Biomedical subjects

S R Bates

Publications and source records attributed to S R Bates.

At least 19 recordsLinked to original sources

PubMed2000

Lamellar body membrane turnover is stimulated by secretagogues.

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. (125)I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

Adenosine Triphosphate↗

PubMed2000

Surface-expressed lamellar body membrane is recycled to lamellar bodies.

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.

Animals↗

PubMed1998

Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells.

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of 125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of 125I-A2R and 125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of approximately 32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was approximately 210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Exclusion probabilities of 22 bovine microsatellite markers in fluorescent multiplexes for semiautomated parentage testing.

Six multiplexes developed for semiautomated fluorescence genotyping were evaluated for parentage testing. These multiplexes contained primer pairs for the amplification of 22 microsatellites on 17 bovine autosomes. Exclusion probabilities were determined from genotypes of 1022 Holstein cattle and 311 beef cattle belonging to five breeds. Two cases were considered: case 1, genotypes are known for an alleged parent and an offspring but genotypes of a confirmed parent are unknown; and case 2, genotypes are known for an alleged parent, a confirmed parent and an offspring. If the alleged parent is not the true parent, then the 22 markers will exclude the alleged parent with a probability of > 0.9986 for case 1 and with a probability of > 0.99999 for case 2. On the basis of these exclusion probabilities, the probability that an alleged parent will be falsely included as a parent is in the range of 1/716 to 1/2845 for case 1 and 1/1.2 million to 1/159753 for case 2. In addition to these results, a rapid and efficient non-organic method for extraction of DNA from semen is described.

Animals↗

PubMed1997

Macrophages primed by overnight culture demonstrate a marked stimulation of surfactant protein A degradation.

The current study examined whether long-term culture of macrophages affects their metabolism of surfactant components. Compared with freshly isolated resting macrophages in culture for 1 h, macrophages attached to plastic dishes for 24 h showed evidence of conversion to a "primed" state with 1) an altered morphology characterized by a larger size, ruffled membranes, lamellipodia, and a "foamy" appearance after attachment to glass and 2) a fivefold greater respiratory burst in response to phorbol 12-myristate 13-acetate stimulation. On incubation with iodinated surfactant protein A (SP-A), the 24-h alveolar or tissue macrophages showed a 5- or a 23-fold greater increase in SP-A degradation, respectively, than macrophages cultured for 1 h. Conditioned media experiments demonstrated that the elevated rate of SP-A degradation after prolonged culture was not a result of proteases secreted by the macrophages. Incubation of cells with NH4Cl reduced the degradation of SP-A to a similar extent (to 33% of control values) in resting and primed tissue macrophages. On the other hand, length of time of cell culture did not affect macrophage uptake and degradation of [3H]dipalmitoylphosphatidylcholine in mixed unilamellar liposomes. Thus freshly isolated resting tissue and alveolar macrophages can be primed to specifically increase their rate of SP-A degradation. Activation of macrophages associated with lung disease may be important for SP-A metabolism and surfactant function.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Bromism: intoxication from a rare anticonvulsant therapy.

Bromide, the first effective therapy for epilepsy, is not commonly prescribed today but has been advocated by some pediatric neurologists for the treatment of intractable seizures in children. We report a 17-year-old female patient with intractable epilepsy who insidiously developed bromism while on treatment with triple bromide elixir. We review the clinical presentation, diagnosis, pathophysiology, and management of bromism.

Adolescent↗

PubMed1996

Secretagogues increase the expression of surfactant protein A receptors on lung type II cells.

