Biomedical subjects

K J Kim

Publications and source records attributed to K J Kim.

At least 163 records · Page 9Linked to original sources

PubMed1995

Proximal migration and subcutaneous coiling of a peritoneal catheter: report of two cases.

The authors report two cases with a rare complication of ventriculoperitoneal shunt. The peritoneal catheters migrated upward to the scalp and to the clavicular area. In one case, the whole length of the peritoneal catheter was pulled out from the peritoneal cavity and was coiled subcutaneously. Both of the patients had the habit of head rotation and one of the patients had a history of subcutaneous fluid collection at the site of coiling. At surgery, whitish inflammatory granulation tissues were noted around the catheter in both cases, which might act as an anchoring point for the "windlass effect".

Child, Preschool↗

PubMed1995

Possible existence of Na(+)-coupled amino acid transport in the pigmented rabbit conjunctiva.

The objective of the present study was to determine whether Na(+)-amino acid cotransport contributed to the short-circuit current (ISC) in the isolated pigmented rabbit conjunctiva. Glycine, L-arginine, D-arginine, and L-glutamic acid were the amino acids tested, and bioelectric measurements were made in the Using chamber. The ISC was increased from 4% (L-glutamic acid) to 44% (L-arginine). The EC50 was 0.35 mM for glycine, 0.06 mM for L-arginine, 0.16 mM for D-arginine, and 1 mM for L-glutamic acid. No elevation in ISC was seen in the absence of Na+ in the mucosal bathing fluid. The above findings are consistent with the possible existence of a Na(+)-amino acid cotransport process on the apical side of the pigmented rabbit conjunctiva.

Amino Acids↗

PubMed1995

Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies.

PURPOSE: The objective of this study was to investigate how culture conditions would affect the morphological, functional, and permeability characteristics of rabbit tracheal epithelial cell layers being considered for drug transport studies. METHODS: Rabbit tracheocytes were isolated by protease treatment and plated onto collagen-treated permeable supports at 1.3 x 10(6) cells/cm2. After 24 hr, cell layers were cultured either air-interfaced (AIC) on their apical surface or under conventional liquid covered conditions (LCC). RESULTS: Scanning electron microscopy revealed mature cilia in AIC cell layers and ciliated cells denuded of cilia in LCC cell layers. Compared with LCC, AIC cell layers (n = 20) achieved a significantly higher peak equivalent short-circuit current (74.1 +/- 6.5 vs. 51.6 +/- 3.4 microA/cm2), a higher potential difference (70.9 +/- 2.8 vs. 60.5 +/- 3.0 mV), and a lower peak transepithelial electrical resistance (1.1 +/- 0.03 vs, 1.5 +/- 0.02 kohms.cm2). About 70% of the short-circuit current in AIC was amiloride-sensitive whereas < 10% was furosemide-sensitive, similar to that found in native tissue. The corresponding values in LCC were 50% and 46%. The permeability of both AIC and LCC to lipophilic solutes (dexamethasone and propranolol) was similar, whereas the permeability of hydrophilic solutes (mannitol, sucrose, and albuterol) in AIC was only half that in LCC. CONCLUSIONS: Given the striking similarity in morphological and functional characteristics of the AIC to those in the in vivo situation, the AIC is favored as an in vitro model for future drug transport studies.

Animals↗

PubMed1995

Dual effect of nitrogen dioxide on barrier properties of guinea pig tracheobronchial epithelial monolayers cultured in an air interface.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.

Animals↗

PubMed1995

Single electron transfer by an extracellular laccase from the white-rot fungus Pleurotus ostreatus.

Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.

Amino Acid Sequence↗

PubMed1995

Nonspecific interstitial pneumonia with fibrosis: radiographic and CT findings in seven patients.

