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R S Redman

Publications and source records attributed to R S Redman.

At least 37 records · Page 2Linked to original sources
PubMed1998

In vivo use of adenoviral vectors: effects on salivary gland structure.

Recently there has been considerable progress in the development of in vivo gene transfer technology. By this means, new genetic information may be introduced directly to cells, while the cells remain in their natural milieu. The ability to express exogenous proteins makes it possible to explore the functions of native or altered proteins and thereby develop new insights into cell function and dysfunction. We have demonstrated that the major salivary glands are efficiently infected by recombinant adenovirus vectors. These vectors are capable of expressing transgenes in both acinar and ductal cell types. Recently, we have developed vectors that contain cell-specific promoters so that proteins may be expressed in selected subpopulations of salivary cells. Early generations of adenoviral vectors elicited potent immune responses in vivo. However, modified vectors and adjunctive measures have improved the safety of gene transfer to salivary glands. Future studies will aim to increase the duration of adenovirus-based gene expression and to produce vector systems that are not toxic to the host.

Adenoviridae↗
PubMed1997

Opposing effects of phorbol esters on transmitter release and calcium currents at frog motor nerve endings.

1. Phorbol esters activate protein kinase C (PKC) and also increase the secretion of neurotransmitter substances by an unknown mechanism. To evaluate whether the stimulatory effects of such agents on acetylcholine (ACh) secretion occur as a consequence of stimulation of Ca2+ entry, we made electrophysiological measurements of ACh secretion (i.e. endplate potentials, EPPs) and the component of the prejunctional perineural voltage change associated with nerve terminal calcium currents (perineural calcium current) at frog neuromuscular junctions. 2. In the first series of experiments, modest concentrations of K+ channel blockers were employed so that simultaneous measurements of EPP amplitudes and perineural calcium currents could be made. In these experiments, 12-O-tetradecanoylphorbol 13-acetate (TPA; 162 nM) and phorbol 12,13-dibutyrate (PDBu; 100-200 nM) each increased ACh release but simultaneously decreased the calcium component of the prejunctional perineural current TPA and PDBu also inhibited perineural calcium currents in the presence of higher concentrations of K+ channel blockers. 3. Blockade of Ca2+ channels by Cd2+ prevented the action of PKC stimulators on perineural waveforms. 4. The inactive compound 4-alpha-phorbol 12-myristate 13-acetate (150 nM) did not affect EPP amplitudes or perineural currents. 5. The extracellular [Ca2+]-ACh release relationship was increased in maximum by PDBu without any change in the potency of Ca2+ to support evoked ACh release. 6. The results demonstrate that phorbol esters increase neurotransmitter secretion whilst simultaneously decreasing the nerve ending calcium currents that promote evoked release. The results, which suggest that the optimal control point for secretion might not be the calcium channel but rather a component of the secretory apparatus, are discussed in conjunction with the possible target sites for phorbol esters in the nerve ending.

Acetylcholine↗
PubMed1997

Cholesterogenesis, lipogenesis, cholesterol degradation to bile acids, and histopathology of the liver in LA/N-cp obese rats.

Various lipid parameters were determined in lean control and LA/NIH-corpulent (LA/N-cp) rats, a normotensive strain showing metabolic characteristics associated with human Type IV hyperlipidemia. Hepatic and plasma total cholesterol, high density lipoproteins (HDL) cholesterol and triglycerides were significantly higher in the obese group than in the control group. Depending upon whether the data were expressed as per gram tissue or per organ, the rates of de novo fatty acid synthesis in the liver and adipose tissue were higher by 61% to 127% (P < .05) and 79% to 355% (P < .05), respectively, in the obese group compared with the lean control group. Similarly, hepatic rate of cholesterol synthesis was higher by 46% to 107% (P < .05) in the obese animals compared with the lean ones. In vivo hepatic rate of HDL2 cholesterol degradation to bile acids was lower in the obese group by 48% to 63% (P < .05). This was confirmed in the perfused liver in spite of the fact that cholesterol uptake from HDL2 was 3- to 4-fold higher in the obese group. These changes in lipid parameters of the obese animals were neither caused by hyperphagia because they were pair-fed with the control group nor caused by increased rate of food consumption because they were meal-fed. At the same time, all these lipid parameters were 17% to 20% higher in ad libitum-fed obese than in pair-fed obese group. Histopathological evaluation of the livers in the obese and control groups also showed prominent lipid droplets in the cytoplasm of the obese liver but not in the lean control liver. Thus, the possible causes of obesity in the LA/N-cp obese rats are higher synthetic rates of lipids coupled with lower rate of degradation of cholesterol to bile acids.

