Biomedical subjects

K W Dunn

Publications and source records attributed to K W Dunn.

18 recordsLinked to original sources

PubMed1997

Bafilomycin A1 treatment retards transferrin receptor recycling more than bulk membrane recycling.

Treatment of Chinese hamster ovary cells with the vacuolar proton pump inhibitor bafilomycin A1 causes a 2-fold retardation in the rate of recycling of transfected human transferrin receptors back to the cell surface as measured using biochemical assays (Johnson, L. S. , Dunn, K. W., Pytowski, B., and McGraw, T. E. (1993) Mol. Biol. Cell 4, 1251-1266). We have used quantitative fluorescence microscopy to determine which step(s) in the endocytic recycling pathway are affected. We show that removal of transferrin from sorting endosomes and accumulation in the peri-centriolar endocytic recycling compartment takes place normally in bafilomycin A1-treated cells. However, the rate constant for exit of transferrin receptors from recycling endosomes (ke) is reduced from 0.063 min-1 in untreated cells to 0.034 min-1 in the presence of bafilomycin A1. This retardation appears to be dependent on the presence of internalization motifs in the cytoplasmic domain since modified receptors lacking these oligopeptide motifs do not show as large a decrease in recycling rate in the presence of bafilomycin A1. Bulk membrane recycling (measured by efflux of an internalized fluorescent lipid analog, 6-[N-[7-nitrobenzo-2-oxa-1, 3-diazol-4-yl--amino-hexoyl- sphingosylphosphorylcholine) is slowed from an exit rate constant of 0.060 min-1 without drug to 0.046 min-1 in the presence of bafilomycin A1. We conclude that bafilomycin A1 slows bulk membrane flow, but it causes additional inhibition of receptor recycling in a manner that is dependent on a peptide motif on the cytoplasmic domain.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Transferrin receptor containing the SDYQRL motif of TGN38 causes a reorganization of the recycling compartment but is not targeted to the TGN.

The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter molecule include the ability to monitor trafficking, in both biochemical and microscopy experiments, using the natural ligand transferrin. When expressed in CHO cells, the SDYQRL-TR construct accumulated in juxtanuclear tubules and vesicles that are in the vicinity of the TGN. The SDYQRL-TR-containing structures, however, do not colocalize with TGN markers (e.g., NBD ceramide), and therefore the SDYQRL motif is not sufficient to target the TR to the TGN. The morphology of the SDYQRL-TR-containing juxtanuclear structures is different from the recycling compartment found in cells expressing the wild-type TR. In addition, the SDYQRL-TR-containing juxtanuclear compartment is more acidic than the recycling compartment in cells expressing the wild-type TR. The juxtanuclear compartment, however, is a bona fide recycling compartment since SDYQRL-TR was recycled back to the cell surface at a rate comparable to the wild-type TR, and sphingomyelin and cellubrevin, both of which label all compartments of the endocytic recycling pathway, colocalize with SDYQRL-TR in the juxtanuclear structures. These findings demonstrate that expression of the SDYQRL-TR construct alters the morphology and pH of endocytic recycling compartments rather than selectively affecting the intracellular trafficking pathway of the SDYQRL-TR construct. Therefore, the SDYQRL trafficking motif is not simply a molecular address that targets proteins to the TGN, but it can play an active role in determining the physical characteristics of endosomal compartments.

Acids↗

PubMed1995

Intraosseous infusion for burns resuscitation.

Two examples of resuscitation of a scalded child by intraosseous infusion, following failed peripheral venous cannulation, are presented. The technique of intraosseous infusion is reviewed and it is concluded that this technique provides a valuable second-line technique for establishing emergency venous access in children. It is quick, reliable and enables the rapid infusion of intravenous fluids and drugs into the systemic venous system. Complications are rare but can be serious and close observation of the infusion site is advisable.

Burns↗

PubMed1995

Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.

Anti-Bacterial Agents↗

PubMed1994

Regulation of endocytic trafficking and acidification are independent of the cystic fibrosis transmembrane regulator.

Cystic fibrosis is associated with mutations of the cystic fibrosis transmembrane regulator (CFTR), a cAMP-regulated plasma membrane Cl- channel. Mutations in the CFTR have been reported to not only impair Cl-transport at the plasma membrane but also inhibit organelle acidification and disrupt cAMP-regulated plasma membrane recycling. Comparisons of cultured pancreatic adenocarcinoma cells expressing the delta 508 mutant CFTR (CFPAC-1 cells) with genetically matched CFPAC-1 cells transfected with the wild-type CFTR demonstrate that expression of wild-type CFTR restores cAMP-mediated plasma membrane Cl- transport, but has no effect on pH regulation of endosomes labeled with either transferrin or wheat germ agglutinin. Endosome pH was, in all cases, similar in the two cell lines and unaffected by treatment with the cAMP agonist forskolin. Forskolin treatment had no effect on transferrin internalization, but increased recycling by 30-40% in both cell lines. The kinetics of wheat germ agglutinin recycling were negligibly affected by forskolin in either cell line. These results demonstrate that endocytic acidification and cAMP-mediated endocytic protein redistribution are similar in genetically matched CFPAC-1 cells which differ only in the expression of mutant or wild-type CFTR.