PURPOSE: To describe the radiographic and computed tomographic (CT) findings in seven patients with nonspecific interstitial pneumonia with fibrosis (NIP). MATERIALS AND METHODS: NIP was proved pathologically in all patients. Findings at radiography and initial and 1-15-month follow-up CT were reviewed. RESULTS: The predominant radiographic abnormalities were areas of patchy parenchymal opacification present bilaterally in the middle and lower lung zones in six patients; in one patient, the chest radiograph depicted no abnormality. The most common finding observed on initial thin-section CT scans was bilateral, patchy areas of ground-glass attenuation present alone or with areas of consolidation in five patients (71%) or irregular lines in two (29%). At follow-up CT, the initial parenchymal abnormalities had resolved completely in three patients, improved in another three, and persisted in one. CONCLUSION: Bilateral patchy areas of opacity depicted radiographically and good clinical response to treatment may differentiate NIP from other interstitial pneumonias.

Adult↗

PubMed1995

Two concentrations of topical tretinoin (retinoic acid) cause similar improvement of photoaging but different degrees of irritation. A double-blind, vehicle-controlled comparison of 0.1% and 0.025% tretinoin creams.

BACKGROUND AND DESIGN: The efficacy of topical tretinoin (all-trans-retinoic acid) in treating photoaging is well established. Questions that remain are (1) whether irritation causes all or part of the improvement; (2) the concentration of tretinoin that maximizes clinical response with minimal side effects; and (3) the effects of long-term treatment on components of the cutaneous immune system. To address these issues, 99 photoaged patients completed a 48-week study using 0.1% tretinoin cream (n = 32), 0.025% tretinoin (n = 35), or vehicle (n = 32) once daily in a double-blind manner. Before and after treatment, we assessed histologic features, keratinocyte expression of HLA-DR and intercellular adhesion molecule-1, numbers of epidermal Langerhans' cells and epidermal and dermal T lymphocytes, and vascularity as measured by dermal endothelial cell area. RESULTS: Both 0.1% and 0.025% tretinoin produced statistically significant overall improvement in photoaging of the face compared with vehicle; there were no clinically or statistically significant differences in efficacy between the two concentrations of tretinoin. After 48 weeks, 0.1% and 0.025% tretinoin produced similar statistically significant epidermal thickening (by 30% and 28%, respectively) compared with vehicle (11% decrease) and increased vascularity (by 100% and 89%, respectively) compared with vehicle (9% decrease). By various analyses, irritant side effects (erythema and scaling) were statistically significantly greater with 0.1% tretinoin than with 0.025% tretinoin. No significant changes occurred in any immunologic markers when tretinoin and vehicle treatments were compared. CONCLUSIONS: Tretinoin 0.1% and 0.025% produce similar clinical and histologic changes in patients with photoaging, despite significantly greater incidence of irritation with the higher concentration. The separation between clinical improvement and irritation suggests that mechanisms other than irritation dominate tretinoin-induced repair of photoaging in humans.

Aged↗

PubMed1994

Monoclonal antibodies detect different distribution patterns of IL-8 receptor A and IL-8 receptor B on human peripheral blood leukocytes.

mAbs previously reported to be specific for IL-8R type A (IL-8R-A) and mAbs specific for IL-8R type B (IL-8R-B), which are described in this paper, were used to investigate the expression of each receptor on various types of cells. We generated mAbs specific for IL-8R-B, 4D1, and 10H2 by immunizing mice with 293 cells that expressed IL-8R-B and by selecting hybridoma cell lines that secreted mAbs that bind to human neutrophils. Flow cytometry showed that mAbs 4D1 and 10H2 were specific for IL-8R-B, as determined by their exclusive binding to 293-27 cells that expressed IL-8R-B, but not to 293-71 cells that expressed IL-8R-A. Epitopes recognized by these IL-8R-B-specific mAbs were shown to be within the N-terminal residues 1-18 of the IL-8R-B on the basis of their binding to various N-terminal peptides, as measured by ELISA. These IL-8R-B-specific mAbs were able to inhibit up to 90 and 50% of the 125I-labeled IL-8 binding to 293-27 cells and human neutrophils, respectively. The combination of mAb 9H1 (anti-IL-8R-A) and mAb 10H2 (anti-IL-8R-B) inhibited approximately 70% of 125I-labeled IL-8 binding to human neutrophils. Flow cytometry showed a wide range of donor variation in the expression levels of IL-8R-A and IL-8R-B on various human peripheral blood leukocytes. All neutrophils, all monocytes, and 5 to 25% of total lymphocytes (CD8+ T cells and CD56+ NK cells) expressed IL-8R. Neutrophils expressed the highest level of both IL-8R-A and IL-8R-B, at an approximately equal ratio, whereas monocytes and IL-8R+ lymphocytes expressed higher levels of IL-8R-B than IL-8R-A. Double-color flow cytometric analysis showed that 7 to 42% of CD8+ T cells and 39 to 76% of CD56+ NK cells, but no CD 20+ B cells or CD4+ T cells, expressed IL-8R.