Since secretagogues have been shown to increase the internalization of surfactant phospholipid and protein by lung cells, we postulated that their action occurred through a mechanism involving increased surfactant protein A (SP-A) receptor density. Therefore, we evaluated the influence of secretagogues on the binding of iodinated SP-A to alveolar type II cells. Type II cells were isolated from rat lung and maintained in primary culture for 18 h on Transwell membranes. Upon exposure to 8-bromo-cyclic AMP (cAMP, 0.1 mM), phorbol 12-myristate 13-acetate (PMA, 10 nM), terbutaline (0.1 mM), or ATP (1 mM), the binding of SP-A increased 1.5-2-fold. This stimulation was cell substrate-dependent since type II cells plated on plastic dishes did not show this effect. A time course of the stimulation of SP-A binding due to secretagogues showed that both cAMP and PMA increased SP-A binding by 2-fold after 20 min. With cAMP, binding remained elevated for 2 h, while binding in the presence of PMA had returned to control values. The effects of submaximal concentrations of cAMP and PMA on binding were additive. Inhibition of cellular protein synthesis with cycloheximide did not alter the increase of SP-A binding stimulated by the secretagogues. Type II cells pretreated with PMA responded to subsequent treatment with cAMP by increasing SP-A binding, while these cells were refractory to subsequent treatment with PMA. Both constitutive and regulated binding of SP-A to type II cells were sensitive to trypsin. The binding of SP-A to type II cells showed saturation at a concentration of 1 microg/ml SP-A under control and secretagogue-stimulated conditions, with both total and calcium-dependent binding showing a 2-fold increase upon secretagogue exposure. The data are consistent with the hypothesis that secretagogues stimulate surfactant uptake, at least in part, through recruitment of SP-A receptors to the type II cell surface, resulting in an increase in the number of SP-A binding sites.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

The economics of infertility: developing an infertility managed-care plan.

Infertility is a natural disorder for developing a managed-care plan. Infertility has a clearly defined end point. The treatment regimens and protocols used for treating infertile couples have predictable outcomes; it is possible to develop rational treatment plans over a limited period of time. It is not unreasonable for infertile couples to share the cost of treatment and for the physician-providers to share the economic risk by providing a bundle of optimal services at a fixed price. We present the theoretic basis for an infertility managed-care plan and the requirements for developing a plan that can be sold in the marketplace. Finally, we discuss some of the consequences of an infertility managed-care plan on physician relationships, physician manpower, research, and education.

Clinical Protocols↗

PubMed1996

Surfactant protein A is degraded by alveolar macrophages.

The metabolism of iodinated lung surfactant protein A (SP-A) by alveolar macrophages in primary culture was examined to determine the role these cells play in the degradation of this surfactant protein. SP-A was isolated from lung lavage obtained from normal bovines, patients with alveolar proteinosis, and silica-treated rats. SP-A (0.5 microgram/ml) was incubated for 3 h with rat alveolar macrophages obtained by lung lavage. Cell association and degradation of human and rat SP-A was three times greater than that of bovine SP-A. During the 3-h period, 50% of total macrophage-associated SP-A was degraded. Degradation was time-, temperature-, and concentration-dependent after a 1-h lag period. SP-A degradation was intracellular, since NH4Cl inhibited degradation > 50%, and macrophage-conditioned medium was ineffective. Tenfold more SP-A was degraded by macrophages than by type II cells isolated after elastase digestion of rat lungs. There was little degradation of SP-A by HeLa cells. We conclude that alveolar macrophages take up and degrade SP-A and thus could contribute to the catabolism of SP-A in the lung.

Animals↗

PubMed1996

Infertility services in a managed care environment.

Managed care schemes are replacing traditional fee-for-service reimbursement to physicians and hospitals in the United States. Managed care schemes take the form of discounted fee-for-service, utilization review, global fee reimbursement, and capitated reimbursement schemes with funds to be distributed among providers. Reimbursement for infertility services has been excluded from many managed care plans as infertility is viewed as a social condition, not a medical condition, and coverage for infertility diagnosis and treatment is viewed as unnecessary in the bundle of services offered by insurers and other managed care organizations. However, some states mandate infertility coverage and some managed care organizations realize that provision of care for infertile couples makes their product more attractive. Large managed care organizations such as Blue Cross/Blue Shield of illinois and some entrepreneurial organizations are developing managed care plans that incorporate infertility services. Comprehensive services--including in-vitro fertilization--can be offered at a lower cost than traditional fee-for-service care. Newer technologies such as in-vitro fertilization are replacing fallopian tube surgery and surgical treatment for male infertility. These can be implemented at a lower cost and with better outcome for infertile couples than traditional services.

Comprehensive Health Care↗

PubMed1995

Clinical, neurodiagnostic, and MR findings in children with spinal and brain stem multiple sclerosis.

