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Biomedical subjects

R C Hunt

Publications and source records attributed to R C Hunt.

At least 19 recordsLinked to original sources
PubMed1992

Loss of one asparagine-linked oligosaccharide from human transferrin receptors results in specific cleavage and association with the endoplasmic reticulum.

The coding sequence for the human transferrin receptor gene has been mutated in order to abolish the attachment of the oligosaccharide closest to the transmembrane sequence. Expression of the mutant receptor in Chinese hamster ovary cells resulted, after analysis under nonreducing conditions, in an 85- and a 73-kDa receptor species. After reduction, the 85-kDa receptors were mostly converted to 73 kDa, although with short labeling periods some 85-kDa receptor remained suggesting that the mutated gene was capable of coding for the entire polypeptide which was then proteolytically processed. This was supported by in vitro translation of mRNA from either wild type or mutant cells which in both cases yielded an 80-kDa protein, the full size of the nonglycosylated protein. The solubility characteristics of the mutant receptor suggest that it contains the COOH-terminal extracellular domain but not the transmembrane sequence. This is supported by the endoglycosidase H sensitivity of the 73-kDa protein which is compatible with the retention of two of the original three high mannose oligosaccharides. The receptor that lacks one oligosaccharide is unable to form intermonomer disulfide bridges or to migrate to the cell surface and is located in the endoplasmic reticulum where it is further degraded after a lag period of about 30 min.

Animals↗
PubMed1992

Release of iron by human retinal pigment epithelial cells.

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, take up iron in association with transferrin by a typical receptor-mediated mechanism (Hunt et al., 1989. J. Cell Sci. 92:655-666). This iron is dissociated from transferrin in a low pH environment and uptake is sensitive to agents that inhibit endosomal acidification. The dissociated iron enters the cytoplasm as a low molecular weight (less than 10 kD) component and subsequently binds to ferritin. No evidence for recycling of iron in association with transferrin was found. Nevertheless, much of the iron that is taken up is recycled to the extracellular medium, primarily from the low molecular weight pool. This release of iron is not sensitive to inhibitors of energy production or of vesicular acidification but is increased up to a maximum of about 40% of the total 55Fe incorporated when cells are incubated with serum or the medium is changed. When a short loading time for 55Fe from 55Fe-transferrin is used (i.e., when the low molecular weight pool is proportionately larger), a much larger fraction of the cell-associated radiolabel is released than when longer loading times are used. The data suggest that a releasable intracellular iron pool is in equilibrium with the externalized material. The released iron may be separated into a high and a low molecular weight component. The former is similar on polyacrylamide gel electrophoresis to ferritin although it cannot be immune precipitated by anti-ferritin antibodies. The low molecular weight 55Fe which is heterogeneous in nature can be bound by external apo-transferrin and may represent a form that can be taken up by cells beyond the blood-retinal barrier.

Biological Transport↗
PubMed1991

Inability to assess breath sounds during air medical transport by helicopter.

This study assessed the capabilities of a traditional and an amplified stethoscope used by flight nurses to assess breath sound during air medical transport in an MBB BO-105 helicopter. We developed a normal breath sound model using a prerecorded tape of breath sounds interspersed with segments without breath sounds; the recorder had been placed in the chest wall of a resuscitation training manikin. Flight nurses completed control listening sessions in a quiet environment and experimental sessions during flight using a traditional stethoscope for half of the sessions and an amplified stethoscope for the remaining half. In the quiet environment, flight nurses accurately reported the presence or absence of breath sounds in 110 (92%) of 120 trials. During helicopter flight, none of the flight nurses heard breath sounds during any of the recorded segments with either the traditional stethoscope or the amplified stethoscope. We conclude that flight nurses are unable to hear normal breath sounds using a traditional or amplified stethoscope during flight in a medically configured MBB BO-105 helicopter. Improved stethoscopes, innovative methods of listening, and reduction of aircraft noise are potential solutions to the problems of breath sound assessment during air medical transport.

Aircraft↗
PubMed1990

Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro.

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin-containing intermediate filaments that react with a combination of AE1 and AE3 anti-keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti-keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non-proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non-proliferating RPE cells. In contrast to the presence of keratin-containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin-containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.

Antibodies, Monoclonal↗
PubMed1990

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems.

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.

Animals↗
PubMed1990

Selective inhibition of growing pigment epithelial cells by a receptor-directed immunotoxin.

An immunotoxin conjugate of a murine monoclonal antibody against human transferrin receptors and the A chain of ricin was examined for its potential to inhibit selectively the growth of retinal pigment epithelial (RPE) cells which grow in an uncontrolled manner in proliferative vitreoretinopathy. The probable efficacy of such an agent in vivo stems from the observation that actively proliferating cells possess many more transferrin receptors than normal quiescent cells. The authors showed in vitro that the immunoconjugate (454A12 MAB-rRA) inhibits protein synthesis in actively dividing RPE cells but has a smaller or no effect on protein synthesis by confluent, nondividing RPE cells. The effect was specific in that neither the free ricin A chain (rRA) nor the monoclonal antibody (454A12 MAB) alone has any inhibitory effect. Furthermore, the antibody competes with the immunotoxin and suppresses the latter's toxicity. This immunotoxin has applications for therapy in conditions in which the pathologic proliferation of RPE cells occurs.