Animals↗
PubMed1997

Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colltotrichum musae (Berk. & Curt.) Arx.].

A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

Ascomycota↗
PubMed1997

Development and characterization of SV40 immortalized rat submandibular acinar cell lines.

Rat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

Animals↗
PubMed1997

Effect of substance P and receptor antagonists on secretion of lingual lipase and amylase from rat von Ebner's gland.

Substance P (SP, 1 microM) when incubated with minced von Ebner's glands for 15, 30, and 60 min, stimulated secretion of lingual lipase (12.14% +/- 0.90) and amylase (8.30% +/- 0.42). Only 10 microM of the SP receptor antagonist CP-96,345 significantly inhibited SP-evoked secretion. D-Pro2-D-Phe7-D-Trp9-SP (Ia), D-Pro2-D-Trp7,9-SP (Ib), D-Arg1-D-Trp7,9-D-Leu11-SP (Ic), or 1 microM CP-96,345 were not effective, suggesting that the SP receptor of von Ebner's gland might be an isoform. Propranolol and timolol, beta 1/beta 2-adrenergic receptor antagonists were not effective and the cholinergic receptor antagonist, atropine, was effective in only slightly reducing amylase secretion but not lingual lipase. Differential secretion of the two enzymes was observed for basal and stimulated secretion. Thus, exocytosis may not be the only pathway involved in SP-evoked protein secretion.

Amylases↗
PubMed1997

Salivary glands: a paradigm for diversity of gland development.

The major salivary glands of mammals are represented by three pairs of organs that cooperate functionally to produce saliva for the oral cavity. While each type of gland produces a signature secretion that complements the secretions from the other glands, there is also redundancy as evidenced by secretion of functionally similar and, in some cases, identical products in the three glands. This, along with their common late initiation of development, in fetal terms, their similarities in developmental pattern, and their proximate sites of origin, suggests that a common regulatory cascade may have been shared until shortly before the onset of overt gland development. Furthermore, occasional ectopic differentiation of individual mature secretory cells in the "wrong" gland suggests that control mechanisms responsible for the distinctive cellular composition of each gland also share many common steps, with only minor differences providing the impetus for diversification. To begin to address this area, we examine here the origins of the salivary glands by reviewing the expression patterns of several genes with known morphogenetic potential that may be involved based on developmental timing and location. The possibility that factors leading to determination of the sites of mammalian salivary gland development might be homologous to the regulatory cascade leading to salivary gland formation in Drosophila is also evaluated. In a subsequent section, cellular phenotypes of neonatal and adult glands are compared and evaluated for insights into the mechanisms and lineages leading to cellular diversification. Finally, the phenomena of proliferation, repair, and regeneration in adult salivary glands are reviewed, with emphasis on the extent to which the cellular diversity is reversible and which cell type other than stem cells has the ability to redifferentiate into other cell types.

Animals↗
PubMed1997

Relationship between the cellular distribution of the alpha(v)beta3/5 integrins and adenoviral infection in salivary glands.