Animals↗

PubMed1994

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU.

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU+ strain fimbriation did not require the fimU gene.

Agglutination↗

PubMed1994

Production of the RANTES chemokine in delayed-type hypersensitivity reactions: involvement of macrophages and endothelial cells.

To understand the selective accumulation of memory T helper lymphocytes and of macrophages in delayed-type hypersensitivity (DTH) granulomas, we studied the in situ production of RANTES, a chemokine initially characterized on the basis of its in vitro chemotactic properties for each of these cell populations. RANTES gene expression was studied by in situ hybridization in 15 human lymph nodes presenting typical DTH lesions related to either sarcoidosis or tuberculosis. A positive signal was detected in all cases. Labeling was specific for the DTH lesions, as very few if any positive cells were detected in the normal residual lymphoid tissue surrounding them or in reactive lymph nodes involved in a B lymphocyte response. RANTES gene expression was associated with the production of the protein, which was detected by immunochemistry in DTH lymph nodes. The morphological characteristics and distribution of positive cells in in situ hybridization and immunochemical experiments indicated that macrophages and endothelial cells, two cell populations not previously reported to produce RANTES, contributed to its production in DTH reactions. The ability of macrophages and endothelial cells to produce RANTES was confirmed by in vitro studies with alveolar macrophages and umbilical vein endothelial cells. In view of the chemotactic properties of RANTES for a limited range of cell populations, these results suggest that RANTES production in DTH granulomas may play a role in the selective accumulation of macrophages and memory T helper lymphocytes characterizing this type of cell-mediated immune reaction, and that macrophages and endothelial cells are involved in this production.

Cells, Cultured↗

PubMed1994

Leukemia inhibitory factor in long-term adherent layer cultures: increased levels of bioactive protein in leukemia and modulation by IL-4, IL-1 beta, and TNF-alpha.

In the current study, we used a monoclonal antibody-based enzyme-linked immunosorbent assay and bioassay to assess leukemia inhibitory factor (LIF) protein levels, activity, and function in supernatants of 59 adherent layers derived from acute and chronic myelogenous leukemia, myelodysplastic syndrome, and hairy cell leukemia patients and from normal controls. We demonstrate that biologically active LIF protein is constitutively produced and secreted by cultured bone marrow stromal cells from all of the studied subjects. Furthermore, various cytokines can alter endogenous LIF protein levels. Twenty-four h of exposure to recombinant human (rh) interleukin (IL) 4 (100 units/ml) significantly decreased LIF protein levels in adherent layer conditioned media [median base line level, 2.6 ng/ml; range, 1.6-8.0 ng/ml; median post rhIL-4 exposure levels, 1.9 ng/ml; range, 0.9-5.8 ng/ml (n = 7; P = 0.022)]. In contrast, rhIL-1 beta and rh tumor necrosis factor alpha consistently increased LIF protein levels. In the samples exposed to 50 units/ml rhIL-1 beta, median base line LIF level was 2.6 ng/ml; median post-LIF level was 9.0 ng/ml (n = 8; P = 0.014). In the two samples exposed to rh tumor necrosis factor alpha (200 units/ml), LIF levels increased from baseline levels of 2.6 and 2.7 ng/ml to postexposure levels of 7.7 and 12.2 ng/ml, respectively. Finally, the presence of LIF may be relevant to both normal and malignant hematopoietic processes as evidenced by: (a) LIF protein levels in adherent layer conditioned media were significantly elevated in samples from patients with a spectrum of hematological neoplasms [acute myelogenous leukemia: median level, 3.0 ng/ml (range, 1.6-11.0 ng/ml); myelodysplastic syndrome: median level, 4.5 ng/ml (range 1.4-15.5 ng/ml); hairy cell leukemia; median level, 3.5 ng/ml (range 2.2-10.3 ng/ml); chronic myelogenous leukemia-chronic phase: median level, 4.35 ng/ml (range 0.3-19.0 ng/ml); and chronic myelogenous leukemia-blast crisis: median level, 6.25 ng/ml (range 0.7-20.3 ng/ml)] as compared to samples from normal individuals (median level, 2.0 ng/ml; range, 0.7-4.6 ng/ml; P < 0.05); and (b) in normal controls, in vitro abrogation of endogenous LIF bioactivity by neutralizing antibody decreased the number of committed granulocyte-macrophage hemopoietic progenitors.

