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Biomedical subjects

S Chien

Publications and source records attributed to S Chien.

At least 19 recordsLinked to original sources
PubMed1992

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Amino Acid Sequence↗
PubMed1992

Deformability measurements on individual sickle cells using a new system with pO2 and temperature control.

Although the rheologic behavior of sickle erythrocytes (SS cells) is highly dependent on oxygen tension (pO2) and temperature, very little data exist regarding the effects of deoxygenation and reoxygenation on the rheology of "individual" SS cells at body temperature. We have devised and assessed a new experiment system, in which micropipette aspiration can be performed on individual cells in a constant-temperature chamber that has ports for changing media with different pO2 (effected in 30 to 120 seconds) and sensing probes for monitoring pO2 and temperature. This system enabled us to simultaneously alter and monitor pO2 at 37 +/- 0.5 degrees C, and to monitor and study a single cell under microscopic observation. The static rigidity (E) and dynamic rigidity (eta) of individual SS cells were determined by repeated aspirations of the same cell under various pO2. With stepwise reductions in pO2, E and eta showed no significant changes before sickling, but once sickled, their values markedly increased by 10(2)- to 10(3)-fold concomitantly with morphologic alteration of the cell. Thus, the deformability of a single SS cell behaves in an "all or none" manner at a critical pO2, and earlier studies on the effect of deoxygenation on the rheology of SS cell suspensions probably reflect the overall behavior of SS cells with widely distributed critical pO2.

Anemia, Sickle Cell↗
PubMed1992

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E1 (PGE1) on platelets is mediated through the PGE1 receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE1 and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 microns. The time course of the extension length (Dp, in microns) of the platelets in response to aspiration with a negative pressure (delta P) of 5 cm H2 O (delta P x Rp = 0.15 dynes/cm) was analyzed. PGE1 treatment (0.1 microM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE1-treated)/Dp/Rp (control), was significantly reduced to 0.90 +/- 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE1-treated platelets had a higher [cAMP]i than controls. Platelets treated with PGE1 or dbcAMP showed a reduced [Ca2+]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE1 and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]i and low [Ca2+]i after PGE1 treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.

Alprostadil↗
PubMed1992

Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.

Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.

Amino Acid Sequence↗
PubMed1992

The Benjamin W. Zweifach Award Lecture. Blood cell deformability and interactions: from molecules to micromechanics and microcirculation.

UNLABELLED: RBC deformability and PMN-endothelial interaction have been used as two examples to illustrate how recent investigations have generated information from molecules to micromechanics and the microcirculation, and how some of the results can be used to understand the physiology and pathophysiology in man. While this presentation on the molecular basis of microcirculatory events is focused on micromechanics, active work is being conducted to establish the molecular basis of many other microcirculatory processes. These include endothelial transport, vascular smooth muscle activity, neurohumoral control of the microcirculation, and angiogenesis, as well as some of the disease states such as ischemia, shock, and cancer. The field is still in its infancy, and we are only seeing the tip of the iceberg. Further developments in this fertile interdisciplinary field will allow us to gain further understanding of the molecular basis of the microcirculatory processes in health and disease. IN CONCLUSION: (i) The deformability and interactions of blood cells play a significant role in microcirculatory dynamics. (ii) Modern biological approaches have provided insights into the molecular bases of blood cell deformability and interactions. (iii) Understanding of the physiology and pathophysiology of the microcirculation requires the application of knowledge derived from molecular and cell biological studies to the in vivo microcirculatory preparations. (iv) Bridging of the new biology and in vivo microcirculatory investigations represents a great challenge and a golden opportunity for microcirculation researchers.

Anemia, Sickle Cell↗
PubMed1992

Locomotion forces generated by a polymorphonuclear leukocyte.

There have been very few studies which have measured the physical forces generated by cells during active movements. A special micropipette system has been designed to make it possible to observe cell motion within the pipette and to apply a pressure to counter the chemotactic migration of the cell. This provides a direct measure of the locomotion force generated by the cell. The average velocity of forward motion is 0.33 microns/s in the absence of counter-pressure. The application of a positive counter-pressure (C-P) causes a decrease in the velocity of the forward motion of the cell. At 17 cm H2O of C-P, the cell velocity drops to zero and even moves backward with a higher C-P. The results show that the decrement of velocity is linearly related to the magnitude of the C-P with a complete stoppage at a pressure of 17 cm H2O which corresponds to a force of 0.003 dyn. The maximum work rate of the cell is approximately 2.5 x 10(-8) erg/s.

Biomechanical Phenomena↗
PubMed1992

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules.