This study compared the localization of the alpha(v)beta3/5 integrins in the different cell types of the rat submandibular gland with the susceptibility of these cells to infection by an intraductally administered replication-deficient recombinant type 5 adenovirus coding for rat aquaporin 5 (termed AdrAQP5). We used a panel of antibodies and immunohistochemical procedures to determine the luminal membrane distribution of the integrins. The alpha(v)beta3/5 integrin subunits were present in luminal membranes of all ductal cell types and acini; however, the alpha(v) subunit was found to a lesser extent in the acini. After AdrAQP5 infection, the expression of AQP5 exhibited a similar, though not identical, cellular localization to that seen for alpha(v)beta3/5 integrins. AdrAQP5 infected all cell types in these glands, except excretory ducts, after intraductal administration, directing the transient expression of AQP5. In addition, the localization of alpha(v)beta3/5 integrins was examined in rabbit, monkey, and human submandibular and parotid glands. Although there were some interspecies differences, glands generally displayed the presence of these integrin subunits on luminal membranes of ductal cells and most acinar cells. These findings show that the presence of alpha(v)beta3/5 integrins on the luminal membranes of rat salivary epithelial cells is associated with the susceptibility of these cells to act as targets for adenoviral-mediated gene transfer. However, the abundance of alpha(v)beta3/5 integrin expression was not necessarily predictive of the extent of transgene expression in a particular cell type. Furthermore, because the salivary glands of rabbits, monkeys, and humans show a similar luminal membrane distribution of these integrins, it is likely that recombinant type 5 adenoviruses may be able to mediate in vivo gene transfer to salivary glands in these species.

Adenoviridae↗
PubMed1996

Autoimmune Sjögren's-like lesions in salivary glands of TGF-beta1-deficient mice are inhibited by adhesion-blocking peptides.

The targeted disruption of the TGF-beta1 gene in mice (TGF-beta1 -/-) leads to extensive inflammation in vital organs, cachexia, and death within 3 to 4 wk. Significant inflammatory lesions develop initially in the periductal regions of the salivary glands and escalate as the animals become symptomatic. These inflammatory sites, characterized by lymphocytic infiltration and increased proliferation, cytokine mRNA expression, and IgG-positive cells, resemble lesions of Sjögren's syndrome. Moreover, the inflammatory pathology, enhanced MHC expression, and Ab production are consistent with an autoimmune-like etiology. Glandular atrophy and loss of acini with reduced saliva production appear to contribute to the wasting syndrome characteristic of the TGF-beta1 -/- mice. To determine whether the structural and functional defects were developmental due to the absence of TGF-beta1 or secondary to the inflammation, TGF-beta1 -/- mice were treated with synthetic fibronectin peptides, which block leukocyte infiltration. Daily systemic injections of RGD, CS-1, and/or peptides derived from the heparin-binding region of the A chain not only prevented leukocyte infiltration in the salivary glands of the TGF-beta1 -/- mice, but also reversed the acinar and ductal derangements. These data suggested that salivary gland development is not jeopardized in the absence of TGF-beta1, but that the extensive infiltration of inflammatory cells compromises glandular structure and function. The essential nature of TGF-beta1 in controlling inflammatory and immune processes is confirmed by these studies. Moreover, these TGF-beta1 -/- mice provide an important model of autoimmune disease that can be used in the design of therapeutic interventions.

Animals↗
PubMed1996

Immediate inflammatory responses to adenovirus-mediated gene transfer in rat salivary glands.