Blast Crisis↗

PubMed1994

Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy.

Although the potent neutrophil chemotaxin, IL-8, is a known product of endotoxin-stimulated cells in vitro, the contribution of IL-8 to neutrophil recruitment in Gram-negative endotoxin inflammation in vivo is unknown. To determine whether neutralization of IL-8 would decrease endotoxin-induced neutrophil influx, we generated neutralizing mAbs to rabbit rIL-8 for use in our rabbit model of endotoxin-induced pleurisy. One mAb, ARIL8.2, specifically inhibited both rabbit rIL-8-induced chemotactic activity and activation of the rabbit IL-8 receptor transfected in 293 cells. Anesthetized rabbits with in-dwelling pleural catheters received either neutralizing mAb (ARIL8.2; 1 mg/kg) or irrelevant isotype-matched mAb (anti-HIV gp120) i.v. 1 h before as well as intrapleurally (20 micrograms/ml) at the time of intrapleural instillation of Escherichia coli endotoxin (200 ng bilaterally). ARIL8.2 blocked 77% of endotoxin-induced neutrophil influx (21 +/- 2 (SE) x 10(6) (ARIL8.2) vs 91 +/- 15 x 10(6) (anti-gp120) (p < 0.0001)). By Western analysis, a band corresponding to rabbit IL-8 was detected in the pleural liquid of rabbits in both groups. By ELISA, however, the concentration of free, unbound IL-8 in the pleural liquid was significantly less in the ARIL8.2 group than in the anti-gp120 group for at least 4 h, confirming that ARIL8.2 bound the IL-8 generated in vivo during that time. We conclude that neutralization of IL-8 profoundly inhibits neutrophil recruitment in endotoxin-induced pleurisy indicating that IL-8 is a major chemotactic factor in this model of acute inflammation.

Animals↗

PubMed1994

Neutralizing monoclonal antibodies to human IL-8 receptor A map to the NH2-terminal region of the receptor.

