Biomedical subjects

T Barka

Publications and source records attributed to T Barka.

At least 19 recordsLinked to original sources

PubMed2000

Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-beta-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for beta-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

Animals↗

PubMed1998

Effect of sodium butyrate on the expression of genes transduced by retroviral vectors.

We have studied the effects of sodium butyrate (NaBu) on the expression of genes transduced by retroviral vectors and stably expressed in two salivary gland-derived cell lines, A5-DAP and A5-BAG, established earlier. These cell lines were obtained by infecting A5 cells with the retroviral vectors DAP and BAG, respectively, and by selecting neomycin-resistant transduced cells. A5-DAP cells express human placental alkaline phosphatase (PLAP) and A5-BAG cells bacterial beta-galactosidase, both under the control of the viral long terminal repeat (LTR) enhancer-promoter. NaBu in the concentration of 2-8 mM inhibited the growth of A5-DAP cells, and induced the expression of heat-stable PLAP. These effects of NaBu were dose-dependent. Induction of PLAP in clones of A5-DAP cells that express different basal levels of the enzyme was not correlated with the relative inducibilty by NaBu. Exposure to 4 mM NaBu for 48 h increased the PLAP mRNA level by 31%. A5-DAP cells released, in a time-dependent manner, PLAP into the culture medium. Cells treated with NaBu released more PLAP than untreated cells in proportion to their elevated level of the enzyme. The parent A5 cells also express a low level of tissue non-specific type alkaline phosphatase, which was also induced by NaBu. NaBu inhibited the growth of A5-BAG cells also, and increased the beta-galactosidase level. These data indicate the genes transduced by retroviral vectors can be induced by NaBu, which most likely interacts with the viral LTR.

Alkaline Phosphatase↗

PubMed1997

Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo.

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the beta-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector.

Alkaline Phosphatase↗

PubMed1996

Retrovirus-mediated gene transfer into salivary glands in vivo.

In the present report, we show prolonged expression of beta-galactosidase (beta-Gal) in the acinar cells of the submandibular and sublingual glands of rats following retrograde ductal injection of the retroviral vector BAG. To facilitate integration of viral DNA, cell division in the gland was induced by the administration of the beta-adrenergic agonist isoproterenol prior to the delivery of the vector. The frequency of cells stained for beta-Gal was higher if the virus was injected 4-20 hr after the two injections of isoproterenol given 24 hr apart than after the injection of only one dose of the drug. Without stimulation of cell division, no integration of the viral DNA was observed. Expression of the marker enzyme was observed up to 43 days, the limit of the observation period. The data indicate that salivary glands are potential targets of retrovirus-mediated gene transfer for somatic gene therapy.

Adrenergic beta-Agonists↗

PubMed1994

Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye.

BACKGROUND: Cystatin C, a naturally occurring inhibitor of cysteine proteinases, belongs to family 2 of the cystatin superfamily. While cystatins in general, and cystatin C specifically, are expressed in various cell types and found in biological fluids, cystatins in ocular structures have not been investigated. In the present study, the expression of cystatin C mRNA in the eye of the rat was studied. METHODS: Total RNA was extracted from eyes as well as from pooled corneae, retinas, lenses, sclerae, and corneae of adult rats. Cystatin C mRNA was detected in the RNA samples by reverse transcriptase--polymerase chain reaction and Northern blot hybridization. In addition, in situ hybridizations of formalin-fixed cryostat sections were carried out using a digoxigenin-labeled cystatin C probe. RESULTS: Cystatin C mRNA was demonstrated in total RNAs extracted from the eye, sclera, and retina, but not in RNAs isolated from the cornea and lens. In situ hybridizations revealed cystatin C mRNA in most of the stromal cells of the sclera. In the retina, a strong signal was localized in the outer nuclear layer. The distribution of the reaction product suggested that in the retina Müller cells and rod cells are the primary sites of expression of cystatin C. In addition, some glial cells in the inner nuclear and ganglion cell layers were stained. No specific signal for cystatin C mRNA was detected in the cornea, lens, iris, ciliary body, and choroid. CONCLUSIONS: In the eye of the rat, significant levels of cystatin C mRNA are detected in the sclera and retina. In the sclera cystatin C may play a role in modulating the activities of cysteine proteinases, mostly cathepsins, involved in the turnover and remodeling of the stroma. In the retina, cystatins synthesized and presumably released by Müller cells and rod cells may have a protective function against the harmful effects of cysteine proteinases released under physiologic and pathologic conditions.

Animals↗

PubMed1994

Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland.

Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot hybridization and in situ hybridization with digoxigenin-labelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the beta-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating beta-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established.

Animals↗

PubMed1994

Lack of expression of T-kininogen gene in the hearts of untreated and turpentine-injected rats.