Adenocarcinoma↗

PubMed1994

Spontaneous pneumothorax in association with a major burn.

We report a case of a spontaneous pneumothorax in association with a major burn. The pneumothorax remained undetected for 4 days and the patient underwent a 2 h aeromedical transfer which could potentially have been hazardous. We strongly recommend that arterial blood gas analysis is performed before air transport of moderate or severely burned patients. If blood gases are abnormal a chest X-ray should also be performed. In practice a strong case can be made for routinely obtaining a chest X-ray in these patients. In-flight monitoring should include pulse oximetry.

Adult↗

PubMed1994

Applications of ratio fluorescence microscopy in the study of cell physiology.

Quantitative fluorescence microscopy is becoming an increasingly important tool in the study of cell biology. Fluorescence microscopy has long been used for qualitative characterizations of subcellular distributions of proteins, lipids, nucleic acids, and ions, but quantifying these distributions is complicated by a variety of optical, biological, and physical factors. Many factors that complicate quantification of fluorescence in cells can be circumvented by analyzing fluorescence ratios derived from pairs of fluorescence images. In this review we will discuss the factors that affect fluorescence quantification, the advantages of quantifying fluorescence as a ratio, and give examples of how fluorescence ratio microscopy is being applied in studies of cell biology.

Animals↗

PubMed1993

Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment.

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with morphologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking temperature-induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40 degrees C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild-type endocytic compartment is closely aligned with the microtubule-organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopied in wild-type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104-5 cells appears normal in immunofluorescent staining. B104-5 cells, unlike the parental cells, do not proliferate at 40 degrees C. The mutation in B104-5 cells is recessive, as fusion with wild-type cells results in a reversion of the B104-5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104-5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines.

Animals↗

PubMed1993

Entrapment against radiators--an underestimated burn hazard to the elderly.

Five cases of burns caused by residents of nursing homes falling out of bed and becoming trapped against radiators or hot pipes are reported. The current guidelines for nursing homes in relation to heating surfaces are reviewed and recommendations are made on how this hazard can be avoided.

Accidental Falls↗

PubMed1993

Coping modeling problem solving versus mastery modeling: effects on adherence, in-session process, and skill acquisition in a residential parent-training program.

This trial compared two approaches used to introduce parenting skills in a residential staff training program. Fifty staff were randomly assigned to: mastery modelling in which videotaped models demonstrated new skills, coping modelling problem solving (CMPS) in which participants formulated their own solutions to the errors depicted by videotaped models, or a waiting-list control group. In both, leaders used modelling, role playing, and homework projects to promote mastery and transfer of new skills. The skills of all groups improved, but CMPS participants attended more sessions, were late to fewer sessions, completed more homework, engaged in more cooperative in-session interaction, rated the program more positively, and reported higher job accomplishment scores. These data suggest that CMPS allowing participants to formulate their own solutions may enhance adherence and reduce the resistance observed in more didactic programs.

Adaptation, Psychological↗

PubMed1993

The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected.

We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min-1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane.

4-Chloro-7-nitrobenzofurazan↗

PubMed1993

Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif.

To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process.

Adaptor Proteins, Vesicular Transport↗

PubMed1992

Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes.

After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.

To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.

Animals↗

PubMed1988

Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation.

In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24-substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric-Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA-induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.

Animals↗

PubMed1987

Self-control and reinforcement in the management of a cerebral-palsied adolescent's drooling.

Self-control of swallowing and positive reinforcement were used to eliminate drooling of a non-vocal 16-year-old with severe spastic quadriplegia. Prompts for self-controlled swallowing appeared to be more effective than positive reinforcement. The effects of treatment were maintained at three-month follow-up, but not at six months. Drooling was eliminated again by prompts for self-controlled swallowing, and had not recurred one year after treatment had ended.

Adolescent↗

PubMed1986

Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima.

In observations on three strains of green hydra, the host and the algal mitotic index is closely coordinated only for the smallest. As the hydra strain size increases the coordination of host and algal mitosis progressively breaks down, first in timing for a medium-sized strain and then in rate for a large strain. Despite disparities between host and algal mitotic index, the number of algae per host cell remains constant in all strains during the interval measured. To account for this constancy we suggest that the hydra may either prolong the duration of the algal tetraspore stage or cull excess algae.

Animals↗

PubMedDate not supplied

Breast implant materials: sense and safety.

Cosmetic breast augmentation, and post-mastectomy breast reconstruction surgery using synthetic implants, have become established in surgical practice over more than 20 years. The operative technique for implant placement have changed somewhat during this time, as many different implant presentations have become available, but the same basic materials have remained in use. We have reviewed the present state of knowledge about breast implant materials with particular reference to the possible connection between polydimethylsiloxane and polyurethane to the so-called "Human Adjuvant Disease", and to carcinogenesis. Problems related to capsular contracture and mammography are also discussed.

Animals↗