mAbs against human IL-8 receptor A (IL-8R-A) were generated by immunizing mice with either: 1) peptides corresponding to various extracellular domains of IL-8R-A or 2) transfected 293 cells expressing IL-8R-A (293-71 cells). Among the seven peptides used for immunization, only the peptide corresponding to residues 2-19 of IL-8R-A produced Abs capable of recognizing native IL-8R-A on 293-71 cells. We screened for hybridomas secreting mAbs capable of binding strongly to peptide 2-19 in an ELISA and capable of recognizing native IL-8R-A in flow cytometry experiments with 293-71 cells. Two clones secreting mAbs capable of recognizing native IL-8R-A were selected for further characterization. None of these mAbs were able to block the binding of 125I-IL-8 to 293-71 cells. We also screened hybridomas derived from mice immunized with the intact receptor expressed on transfected 293-71 cells and identified several clones secreting mAbs capable of recognizing native IL-8R-A in flow cytometry experiments. Two of these mAbs were capable of blocking the binding of 125I-IL-8 to 293-71 cells. The epitopes, recognized by these blocking mAbs and by the other nonblocking mAbs derived from the peptide immunization, mapped to the NH2-terminal region of IL-8R-A using an ELISA against synthetic peptides: the two blocking mAbs mapped to residues 2-14 of IL-8R-A, whereas the nonblocking mAbs mapped to residues 2-11. Furthermore, flow cytometry analysis of IL-8 receptor mutants showed that Asp6 plays an important role in the binding of the blocking mAbs but not in the binding of the nonblocking mAbs. Conversely, a mutation of Asp11 to Lys disrupts the binding of one of the nonblocking mAbs (4C8) but has no effect on recognition by others. Analysis of the affinity of these mAbs for IL-8R-A demonstrated that blocking mAbs have affinities at least sevenfold higher than the nonblocking mAbs.

Amino Acid Sequence↗

PubMed1994

Consensus repeat domains of E-selectin enhance ligand binding.

To study the structural characteristics of E-selectin necessary for mediating cell adhesion, we examined the role of the consensus repeat (CR) domains in E-selectin function. Soluble constructs containing different numbers of CR domains were stably expressed in Chinese hamster ovary cells, purified to homogeneity, and characterized. The minimum functional unit of soluble E-selectin consisted of the lectin (Lec) and epidermal growth factor (EGF) domains alone (Lec-EGF) as indicated by its ability to mediate in vitro HL-60 cell adhesion. However, E-selectin containing all six CR domains (Lec-EGF-CR6) at its COOH terminus was the most potent in blocking neutrophil or HL-60 cell adhesion to either immobilized E-selectin or cytokine-stimulated human umbilical vein endothelial cells. This increased potency of Lec-EGF-CR6 in blocking cell adhesion was not due to CR-mediated oligomerization of the protein. Lec-EGF-CR6 was most likely monomeric in solution, as judged by gel filtration fast protein liquid chromatography, membrane ultrafiltration, and chemical cross-linking analysis. Therefore, although the lectin and EGF domains are necessary and sufficient for mediating cell adhesion, the additional six CR domains, present in native E-selectin, contribute to the enhanced binding of E-selectin to its ligand.

Base Sequence↗

PubMed1994

Identification of actinomycins by high performance liquid chromatography and fast atom bombardment mass spectrometry.

An actinomycin complex isolated from culture broth of a soil microorganism, SNUS 9305011 has been examined by High performance liquid chromatography (HPLC). From the analysis of the fractions obtained by column chromatography of the ethyl acetate extract, three actinomycin components are confirmed. The HPLC analysis is carried out with a CN-bonded nucleosil column. Comparison of the retention times of the components with those of actinomycin D, C complex X0 beta, and V suggests that they are different actinomycins. FAB mass spectra of the components also shows different molecular ions from those of standards and other reported actinomycins. The present work was has demonstrated that actinomycin components can be separated by a CN-bonded HPLC column, and that comparison of their HPLC chromatograms with authentic samples and information on their molecular ions can be successfully employed for identification of actinomycins.

Anti-Bacterial Agents↗

PubMed1994

Transport of thyrotropin-releasing hormone across rat alveolar epithelial cell monolayers.