Cystatins, inhibitors of cysteine proteinases, are present in rat heart. However, the controls of genes coding for various cystatins in the heart, and the cellular sites of expression of these genes are not known. With a sensitive reverse transcriptase--polymerase chain reaction T-kininogen mRNA was readily detected in the submandibular glands and livers, but not in the hearts, of control or turpentine-injected rats. Immunocytochemical observations employing a monoclonal antibody to bradykinin, which reacts with kininogens in general, revealed no specific staining in cardiac structures, but a weak staining was apparent in blood vessels and on the surface of endothelial cells of both control and turpentine-injected rats. The monoclonal antibody revealed the presence of kininogens in the acinar cells of the submandibular gland, and, in acute inflammation, in the hepatocytes. These findings suggest that the T-kininogen gene is not expressed in the heart, and the T-kininogen demonstrable in heart extracts derives from the blood. Circulating kininogens are likely bound to endothelial cells, and may be a local source of kinins. In addition, kininogens, as potent inhibitors of cysteine proteinases, may play a role in pathologic conditions of the heart by controlling the deleterious effects of cathepsins released from lysosomes or secreted by macrophages.

Animals↗

PubMed1993

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes.

Animals↗

PubMed1992

Immunofluorescence localization of cystatins in human lacrimal gland and in the exorbital lacrimal gland of the rat.

Cystatins are widely distributed natural inhibitors of cysteine proteinase. They occur both intra and extracellularly in various cells and tissue fluids including tears. Using an immunofluorescence technique with antibodies against rat cystatin S, an inhibitor of submandibular gland origin, cystatin-like immunoreactive material was demonstrated in the acinar cells of the exorbital lacrimal gland of the rat. Administration of the cholinergic agonist carbachol caused a depletion of cystatin from the acinar cells. This depletion was followed by a partial restitution in 6-8 h. Administration of the beta-adrenergic agonist isoproterenol for 4 days, which caused a marked hypertrophy of the submandibular gland, had no effect on the structure, weight, or cystatin content of the exorbital lacrimal gland. After such treatment, however, single large cells with intense staining for cystatin were encountered. Cystatin-like immunoreactive material was also demonstrated in human lacrimal gland using antibodies against human cystatin S. These data suggest the notion that tear cystatins are secreted by the lacrimal glands.

Animals↗

PubMed1992

Cysteine proteinase inhibitor in cultured human medullary thyroid carcinoma cells.

The TT cell line of human medullary thyroid carcinoma, that retains some of the differentiated functions of thyroid C cells including the synthesis and secretion of calcitonin, was found to contain and release into the culture medium cysteine proteinase inhibitor(s), cystatin(s). The major inhibitor, which is similar to, if not identical with, cystatin C, is constitutively released, or secreted, by TT cells. The rate of secretion of cystatin, quantified by titration of inhibition of papain, was stimulated by dibutyryladenosine 3':5'-cyclic monophosphate, forskolin, the calcium ionophore A 23187, and by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Neither forskolin nor TPA had, however, an effect on the level of the inhibitor in TT cells. Treatment with n-butyrate strongly inhibited the proliferation of TT cells, and led, in 4 to 7 days, to a doubling of the intracellular concentration of cystatins. Northern blot hybridizations to a 32P-labeled riboprobe complementary to human cystatin C cDNA indicated that cAMP, forskolin, and TPA had no effect on the steady-state levels of cystatin C mRNA. These data indicate that release of cystatin(s) from TT cells is regulated by cAMP-calcium-protein kinase C mechanisms that appear to be similar to those that regulate the secretion of calcitonin from these cells. However, in contrast to the calcitonin gene, the expression of the cystatin C gene in these cells is not regulated by cAMP or TPA. By a combination of acetone fractionation, affinity chromatography on Cm-papain-Sepharose, and gel exclusion chromatography a protein of approximately 14 kilodaltons was isolated from TT cells that reacted with antibodies against human cystatin C, and strongly inhibited papain. Cystain secreted by TT cells also had a molecular weight of 14 kilodaltons, and reacted with anti-human cystatin C antibodies. The physiologic and pathologic roles of cystatins in different cell types remain to be established. The TT cells provide a suitable cell type to study the regulation of the expression of the cystatin gene and the mechanism of cystatin release.

Blotting, Northern↗

PubMed1991

Cystatins in human tear fluid.

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.

Adrenergic beta-Agonists↗

PubMed1989

Beta-adrenergic induction of a cysteine-proteinase-inhibitor mRNA in rat salivary glands.

Transcripts encoding the cysteine-proteinase inhibitor rat cystatin S are induced in submandibular and parotid glands by the beta-adrenergic agonist isoproterenol (isoprenaline). High levels of cystatin S mRNA persist in glands of chronically treated animals for 6 days after discontinuation of the catecholamine, indicating a long half-life of the mRNA. Post-transcriptionally the size of the mRNA decreases, owing to a shortening of the poly(A) tail.

Animals↗

PubMed1989

Adrenergic regulation of c-fos expression in cultured BC3H1 muscle cells.