Antibodies, Monoclonal↗
PubMed1989

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand.

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.

Alkaline Phosphatase↗
PubMed1989

Influence of emergency medical services systems and prehospital defibrillation on survival of sudden cardiac death victims.

This article reviews the influence of emergency medical systems and prehospital defibrillation on survival of sudden cardiac death. The historical perspective and epidemiologic considerations of prehospital sudden cardiac death are highlighted. Factors predictive of successful resuscitation and impact of community activity on sudden death are discussed. Influences of emergency medical services on outcome of prehospital cardiac arrest are reviewed, with emphasis on the role of dispatchers, emergency medical technicians, and paramedics. The recent emergence of prehospital automatic defibrillation by emergency medical technicians, first responders, and lay persons is discussed in depth, as it has great potential to positively influence outcome of prehospital sudden cardiac death.

Death, Sudden↗
PubMed1989

Pharyngeal tracheal lumen airway training: failure to discriminate between esophageal and endotracheal modes and failure to confirm ventilation.

The pharyngeal tracheal lumen (PTL) airway is a new airway control device for emergency medical technicians (EMTs) and paramedics and functions as either an esophageal obturator or an endotracheal tube. We developed a ten-step PTL airway training protocol that included proper airway insertion, patient ventilation, and confirmation of tube placement by auscultation. We then prospectively evaluated the effectiveness of training by testing the ability of 32 EMTs and paramedics to discriminate between esophageal and endotracheal mode placement. Training consisted of a group demonstration followed by supervised individual practice on a Laerdal Adult Intubation Model. All 70 insertion attempts resulted in esophageal obturator mode placement. As a result, trainees did not have an opportunity to practice discrimination between esophageal and endotracheal placement. Six weeks after initial training, 19 of the entry group were randomly and blindly assigned to ventilate each of two Laerdal models that had been previously intubated with the PTL, one in esophageal mode and the other in endotracheal mode. In the esophageal mode, 15 of 19 trainees (79%) correctly selected the esophageal port to ventilate, but four (21%) could not decide and abandoned the airway. In the tracheal mode, 16 of 19 trainees (84%) correctly selected the tracheal port to ventilate, one (5%) was unable to decide, and two (10.5%) selected the wrong tube and attempted ventilation with the esophageal port even though the tracheal balloon was completely occluding the airway. Ventilation was confirmed with auscultation in only 50% of the attempts (19 of 38). Our study indicates that the training protocol was inadequate to teach critical decision-making in the use of the PTL airway.(ABSTRACT TRUNCATED AT 250 WORDS)

Allied Health Personnel↗
PubMed1989

Transferrin receptors on the surfaces of retinal pigment epithelial cells are associated with the cytoskeleton.

Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93,000. These receptors internalize 125I-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100,000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surface receptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures have no apparent association with cytoskeletal elements.

Cell Division↗
PubMed1986

Internalization and recycling of transferrin and its receptor. Effect of trifluoperazine on recycling in human erythroleukemic cells.

When human erythroleukemic (K562) cells were incubated with 25 microM trifluoperazine (TFP), a drug that inhibits both calmodulin-dependent and calcium-activated phospholipid-dependent kinases, the number of transferrin receptors detected on the cell surface was reduced to approximately half with no change in the affinity of the remaining surface receptors. Removal of the TFP from the incubation medium reversed the loss of surface receptors and they returned to the cell surface in an apparently synchronous manner. As a result, the number of receptors detected on the cell surface exceeded the original level but later returned to normal. Measurements of the total number of receptors available to transferrin in TFP-treated cells suggested that the lost receptors were not participating in the internalization and recycling pathway but instead were probably trapped at an intracellular location. However, those receptors that remained on the cell surface continued to internalize transferrin and to recycle apotransferrin to the cell surface albeit more slowly than in cells that had not been treated with TFP. Using transferrin that had been labeled with iron-59, it was found that although iron uptake was reduced in line with the diminished number of surface receptors, iron still accumulated within TFP-treated cells, suggesting that in the presence of the drug, transferrin-transferrin receptor complexes continued to migrate through an intracellular compartment that contained a low pH.

Cells, Cultured↗
PubMed1986

Preribosomal ribonucleoprotein particles are a major component of a nucleolar matrix fraction.