Although replication-deficient adenoviruses can efficiently transfer genes to the salivary glands, the current vectors precipitate an immediate, transient decrease in salivary function. To study the cause of this salivary hypofunction, 10(6)-10(10) plaque-forming units (pfu) of the vector AdCMV beta gal were delivered by retrograde ductal infusion to the submandibular glands (SMGs) of rats. Microscopic analysis of infected glands showed a dose-related, rapidly developing inflammatory response, which at the highest amount of virus was characterized by a predominantly neutrophil-containing infiltrate, focal necrosis, and edema. Moreover, the glands of nude rats developed similar morphologic changes to those of immunocompetent rats. After 3 days, the volume of stimulated saliva secreted from SMGs receiving AdCMV beta gal (6.75 x 10(9) pfu) was approximately 20% that of controls. UV-inactivated virus caused a similar decrease in saliva output. We evaluated to what extent the anti-inflammatory glucocorticoid, dexamethasone, could suppress inflammation and preserve salivary function. Three days after infusion with a high dose of AdCMV beta gal (6.75 x 10(9) pfu), the glands from dexamethasone-treated animals showed markedly less inflammation and no necrosis. Furthermore, there was no significant difference in the average amount of saliva secreted from the infected glands (105 +/- 17 microliters) compared to the control glands (123 +/- 18 microliters). In addition, dexamethasone extended the expression of beta-galactosidase in the SMGs. These results suggest that the adenovirus-mediated acute inflammation in rat SMG is responsible for diminished gland function and transgene expression. Furthermore, we demonstrate a useful role for glucocorticoids in controlling acute inflammation during experimental gene transfer with current adenovirus vectors.

Adenoviridae↗
PubMed1996

Synchronous release of ATP and neurotransmitter within milliseconds of a motor nerve impulse in the frog.

1. It has been suggested that ATP is released together with the neurotransmitter acetylcholine (ACh) and, after hydrolysis to adenosine, is the primary physiological mediator of prejunctional neuromuscular depression. To evaluate whether ATP is released with sufficient rapidity to mediate prejunctional depression, outside-out patches containing both ATP-gated and ACh-gated ion channels were made from acutely dissociated guinea-pig sympathetic neurons and used to detect the co-release of nucleotide and neurotransmitter in frog cutaneous pectoris nerve-muscle preparations. 2. In a normal bathing solution in which muscle nicotinic receptors were blocked, a single stimulus to the motor nerve produced channel openings in the detector patch characteristic of both ATP and ACh. 3. In the remaining experiments, preparations were treated with sufficient hexamethonium (200 microM) to block nicotinic responses in the detector patch. In these experiments, a single temporally isolated nerve impulse caused the synchronous opening of ATP-gated channels in the detector patch with a latency of < 5 ms when patches were placed within 10 microns of the motor nerve ending. This multichannel phasic response was followed by trail of discrete channel openings characteristic of ATP-gated channels. 4. The selective ATP antagonist suramin (50 microM) reversibly eliminated the response to nerve stimulation. 5. The results suggest that ATP is released synchronously together with the neurotransmitter ACh in response to an individual nerve impulse and with a brief latency characteristic of quantal release from synaptic vesicles.

Acetylcholine↗
PubMed1995

Neurotransmitter release evoked by nerve impulses without Ca2+ entry through Ca2+ channels in frog motor nerve endings.

1. The requirement for extracellular Ca2+ in the process of evoked acetylcholine (ACh) release by nerve impulses was tested at endplates in frog skeletal muscle. Ca(2+)-containing lipid vesicles (Ca2+ liposomes) were used to elevate cytoplasmic Ca2+ concentrations under conditions in which Ca2+ entry from the extracellular fluid was prevented. 2. In an extracellular solution containing no added Ca2+ and 1 mM Mg2+ ('Ca(2+)-free' solution), Ca2+ liposomes promoted the synchronous release of ACh quanta, reflected electrophysiologically as endplate potentials (EPPs), in response to temporally isolated nerve impulses. 3. Motor nerve stimulation generated EPPs during superfusion with Ca2+ liposomes in Ca(2+)-free solutions containing the Ca2+ channel blocker Co2+ (1 mM), and the Ca2+ chelator EGTA (2 mM). As a physiological control for Ca2+ leakage from the liposomes to the extracellular fluid, the effect of Ca2+ liposomes on asynchronous evoked ACh release mediated by Ba2+ was examined. In contrast to the effects of 0.2-0.3 mM extracellular Ca2+, which generated EPPs but antagonized Ba(2+)-mediated asynchronous ACh release, Ca2+ liposomes generated EPPs but did not reduce asynchronous release mediated by Ba2+. The effects of Ca2+ liposomes were thus not due to leakage of Ca2+ from the liposome to the extracellular fluid. 4. Morphological studies using fluorescently labelled liposomes in conjunction with a confocal microscope demonstrate that lipid is transferred from the liposomes to nerve endings and liposomal contents are delivered to the nerve terminal cytoplasm. 5. The results suggest that when intracellular Ca2+ is elevated using liposomes as a vehicle, evoked ACh release can occur in the absence of Ca2+ entry via Ca2+ channels.