PURPOSE: To describe the clinical, neurodiagnostic, and MR findings in seven children with brain stem and spinal multiple sclerosis. METHODS: Spinal or brain stem multiple sclerosis was diagnosed in seven children between 1986 and 1992. All patients had neurologic and MR examinations as well as neurodiagnostic testing, including spinal fluid analysis and brain stem and auditory evoked potentials. RESULTS: Three children had clinical findings and masslike lesions in the brain stem (two) or spinal cord (one) suggestive of neoplasm, which prompted biopsy (two) or radiation therapy (one). Five of six patients with spinal involvement had cord swelling with increased signal on T2-weighted images over at least three cord segments, and two children had essentially holocord involvement. Three children had normal cranial MR at presentation. CONCLUSIONS: Multiple sclerosis involvement of the brain stem and spinal cord may be associated with extensive swelling and MR signal changes suggestive of neoplasm without typical cerebral white matter abnormalities. Serial clinical and neuroimaging examinations may be necessary to make a definitive diagnosis of multiple sclerosis in children.

Adolescent↗

PubMed1994

Inhibition of Trimeresurus flavoviridis phospholipase A2 by lung surfactant protein A (SP-A).

A marked sequence homology has been noted between lung surfactant protein A (SP-A) and an inhibitor of phospholipase A2 (PLA2) isolated from the serum of Trimeresurus flavoviridis (Habu snake). This study evaluated the effect of SP-A on PLA2 activity from several sources. SP-A was isolated from bovine or rat lung surfactant by extraction with 1-butanol and octyl beta-D-glucopyranoside. The addition of SP-A produced a concentration-dependent inhibition of T. flavoviridis PLA2 that indicated non-competitive kinetics with Ki 5 micrograms/ml. Inhibition was reversed by heat inactivation, disulfide bond reduction or alkylation of SP-A, or by the presence of anti-SP-A antibody. Treatment of SP-A with endoglycosidase F or the presence of variation monosaccharides or lectins did not alter SP-A inhibition. Binding of PLA2 to SP-A was shown by ultrafiltration and was abolished by SP-A alkylation or the presence of SDS. The SP-A/PLA2 complex recovered from the ultrafilter had essentially no enzymatic activity, but activity was restored by treatment with mercaptoethanol. SP-A had no effect on activity of PLA2 from Naja naja, Crotalus atrox, or bovine pancreas. These results indicate that surfactant protein A selectively inhibits Trimeresurus phospholipase A2 activity and suggest that binding to the enzyme is the mechanism for inhibition.

Animals↗

PubMed1994

Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes.

We have investigated the internalization of surfactant by type II cells in primary culture. Previously, we demonstrated that type II cells cultured on microporous membranes [Transwell membranes (TM)] maintained the morphological characteristics of lung pneumocytes and took up surfactant protein and phospholipid in a fashion similar to that described for the uptake of surfactant by whole lung. In the present study, cells cultured on TM and exposed to equivalent amounts of phospholipid (PL) as either natural surfactant or liposomes incorporated fivefold greater amounts of natural surfactant PL. The evidence supported an important role for surfactant protein A (SP-A), as the incorporation of surfactant into type II cells on TM was reduced by anti-SP-A antisera and by pretreatment of the surfactant with beta-mercaptoethanol. In addition, the uptake of liposomes into type II cells on TM was augmented by SP-A. With the use of iodinated bovine SP-A reconstituted in rat surfactant, cells cultured on TM showed a 2.6-fold increase in binding and a 3.2-fold stimulation in uptake of SP-A over that seen with cells cultured on plastic. The change in the slope of the total binding curve of 125I-labeled bovine SP-A reconstituted with bovine surfactant to type II cells on the two substrates occurred at the same concentration of SP-A (17 micrograms/mg), but maximal binding was approximately eight times higher for cells on TM than for cells on plastic dishes. Thus, in a more physiological environment, i.e., SP-A in surfactant and cells on microporous membranes, SP-A plays an important role in the uptake of phospholipids by alveolar pneumocytes.

Animals↗

PubMed1993

Degradation of surfactant protein B by alveolar type II cells.