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.

Antigens, CD↗
PubMed1992

Long-term nicotine exposure increases aortic endothelial cell death and enhances transendothelial macromolecular transport in rats.

Repeated endothelial cell injury has been suggested as an initiating factor in atherogenesis. Dying or dead endothelial cells have been shown to make significant contributions to the local enhancement of transendothelial macromolecular transport. Since cigarette smoking is one of the major risk factors for atherosclerosis, we examined the hypothesis that smoking accelerates atherogenesis by increasing the frequency of endothelial cell death and hence transendothelial macromolecular transport. Sixteen male Sprague-Dawley rats were given nicotine at a weight-adjusted dose of 5 mg/kg body wt per day in their drinking water over a period of 6 weeks. A group of 16 age-matched male Sprague-Dawley rats not exposed to nicotine and maintained over the same time period served as the control group. In en face preparations of thoracic aorta, immunoglobulin G-containing dying or dead endothelial cells were identified by the indirect immunoperoxidase method, and endothelial leakage to Evans blue-albumin (EBA) complexes (5 minutes after intravenous injection) was visualized by fluorescence microscopy. The results showed that in nicotine-treated rats, 51% of dead endothelial cells were associated with EBA leakage, which was responsible for 57% of total EBA leaky foci. Both the frequency of endothelial cell death (0.94 +/- 0.11% versus 0.40 +/- 0.04%, p < 0.0001 by two-tailed, unpaired Student's t test) and the number density of EBA leaky foci (6.45 +/- 1.23/mm2 versus 3.30 +/- 0.49/mm2, p < 0.05 by two-tailed, unpaired t test) were significantly greater in nicotine-treated rats than in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Force contribution of the LFA-1/ICAM-1 complex to T cell adhesion.

Little is known in quantitative terms about forces between cells generated during adhesion and recognition, or about the contribution of any one set of molecular associations to the development of these forces. To determine the forces involved in adhesion dependent on lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1), we have measured the junctional avidity between single cell pairs consisting of a cloned T cell that expresses LFA-1 and a fibroblast cell that expresses MHC class II molecules and ICAM-1 after transfection. Micromanipulation was used to induce conjugation of cell pairs and to determine the force required to separate the conjugate. T cell adhesion to three related fibroblast cell lines was compared: the parent line that does not express ICAM-1 or other LFA-1 counter-receptors, and two transfectants that have high and moderate levels of surface ICAM-1 expression. The force needed to separate the conjugates varied with the fibroblast ICAM-1 expression levels. The T cell adhesion to ICAM-1-expressing fibroblasts was strong, and the critical separation stresses measured for the three cell lines were 1.4 x 10(3) dyn/cm2 (1 dyn=10(-5) N) for the ICAM-1-negative fibroblast, 4.98 x 10(3) dyn/cm2 for the fibroblast with a moderate level of ICAM-1 expression, and 6.25 x 10(3) dyn/cm2 for the fibroblast line with the highest ICAM-1 expression. The dependence of adhesion strength on the LFA-1/ICAM-1 complex was confirmed by the use of blocking antibodies, which showed the contribution from the interaction of CD4/MHC class II to be negligible.

Animals↗
PubMed1992

Influence of temperature on rheology of human erythrocytes.

The effect of variation in temperature on the rheological properties of human red blood cells was studied by determining the cell deformation in response to aspiration pressure applied via a micropipette. The time history of the deformation was analyzed by the use of a Voigt viscoelastic model consisting of an elastic element (E) in parallel with a viscous element (eta). Viscosity values were obtained during the initial rapid deformation phase (phase I), the later slow deformation phase (phase II), and the recovery phase. With a rise in temperature from 0 to 45 degrees C, both E and eta values decreased. A thermotropic transition temperature was found at approximately 21 degrees C for phase I viscosity and at approximately 9 degrees C for viscosity in the recovery phase, but not for phase II viscosity. These responses to temperature variations provide insights into the viscoelastic properties of the erythrocyte membrane.

Elasticity↗
PubMed1992

Role of intercellular junctions in the passage of horseradish peroxidase across aortic endothelium.

