desensitization to the ligand over a period of tens of seconds. 3. The prominent openings were associated with a conductance near 50 pS and an open-time constant of 0.5 ms or less. There was also an indication of sub-state openings. 4. The conductance of the large openings appeared to be invariant between -50 mV and +50 mV. However, the macroscopic current-voltage relation measured at a saturating concentration of cyclic GMP showed a slight upward curvature, which we attribute to a voltage dependence in the open probability of the fully liganded channel. 5. The relation between mean current and cyclic GMP concentration had an average Hill coefficient of about 2.4. The Hill coefficient was not affected by membrane voltage, but the conductance was activated by cyclic GMP slightly more readily at depolarizations; this could be adequately explained by a higher open probability of the fully liganded channel at positive voltages. 6. In several experiments, the membrane patch apparently contained a single cyclic GMP-activated channel, in that the measured current never rose above that for a single channel even at high concentrations of cyclic GMP. In these cases, a high concentration of the ligand simply engaged the channel in a literally continuous burst of openings, with an open probability of 0.8-0.9 at between -30 mV and +30 mV. The amplitude distribution of the burst under these conditions could be described by a beta distribution, consistent with the channel switching predominantly between a single closed state and a single open state when fully liganded. 7. Estimates of channel density on the cone membrane ranged from about 2 to 130 microns -2, with an average of 20 microns -2. This observed density is about ten times lower than the density of the homologous channel on rod membrane, being roughly in inverse relation to the tenfold larger surface membrane area of the cone outer segment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Eosinophil chemotactic response to rat CGRP-1 is increased after exposure to trypsin or guinea-pig lung particulate fraction.

Calcitonin Gene-Related Peptide (CGRP) immunoreactive nerve fibres innervate the vasculature and epithelia of the peripheral airways of the guinea-pig lung. Guinea-pig eosinophils exhibit a concentration-dependent chemotactic response to rat Calcitonin Gene-Related Peptide-1 (rCGRP-1). The sequence rCGRP-1-(32-35) (valyl-glycyl-seryl-glutamic acid), which is identical to an eosinophil chemotactic factor of anaphylaxis (ECF-A) previously purified from guinea-pig lung (Goetzl, E. J. and Austen, F. (1975) Proc. Natl. Acad. Sci. USA 72, 4123-4127), was both more potent and more effective than rCGRP-1 in the chemotaxis assay. Products of rCGRP-1 tryptic digestion exhibited increased chemotactic activity compared to the intact peptide. Exposure of rCGRP-1 to a particulate fraction of guinea-pig lung also elevated its chemotactic activity, increasing the resultant potency by one hundred-fold. Resolution of pure and digested peptides by reverse-phase h.p.l.c. revealed evidence of endopeptidase activity in the lung particulate fraction. Elution times of rCGRP-1 cleavage fragments differed mostly from those of tryptic digestion.

Animals↗
PubMed1989

The opiomelanocortin peptide family: neuronal expression and modulation of neural cellular development and regeneration in the central nervous system.

1. Pro-OMLC is amongst a small number of propeptide-encoding genes which are expressed at highest levels in the CNS early in development. 2. The reappearance of the peptide products in injured neurones suggests that they fulfill a function in neuronal growth, differentiation and regeneration. 3. Axonal cues may regulate gene expression in neurones with greater or less functional interaction with their target cells. 4. alpha-MSH and ACTH stimulate the differentiation of neurones by accelerating their energy uptake and axonal growth during its early phases. 5. Their neurotrophic action is mediated through a common N-terminal amino acid sequence. 6. The structure activity requirements of the molecular second messenger responses underlying this action have yet to be conclusively determined. 7. Endorphins may regulate the transition from the mitotic cycle to the onset of differentiation of neurones and glia in the CNS. 8. Little is yet known of the cellular mechanism underlying this response, but the control of peptide processing to favour opiate and non-opiate receptor-mediated responses may be a key factor in determining whether they accelerate or retard neuronal differentiation.

Animals↗