Biochemical and morphological studies were performed on Novikoff hepatoma ascites cell nucleolar matrix fractions prepared by deoxyribonuclease I digestion and high-molarity salt extractions essentially according to a published method [Berezney, R., & Buchholz, L. A. (1981) Exp. Cell Res. 20, 4995-5002]. The nucleolar matrix fraction was enriched in polypeptides of molecular mass of 28, 37.5, 40, 70, 72, 110 (protein C23), and 160 kDa, compared to the nuclear fraction in which polypeptides of molecular mass of 31, 33.5, 43.5, 46, 50, 56, and 59 kDa were predominant. About one-fourth of the protein, half of the RNA, and less than 4% of the DNA originally present in the nucleoli remained in the matrix fraction. Addition of single agents such as ethylenediaminetetraacetic acid, ribonuclease A, or mercaptoethanol during preparation had no significant effect on the polypeptide composition of the nucleolar matrix fraction. However, the combination of mercaptoethanol and ribonuclease A caused most of the RNA and protein to be removed, including protein C23 and the 160-kDa polypeptide, with polypeptides in the range of Mr 30 000-50 000 remaining. Electron microscopy of nucleolar matrix fractions revealed the presence of particles similar in size to the granular elements of nucleoli. However, when ribonuclease A and mercaptoethanol were included in the procedure, only amorphous material remained. Many proteins of nucleolar preribosomal RNP particles were also associated with the nucleolar matrix fraction. RNA from the nucleolar matrix fraction was enriched in sequences from 18S and 28S ribosomal RNA. These results indicate that preribosomal RNP particles are major constituents of a nucleolar matrix fraction prepared by the deoxyribonuclease I-high-molarity salt method.

Animals↗
PubMed1985

Comparison of EMT blood pressure measurements with an automated blood pressure monitor: on scene, during transport, and in the emergency department.

Auscultation or palpation of blood pressure in critically ill patients by emergency medical technicians (EMTs) can be difficult, if not impossible, because of ambient noise, motion artifact, limited access to patients, or weak pulses. Automated blood pressure monitors (ABPMs) have been designed to overcome these problems during field emergencies and patient transport. Our study compared blood pressure measurements taken by EMTs with measurements provided by a Lifestat ABPM. Measurements in emergency patients on scene, during transport, in the emergency department (ED), and in a controlled environment were compared. Measurements in the various sites were obtained from 57 patients, and provision was made for two measurements at each site. Comparison of on-scene systolic blood pressures yielded a mean absolute systolic difference of 10.46 +/- 1.42 mm Hg and a mean absolute diastolic difference of 9.33 +/- 1.32 mm Hg. During transport systolic pressures showed a mean absolute difference of 11.50 +/- 1.72 mm Hg, and diastolic pressures showed a mean absolute difference of 7.59 +/- 1.16 mm Hg. Mean absolute differences in the ED were 11.23 +/- 1.49 mm Hg systolic and 8.37 +/- 1.25 mm Hg diastolic. Ninety comparison measurements in a controlled environment yielded a mean absolute systolic difference of 8.74 +/- 0.87 mm Hg and a mean absolute diastolic difference of 7.97 +/- 0.72 mm Hg. Comparison of mean diastolic pressure differences between EMT and ABPM measurements in various settings revealed some small, but statistically significant, discrepancies that were not considered clinically relevant.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Pressure↗
PubMed1979

The interaction of lectins with the surface of differentiating erythroleukaemic cells.

Friend erythroleukaemic cells can be induced to mature along the erythroid differentiation pathway when an inducing agent such as dimethyl sulphoxide is included in the medium. In the absence of the inducing agent, the 707B line of Friend erythroleukaemic cells is highly agglutinable by the lectins concanavalin A or wheat germ agglutinin. However, 48 h after the induction of differentiation, there is a marked decrease in the agglutination of the cells in the presence of either lectin. This suggests that early in differentiation a change occurs in the cell membrane preceding the onset of globin synthesis which starts approximately 72 h after induction. The change in agglutination by concanavalin A also occurs in the presence of reagents which do not induce haemoglobin synthesis in the 707B line of Friend erythroleukaemic cells but which are able to stimulate the synthesis of this protein in other erythroleukaemic cell lines. The reduction in the agglutinability of the differentiating cells does not seem to result from a reduction in the number of concanavalin A receptors on the cells, nor does it reflect a change in the clustered distribution of concanavalin A receptors in the differentiating cells. Both the control and dimethyl sulphoxide-induced cells show a similar patchy distribution of ferritin-labelled concanavalin A when examined by electron microscopy. Polyacrylamide gel electrophoresis shows little change in the total pattern of protein synthesis by control and differentiating cells when pulse-labelled with [35S] methionine. However, use of 125I-labelled concanavalin A to stain polyacrylamide gels, on which the total proteins of control and differentiating cells had been separated, revealed a profound change in the composition of the concanavalin A-binding proteins. The control, undifferentiated cells contained eleven or more classes of concanavalin A-binding glycoproteins, many of which stained to a lesser degree as the cell density increased. After the onset of differentiation, 2 new concanavalin A-binding glycoproteins appeared within 48 h. One of these proteins has a molecular weight in excess of 180 000 while the other migrated with an apparent molecular weight of approximately 100 000. After erythroid differentiation had progressed for 120 h, these newly synthesized glycoproteins became the major concanavalin A-binding proteins of the erythroleukaemic cells.

Cell Aggregation↗