It has been recently shown that epithelial cell monolayers of rat type II pneumocytes cultivated on tissue culture-treated polycarbonate Transwell filters are tight (> 2,000 ohm-cm2) and exhibit morphological and phenotypic characteristics of in vivo type I pneumocytes. We studied, utilizing these tight monolayers, the transepithelial transport of thyrotropin-releasing hormone (TRH). Either [125I]TRH or [3H]TRH was used to measure the transalveolar epithelial radiolabel fluxes across the monolayer. Radiochromatography was performed, utilizing reverse-phase HPLC techniques, to determine the presence of TRH and its subspecies in dosing, donor and receiver fluids. The apparent permeability coefficient (Papp) estimated from 125I-radiolabel fluxes was approximately 1.7 x 10(-7) cm/sec in both the apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. In contrast, the Papp for 3H-radiolabels was approximately 4.2 x 10(-7) cm/sec in both directions. Radiochromatography results indicated that neither apical nor basolateral receiver fluid collected at the end of 4 h flux studies contained metabolites of [125I]TRH or [3H]TRH. In the presence of 1,000-fold excess of unlabeled TRH in the donor fluid, the Papp of neither [125I]- or [3H]-TRH in either direction was altered. These data taken together provide evidence for restricted diffusional transport of intact TRH across the rat alveolar epithelial cell monolayer, most likely via paracellular pathways. Thus, it appears that TRH delivery via pulmonary alveolar epithelium in the distal airspaces of the mammalian lung may be feasible without significant interference from peptidase activities.

Animals↗

PubMed1994

Effects of protease inhibitors on vasopressin transport across rat alveolar epithelial cell monolayers.

The transepithelial transport of arginine vasopressin (AVP) across cultured rat alveolar epithelial cell monolayers was studied. At 0.1 nM donor [125I]AVP, the radiolabel flux measured in the apical-to-basolateral (AB) direction was about 10 times greater than that in the reverse (BA) direction. HPLC analyses of the basolateral receiver fluid collected at the end of these flux measurements showed that about 97% of the total [125I]label represented subspecies of AVP, whereas the apical receiver fluid contained largely intact AVP (approximately 85% of total [125I]label). Both donor fluids contained virtually no degradation products of AVP (> 99%). In the presence of an excess 0.1 mM unlabeled AVP in the apical donor fluid, the Papp for radiolabeled AVP in the AB direction was decreased by approximately 68%, while the fraction of intact AVP in the basolateral receiver fluid was increased six-fold as compared to that observed at 0.1 nM [125I]AVP alone. Under this condition, the flux of intact AVP was approximately the same in both directions. When the concentration of apical camostat mesylate, an aminopeptidase inhibitor, was varied from 0 to 2 mM, the radiolabeled flux in the AB direction (with 0.1 nM [125I]AVP in the donor fluid) was significantly decreased in a dose-dependent manner, yielding commensurably elevated concentrations of intact AVP in the basolateral receiver fluid. In contrast, leupeptin (0.5 mM), a serine protease inhibitor, was without effect. These data, taken together, suggest that apically-presented AVP undergoes proteolysis (most likely by peptidases localized at apical cell membranes of alveolar epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Structural requirements for adhesion of soluble recombinant murine vascular cell adhesion molecule-1 to alpha 4 beta 1.

This study describes the identification of seven amino acid residues of the vascular cell adhesion molecule (VCAM-1) that influence binding to the alpha 4 beta 1 receptor. Using recombinant murine VCAM-1-IgG, which is bound by both mouse (WEHI 231) and human (Ramos) lymphoid cells, two approaches demonstrated the crucial role of the first two NH2-terminal Ig-like domains in binding: (a) blocking monoclonal anti-mouse VCAM-1 antibodies bound to only truncation variants that included the first two domains; (b) site-direct mutagenesis of the first NH2-terminal domain showed that alanine substitution of the amino acid residues R36, D40, K46, S54, N65, T72, and E81 partially or completely reduced adherence by human and/or mouse cells. Of these D40, when mutated to A, N, or K (but not E), showed complete abrogation of adherence by mouse and human cells, as well as inability to bind blocking anti-murine VCAM-1 antibody MVCAM.A429, while not inducing gross structural perturbations in VCAM-1. By molecular modeling, the D40 residue was located on a beta turn connecting two beta strands defined as C and D. The residues R36, K46, S54, N65, T72, and E81, which perturb cell adherence and caused small changes to gross structure, are conformationally near or adjacent to D40. Although these residues, identified as crucial for cell adhesion, are all located in domain 1, it is evident that there is a structural requirement for domains 1 and 2 to be intact so that cell adhesive function can occur.

Amino Acid Sequence↗