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R B Wellner

Publications and source records attributed to R B Wellner.

28 records · Page 2Linked to original sources
PubMed1989

Beta-adrenergic responsiveness in a human submandibular tumor cell line (A253).

Salivary epithelial functions are regulated by the autonomic nervous system. In this regard, we have been studying the morphology and neuroreceptor composition of A253, an immortal cell line isolated from a human submandibular carcinoma (Giard et al., JNCI, 51:1417-1421, 1973). Phase contrast and electron microscopic observations indicate that A253 cells are of epithelial origin. Physiologically, A253 cells possess beta-adrenergic, but not alpha-adrenergic or muscarinic-cholinergic receptors. The beta-adrenergic receptors (BARs) are composed primarily of a single class of high affinity, beta 2-subtype receptors as judged by [3H]dihydroalprenolol antagonist binding studies. The BARs are functional inasmuch as isoproterenol stimulation increases both intracellular cAMP content and [3H]mannose incorporation into endogeneous glycoproteins. Differences in ultrastructure and neuroreceptor composition between A253 and other immortal salivary tumor cell lines are discussed.

Cell Line↗
PubMed1989

Characteristics of alpha 1-adrenergic receptors in a rat salivary cell line, RSMT-A5.

1. Membrane preparations obtained from RSMT-A5, an established rat submandibular cell line, possess alpha 1-adrenergic receptors as judged by [3H]prazosin binding studies. Scatchard analysis reveals a single class of binding sites with an equilibrium dissociation constant (Kd) of 0.052 nM and a receptor density (Bmax) of 104 fmol/mg protein. 2. These receptors are functional since stimulation by epinephrine results in an increased rate of phosphatidylinositol turnover which is blocked by both phentolamine and phenoxybenzamine. 3. Phenoxybenzamine (10(-5) M) irreversibly blocks 95% of the receptors within 2 min. A kinetic analysis shows that the receptors subsequently reappear at a rate of 6 fmol/mg protein/h. The calculated rate constant for receptor clearance (Kc) is 0.027 hr-1, and the half life (t 1/2) of the receptor is 26 hr. 4. These results are comparable to those reported for alpha 1-adrenergic receptors in the BC3H-1 smooth muscle cell line and suggest that alpha 1-adrenergic receptors in cultured cells, from diverse tissues, display similar characteristics.

Animals↗
PubMed1987

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells.

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.

Animals↗
PubMed1985

Enhancement of ricin cytotoxicity in Chinese hamster ovary cells by depletion of intracellular K+: evidence for an Na+/H+ exchange system in Chinese hamster ovary cells.

Depletion of intracellular K+ has been reported to result in an arrest of the formation of coated pits in human fibroblasts (Larkin, J.M., M.S. Brown, J.L. Goldstein, and R.G.W. Anderson, 1983, Cell, 33:273-285). We have studied the effects of K+ depletion on the cytotoxicities of ricin, Pseudomonas exotoxin A, and diphtheria toxin in Chinese hamster ovary (CHO) cells. The cytotoxicities of ricin and Pseudomonas toxin were enhanced in K+-depleted CHO cells whereas the cytotoxicity of diphtheria toxin was reduced by K+ depletion. The effects of NH4Cl on the cytotoxicities of ricin, Pseudomonas toxin, and diphtheria toxin were found to be similar to those of K+ depletion, and there were no additive or synergistic effects on ricin cytotoxicity by NH4Cl in K+-depleted medium. The enhancement of ricin cytotoxicity by K+ depletion could be completely reversed by the addition of K+, Rb+, and partially by the addition of Cs+, before the ricin treatment, whereas Li+ was ineffective. These protective effects of K+ or Rb+ requires a functional Na+/K+ ATPase. CHO cells grown in K+-depleted media were found to contain 6.3-fold increase in intracellular Na+ level, concomitant with a 10-fold reduction in intracellular K+ level. The enhanced cytotoxicity of ricin in K+-free medium and the increased uptake of Na+ could be abolished by amiloride or amiloride analogues, which are known to be potent inhibitors of the Na+/H+ antiport system. Our results suggest that a depletion of intracellular K+ results in an influx of Na+, which is accompanied by the extrusion of H+. Consequently, there is an alkalinization of the cytosol and the ricin-containing endosomes. As a result, ricin is more efficiently released from the endosomes in-K+-depleted cells. Results from the studies of the binding, internalization, and degradation of 125I-ricin, and the kinetics of inhibition of protein synthesis by ricin in K+-depleted cells are consistent with this working hypothesis.

ADP Ribose Transferases↗
PubMed1984

Genetic and biochemical analysis of mutation(s) affecting ricin internalization in Chinese hamster ovary cells.

We have previously reported the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in the internalization of ricin (Ray, B., and Wu, H.C. (1982) Mol. Cell. Biol. 2, 535-544). These mutants also do not exhibit the enhancement of ricin internalization by nigericin pretreatment at a low concentration, which is observed in the wild-type CHO cells. An analysis of somatic cell hybrids between the mutant and the toxin-sensitive wild-type CHO cell line shows that all of the phenotypes associated with the toxin resistance mutation are dominant in the hybrid cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]palmitic acid-labeled cell extracts from the mutant and toxin-resistant hybrid cell lines has revealed an increased incorporation of [3H] palmitic acid into two proteins with apparent molecular weights near 30,000 in the mutant and hybrid cells as compared to that in the wild-type cell line. Our studies indicate that these two fatty acyl proteins might be related to a dominant mutation(s) which results in a decreased uptake of ricin.

Animals↗
PubMed1984

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.

Animals↗
PubMedDate not supplied

Cationic liposome-mediated gene transfer to rat salivary epithelial cells in vitro and in vivo.

BACKGROUND: Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo. METHODS: Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts. RESULTS: Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed. CONCLUSIONS: The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

Amylases↗
PubMedDate not supplied

Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

Artificial Organs↗