Administration of adrenergic agonists induced c-fos mRNA in the salivary glands of the mouse and in the heart of the mouse, rat, and hamster (Barka et al., 1986, Mol. Cell Biol. 6, 2984-2989; 1987; Oncogene 1, 439-443). To further analyze transcriptional and post-transcriptional control of c-fos expression by adrenergic receptors and the putative role of fos in replication and differentiation pathways, we have examined c-fos expression in BC3H1 cells, a tumor-derived nonfusing muscle cell line. BC3H1 cells possess alpha 1- and beta 2-adrenergic receptors as well as receptors for histamine and acetylcholine. Furthermore, rapidly proliferating BC3H1 cells undergo differentiation toward muscle phenotype when exposed to low serum-containing culture media. Both alpha- and beta-adrenergic agonists and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate caused a rapid, transient increase in the steady-state level of c-fos mRNA. This induction was essentially independent of whether the cells were in the proliferative, relatively quiescent, or differentiated state. Protein synthesis inhibitors cycloheximide and anisomycin also increased markedly the concentration of c-fos mRNA, and in the presence of anisomycin c-fos mRNA was superinduced by the alpha-adrenergic agonist norepinephrine. Run-on transcription assays indicated that the c-fos gene is expressed in both proliferating and differentiated cells, although the steady-state levels of c-fos mRNA were low, or even undetectable, in such cells. The adrenergic agonists and the tumor promoter stimulated the transcription of the c-fos gene in both proliferating and differentiated cells. This stimulation, however, was modest, two- to three-fold compared to controls, in contrast to the marked elevation of the level of c-fos mRNA they caused. Neither the proliferation nor the expression of muscle type creatine kinase activity was influenced by adrenergic agonists. It is suggested that activation of the c-fos gene is a consequence of adrenoreceptor stimulation in diverse cell types, and thus it is involved in pleiotropic cellular responses to adrenergic agonists. Catecholamines may be one of the physiologic regulators of the c-fos gene.

Animals↗

PubMed1988

Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists.

The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists.

Aging↗

PubMed1987

Autoradiographic localization of dihydrotestosterone binding in the major salivary glands and other androgen-responsive organs of the mouse.

Mouse submandibular glands show an androgen-dependent sexual dimorphism, reflected in higher concentrations in males than in females of bioactive peptides, such as epidermal growth factor (EGF), nerve growth factor, and renin in the cells of the granular convoluted tubules (GCT). Biochemical studies have demonstrated androgen receptors in submandibular gland and other androgen-responsive organs in mouse. We have determined the cellular localization of these receptors using steroid autoradiography. Fifteen adult gonadectomized male mice were injected intravenously with 0.13 microgram or 0.26 microgram [3H]-dihydrotestosterone (SA 135 Ci/mM); some animals were pre-treated with cyclocytidine to stimulate secretion by GCT cells. Animals were killed 15 min, 1, 2, or 3 hr after isotope injection. Steroid autoradiographs were prepared, and some were stained immunocytochemically for EGF. Of the different cell types of submandibular gland, the acinar cells most frequently and intensely concentrated [3H]-DHT; GCT cells also concentrated the hormone, as did a small number of striated duct cells. In the other major salivary glands, the only cells that concentrated the androgen were interlobular striated duct cells in sublingual gland. In prostate, anterior pituitary, and brain a large number of cells concentrated androgen, as has been previously reported. Androgen binding by the GCT cells was a predictable finding, since androgen-induced alterations in composition and form of these cells are well documented. The intense androgen concentration by the acinar cells was an unexpected finding and suggests a hitherto unknown androgen regulation of these cells. An incidental finding was intense concentration of [3H]-DHT in the nuclei of the endothelial cells of the post-capillary venules of the cervical lymph nodes.

Animals↗

PubMed1987

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride. Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Animals↗

PubMed1987

Proto-oncogene fos (c-fos) expression in the heart.

Administration of the beta-adrenergic agonist isoproterenol led to a marked rapid increase in the steady-state level of c-fos mRNA in the heart of mice, rats, and Syrian hamsters. Stimulation of c-fos expression by isoproterenol was inhibited by the beta-adrenergic antagonist propranolol. An increase in Ca2+ influx through voltage-dependent calcium channels is probably not required for the activation of the c-fos gene by isoproterenol since the calcium channel blockers verapamil, nifedipine, and diltiazem had no effect on the induction of c-fos by the drug. In the heart of the rat, c-fos expression was also stimulated by the alpha-adrenergic agonist phenylephrine, histamine, and prostaglandin E1. The histamine-induced expression of the c-fos gene was blocked by the histamine H1-receptor antagonist pyrilamine but not by H2-receptor antagonists ranitidine and cimetidine. It is concluded that in the heart, hormones which increase cAMP and cytosolic Ca2+, such as beta-adrenergic agonists and prostaglandin E1, and/or stimulate the turnover of inositol phospholipids, such as alpha-adrenergic agonists and histamine H1-receptor agonists, regulate c-fos gene expression. The fos protein is likely to play a role in the mechanisms of neurotransmitters and hormones that modulate the functioning of the heart and of cardiac hypertrophy, degeneration and necrosis.

Alprostadil↗