BACKGROUND: The manner in which molecules are transported across the arterial endothelial layer has been a subject open to much interpretation and controversy. Further elucidation and clarification of these mechanisms are of primary interest. EXPERIMENTAL DESIGN: To investigate the ultrastructural features of arterial endothelial junctions and to evaluate their functional roles as a transendothelial pathway for macromolecular transport, experiments were performed on the thoracic aortae of adult male Sprague-Dawley rats by using the ultrathin serial sectioning technique and horseradish peroxidase (HRP). The aorta was perfusion-fixed with or without prior intravenous injection of HRP. RESULTS: The intercellular clefts exhibited a great deal of variety in shape, being linear, winding, interdigitated, irregular and/or dumbbell-shaped in appearance. Besides the typical 20-nm width encountered at the uniform region of intercellular clefts, local widenings (up to several hundred nm) were quite common. The arterial endothelial junctions were highly organized. Junctional elements, including tight junctions and gap junctions, were frequently present in the same intercellular cleft, even on the same plane of sectioning. Sometimes, gap junctions were found without tight junctions, but the intercellular clefts were rarely obliterated by tight junctions alone. Some intercellular clefts were not obliterated by either gap or tight junctions, and HRP was found to reach the subendothelial space by passing through these junctionless clefts. Densitometric determination of the HRP concentration profile in such junctionless clefts showed a decreasing gradient from the luminal to the abluminal front. The serial sections provided evidence that the apparently free vesicles were actually plasmalemmal membrane invaginations open to the luminal or abluminal front in the arterial endothelium. CONCLUSIONS: The present study showed that the junctionless normal endothelial clefts, in addition to the transiently open junctions surrounding mitotic cells, might provide a significant pathway in the transendothelial transport of macromolecules with the size of HRP.

Animals↗
PubMed1991

Gender differences in left ventricular anatomy, blood viscosity and volume regulatory hormones in normal adults.

Gender differences in left ventricular (LV) anatomy, whole blood and plasma viscosity, and blood volume regulatory hormones were studied in 110 normotensive employed adults (28 black and 34 white men [mean age 51 +/- 12 years], 20 black and 28 white women [mean age 53 +/- 12 years]). LV mass and wall thicknesses were positively related to whole blood viscosity, primarily because of higher values of both variables in men. LV chamber size was inversely related to hematocrit and to blood viscosity (p less than 0.002) in women but not in men. Whole blood viscosity increased with age in men (p less than 0.01), but tended to decrease in women; older women also had better LV function, larger LV chambers, and a trend toward increasing LV mass. Atrial natriuretic factor increased with age in women but not in men (r = 0.60, p less than 0.001), and plasma renin activity decreased (r = -0.35, p less than 0.02). Thus, in women, increase in LV chamber size with age and associated changes in LV systolic function, atrial natriuretic factor levels and plasma renin activity suggest plasma volume expansion related to the aging process. These findings also suggest that an increase in LV volume load with age may contribute to previously reported increases in LV mass in older women.

Adolescent↗
PubMed1991

Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.

Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that might regulate cardiac genes during growth and hypertrophy. One of the first detectable changes in cardiac gene expression is the activation of a program of immediate early gene expression, which is distinct for the hypertrophic response, and is conserved in multiple models of both in vivo and in vitro hypertrophy. Diverse and distinct hormonal stimuli have been documented to activate several features of the hypertrophic response, including several autocrine and paracrine factors. Although the signaling mechanisms that link these factors with the activation of cardiac gene expression are unclear, recent studies suggest that the activation of protein kinase C may represent one of the most proximal common events in this signaling cascade. The activation of cardiac target genes induces a program of embryonic gene expression, including the atrial natriuretic factor (ANF) gene. The cis sequences that mediate cardiac-specific and inducible expression of an embryonic marker gene (ANF) can be segregated by studies in both cultured cell models and in vivo models of hypertrophy in transgenic mice, suggesting that specific sets of regulatory elements may exist for inducible expression of this class of cardiac gene responses. However, the induction of a constitutively expressed contractile protein gene (MLC-2) is mediated by a set of conserved elements that regulate both cardiac-specific and inducible expression. Finally, a subset of cardiac muscle genes appears to be noninducible during in vivo or in vitro hypertrophy in myocardial cells, demonstrating specificity of transcriptional activation during the hypertrophic process. The development of a bona fide in vivo pressure overload model of hypertrophy in a small animal model that can be genetically manipulated, such as the in vivo murine model recently described, should allow a rigorous analysis of the role of these specific signaling mechanisms in the activation of the responses of cardiac genes during the hypertrophic process in vivo.

Adaptation, Biological↗
PubMed1991

A mathematical model for the receptor mediated cellular regulation of the low density lipoprotein metabolism.