Surfactant protein B (SP-B) metabolism was studied in primary cultures of alveolar type II cells. Iodinated SP-B reconstituted with surfactant was incorporated rapidly into lung pneumocytes and degraded to trichloroacetic acid (TCA)-soluble products after a lag period of 1 h. Cellular degradation of SP-B occurred whether or not phospholipid liposomes or surfactant was added to the phospholipid-poor SP-B. Uptake and degradation of SP-B at 37 degrees C showed a linear increase up to 3 micrograms SP-B/ml after which the slope of the curve became less steep with increasing concentrations of SP-B in the media. After 4 h of incubation with SP-B, 35% of the SP-B processed was recovered as degradation products. Ninety-six percent of the degradation products were in the media and only 4% were recovered in the cell. The bulk of the breakdown of SP-B occurred inside the type II cells since degradation did not occur at 4 degrees C, showed a 1-h lag period, was proportional to the SP-B protein internalized by the cells, was inhibited 47% by ammonium chloride, was unaffected by the addition of protease inhibitors to the medium, and cell-conditioned medium produced only limited SP-B degradation. Alveolar macrophages also degraded SP-B, whereas other cell types degraded SP-B to a lesser extent. Thus the specificity of the metabolism of SP-B may be through the capability of lung cells to degrade SP-B.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1992

Differential extraction for the rapid purification of bovine surfactant protein B.

Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.

Amino Acid Sequence↗

PubMed1992

Binding and uptake of surfactant protein B by alveolar type II cells.

Surfactant protein B (SP-B, mol wt 9,000, reduced) is a low-molecular-weight hydrophobic protein found in organic extracts of lung surfactant. The interaction of iodinated bovine SP-B (125I-SP-B) and isolated rat alveolar type II cells was examined. The association of SP-B with the lung cells was time and temperature dependent; type II cells exhibited time-dependent binding (at 4 degrees C) and uptake (at 37 degrees C) of SP-B. Binding of phospholipid-poor 125I-SP-B was linearly related to the external SP-B concentration from 0.25 to 60 microgram/ml and was not inhibited by a 60-fold excess of unlabeled SP-B. However, the binding of 125I-SP-B reconstituted with bovine surfactant or with phospholipid-containing liposomes occurred through a high-affinity, saturable process and could be inhibited with unlabeled SP-B. By Scatchard analysis, half-maximum binding in the presence of surfactant occurred at 3.1 +/- 0.7 micrograms SP-B/ml. Saturable binding of SP-B reconstituted with surfactant also occurred with other cell types. The results indicate that SP-B was bound and internalized by type II cells. The apparent lack of specificity in the absence of phospholipid may have been due to the self-association of SP-B. The reconstitution of SP-B with phospholipid altered the binding of phospholipid-poor SP-B from a nonspecific process to a high-affinity process consistent with a cell surface binding site.

Animals↗

PubMed1991

Reduction of serum carnitine concentrations during anticonvulsant therapy with phenobarbital, valproic acid, phenytoin, and carbamazepine in children.

We determined four carnitine constituents (total and free carnitine and short- and long-chain fatty acid carnitine esters) in serum from 471 patients treated for convulsions with phenobarbital, valproic acid, phenytoin, and/or carbamazepine. The 471 patients were in eight treatment groups; four were treated with monotherapy and four with polytherapy. The means of all four carnitine constituents were significantly reduced in all treatment groups (except for free carnitine in four groups). Total carnitine was reduced by 23% to 48%, free carnitine by 9% to 45%, short-chain fatty acid carnitine by 46% to 64%, and long-chain fatty acid carnitine by 6% to 29%. Patient frequency of reduction for total carnitine was 20% of all patients (10% for free carnitine), 23% of patients receiving valproate (9% for free carnitine), 36% of those receiving phenobarbital (21% for free carnitine), 12% of those receiving phenytoin (8% for free carnitine), and 8% of those receiving carbamazepine (1% for free carnitine). Only for phenobarbital was there an inverse correlation between the serum concentration of the drug and that of carnitine concentration. One patient receiving carbamazepine had a 59% reduction in the total and a 65% reduction in the free carnitine concentration and a fivefold increase in long-chain fatty acid carnitine, values similar to those seen in neonatal lethal carnitine palmitoyl transferase II deficiency. It remains to be determined whether a reduction in serum carnitine values in patients receiving anticonvulsant therapy is of clinical consequence, whether the reduction is present in some patients before the start of therapy, when and by what mechanism carnitine levels may become reduced during therapy, and whether the reduction exists in the solid tissues of these patients.

Analysis of Variance↗

PubMed1990

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex.

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.

Base Composition↗