Acetylcholine↗
PubMed1995

Proliferative activity by cell type in the developing rat parotid gland.

BACKGROUND: In contrast to the considerable amount of research that has been done on the proliferative activity of the several types of parenchymal cells in the developing submandibular glands of rodents, systematic studies of cellular proliferation in the developing parotid gland have been confined to the acinar cells. The purpose of the present study was to attempt to fill this knowledge gap. METHODS: Tritiated thymidine was parenterally administered to Sprague-Dawley rats at ages representative of the pre- and postnatal development of the parotid gland, and glands were harvested for autoradiography 90 min after injection. Mitotic activity among all cell types was verified by electron microscopy. RESULTS: At all ages, the % labeled cells was much greater among the acini than any other cell type, including well-differentiated cells at 25 and 40 days. However, there were only small alterations in the proportions of cells comprised by the major cell types. CONCLUSIONS: Current theories on the histogenesis of salivary glands and their neoplasms are based on the renewing population model, in which both normal differentiated cells and neoplastic cells arise from undifferentiated stem cells in the ducts. However, these results suggest that most of the migration and redifferentiation in the developing rat parotid gland must be in the opposite direction, i.e., the acinar cells redifferentiate into ductal cells. They also indicate that until there are precise data on the rates of cell death among the several cell types, it remains more appropriate for salivary glands to be categorized as an expanding, rather than renewing, population.

Aging↗
PubMed1995

On the simultaneous electrophysiological measurements of neurotransmitter release and perineural calcium currents from frog motor nerve endings.

Ca2+ currents from the perineural region of motor nerve endings were measured together with evoked acetylcholine (ACh) release (i.e., end-plate potentials EPPs) in frog skeletal muscle in an attempt to define experimental conditions in which simultaneous measurements of both phenomena were feasible. In a solution containing low Ca2+ (0.9 mM), high Mg2+ (10 mM) and modest concentrations of K+ channel blockers (250 microM tetraethylammonium, 100 microM 3,4,-diaminopyridine), reliable measurements of perineural Ca2+ currents were possible. For convenience, this solution will be termed 'Ca2+ current' Ringer. The mean number of ACh quanta released in Ca2+ current Ringer was near the midpoint of the relationship between extracellular [Ca2+] and evoked ACh release observed previously in normal Ringer solutions. Consequently, ACh release in response to low-frequency motor nerve stimulation (0.05 Hz) was well maintained, allowing simultaneous measurements of Ca2+ currents and evoked ACh release to be made. Ca2+ currents and EPPs measured simultaneously in Ca2+ current Ringer were increased or decreased in parallel by increasing or decreasing the extracellular Ca2+ concentrations. Ca2+ channel blockers (Cd2+, 500 microM; omega-conotoxin, 3 microM) eliminated both EPPs and the Ca2+ component of the perineural current. NaF (10 mM), which stimulates ACh release, produced parallel increases in EPPs and perineural Ca2+ currents. NG-cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, inhibits ACh release without effects on perineural currents. The results suggest that the concurrent electrophysiological recording of Ca2+ currents and ACh release in Ca2+ current Ringer is a reliable experimental approach for determining whether drugs or disease states affect ACh release by acting on Ca2+ channels in the presynaptic membrane.

Animals↗
PubMed1994

ATP released together with acetylcholine as the mediator of neuromuscular depression at frog motor nerve endings.

1. The hypothesis that ATP released by presynaptic stimulation is hydrolysed to adenosine and mediates prejunctional neuromuscular depression was tested at vertebrate neuromuscular junctions. Electrophysiological recordings of evoked acetylcholine (ACh) release and perineural ionic currents at motor nerve endings were made using the frog cutaneous pectoris nerve-muscle preparation. Either tubocurarine or alpha-bungarotoxin was used to block muscle contractions. 2. Either alpha,beta-methylene ADP (which inhibits ecto-5'nucleotidases and thus prevents the degradation of ATP to adenosine) or selective adenosine receptor antagonists (8-cyclo-pentyl alkyl xanthines) prevented the inhibitory effects of exogenous ATP on ACh release in response to low-frequency nerve stimulation. These results confirm earlier findings that ATP must be hydrolysed to adenosine to inhibit ACh release. 3. The presence of alpha,beta-methylene ADP completely prevented neuromuscular depression in response to repetitive high-frequency nerve stimulation (0.5-1 Hz). alpha,beta-Methylene ADP had no effect on ACh secretion under conditions where ACh release is well maintained (low-frequency stimulation, 0.05 Hz). 4. Selective adenosine receptor antagonists completely eliminated neuromuscular depression produced by repetitive high-frequency nerve stimulation (1.0 Hz) but had no effect on ACh release at low frequencies of stimulation (0.05 Hz). 5. Exogenous adenosine deaminase (5 i.u. ml-1), which degrades adenosine to its inactive nucleoside inosine, also eliminated neuromuscular depression but had no significant effect on ACh release at frequencies of nerve stimulation too low to produce prejunctional depression. 6. During maximal neuromuscular depression, the effects of exogenous adenosine or 2-chloroadenosine, an adenosine agonist, were occluded. 7. The calcium-sensitive component of perineurial recordings of motor nerve terminal currents did not change during depression or during application of adenosine receptor antagonists and adenosine deaminase, suggesting that neuromuscular depression in this species was not associated with changes in presynaptic Ca2+ currents. 8. These results suggest that, under the conditions of these experiments, endogenous ATP, after hydrolysis to adenosine, causes prejunctional neuromuscular depression. This inhibitory effect of endogenous adenosine occurs at a site distal to the locus of Ca2+ entry in the frog.

Acetylcholine↗
PubMed1994

Myoepithelium of salivary glands.

In salivary glands and other exocrine organs, there are starfish-shaped cells that lie between the basal lamina and the acinar and ductal cells. These have structural features of both epithelium and smooth muscle cells, and so are called myoepithelial cells. Their functions include contraction when the gland is stimulated to secrete, compressing or reinforcing the underlying parenchymal cells, thus aiding in the expulsion of saliva and preventing damage to the other cells. They also may aid in the propagation of secretory and other stimuli. Their common developmental origin with the basal cells of the larger ducts is displayed in the mature glands by shared structural and immunohistochemical features, but most such basal cells do not have the distinguishing features of myoepithelial cells, such as myofibrils. Although myoepithelial cells can be identified by light microscopy through enzyme histochemistry and special stains and immunohistochemistry for their myofibrils, these techniques can be misleading in salivary gland neoplasms. Thus, the most reliable means of identifying neoplastic myoepithelial cells is with a combination of histochemistry and electron microscopy. The extent to which these cells are derived from undifferentiated stem cells in both normal and neoplastic growth is controversial. The presentation here of transmission electron microscopy (TEM) of well-differentiated myoepithelial cells in mitotic division indicates that stem cells are not necessarily the only source of myoepithelial cells in the later stages of salivary gland development or in neoplasia.

Animals↗