A prototype mathematical model for Brown and Goldstein's pioneering studies on the LDL receptor mediated pathway for the regulation of the cellular content of cholesterol has been developed in this paper. In order to analyze the essential features of this complex system quantitatively and still reflect the framework of the total system, six important processes are considered in the model. They are: (1A, B) the hydrolysis and synthesis of the LDL receptor; (2) the binding of LDL to its receptors; (3) the hydrolysis of LDL; (4) the storage of cholesteryl esters; (5) the regulation of de novo synthesis of cholesterol; and (6) the efflux of free cholesterol to the external medium. All these processes form a system to let the cells take up enough cholesterol from the external medium for their utilization and yet avoid the excessive accumulation of the lipid within the cells. The validity of the model is tested by showing that it can predict many of experimental curves obtained for human fibroblasts in tissue culture studies. The main purpose of the model is to determine how the free cholesterol level in the cell is related to the external LDL concentration and the regulatory capacity of the cells to adapt to a changing LDL environment. In addition, the model reveals an important behavior of SMC, i.e., for a slowly increasing LDL concentration in the extracellular medium, the rate of intracellular degradation of LDL will first increase and then become saturated. It is proposed based on these results that the saturation of LDL degradation by SMCs and the subsequent increase in subendothelial LDL levels in regions of high macromolecular permeability might play a vital role in the formation of the early foam cell lesion.

Cells, Cultured↗
PubMed1991

A new view of convective-diffusive transport processes in the arterial intima.

In this paper a new theoretical framework is presented for analyzing the filtration and macromolecular convective-diffusive transport processes in the intimal region of an artery wall with widely dispersed macromolecular cellular leakage sites, as proposed in the leaky junction-cell turnover hypothesis of Weinbaum et al. In contrast to existing convection-diffusive models, which assume that the transport is either 1-D, or convection is primarily in a direction normal to the endothelial surface, the present model considers for the first time the nonuniform subendothelial pressure field that arises from the different hydraulic resistances of normal and leaky endothelial clefts and the special role of the internal elastic lamina (IEL) in modulating the horizontal transport of macromolecules after they have passed through the leaky clefts of cells that are either in mitosis or demonstrate IgG labeling. The new theory is able to quantitatively explain the growing body of recent experiments in which an unexpectedly rapid early-time growth of the leakage spot has been observed and the longer time asymptotic behavior in which the leakage spot appears to approach an equilibrium diameter. The new theory also predicts the observed doubling in macromolecular permeability between EBA labeled blue and white areas when the frequency of leakage sites is doubled. This frequency for doubling of permeability, however, is an order of magnitude smaller than predicted by the author's previous model, Tzeghai et al., in which only convection normal to the endothelial surface was considered and the pressure was uniform in the intima. The longer time model predictions are used to explain the time scale for the formation of liposomes in subendothelial tissue matrix in animal feeding experiments where it has been observed that the extracellular lipid concentration rises sharply prior to the entry of monocytes into the intima.

Arteries↗
PubMed1991

Folding of red blood cells in capillaries and narrow pores.

The geometric features of red blood cells in narrow channels in vivo and in vitro were studied by electron microscopy. In rabbit myocardial capillaries about half of the red cells were folded. In polycarbonate filters with pore diameters of 2.2-4.5 microns approximately one third of the trapped red blood cells were folded. The frequency of folding did not depend on the applied pressure, which ranged from 0.1 to 8.0 cm H2O. The folding of the red blood cells in filter pores was used to estimate the bending stiffness of the membrane. An analysis based on the large deformation theory of bending of an elastic sheet was developed. Using pressures of 0.2 and 1.0 cm H2O, the bending stiffness of human red cell membranes was estimated to be approximately 2.4 - 11.6 x 10(-12) dyn-cm, which is in good agreement with other methods. A limiting radius of curvature of about 85 nm was found at higher pressures.

Animals↗
PubMed1991

Two-day preservation of major organs with autoperfusion multiorgan preparation and hibernation induction trigger. A preliminary report.

A new autoperfusion multiorgan preparation was studied in which the heart and lungs were removed with the liver, pancreas, duodenum, and both kidneys en bloc while being perfused by the heart and oxygenated by the lungs. A respirator with 50% oxygen was used for ventilation. Fresh blood, glucose, electrolytes, mannitol, and antibiotics were given through the portal vein. Fifteen mongrel dogs were used. In the study group (seven dogs), 10 ml of plasma containing hibernation induction trigger, obtained from deeply hibernating woodchucks, was given intravenously 2 hours before the operation, and 4 ml was given every 4 hours during the preservation period. In the control group (eight dogs), no hibernation induction trigger was used. Survival time in the study group ranged from 33 to 56 hours (mean 43.4 +/- 4.1 hours), longer than that of the control group, which was 9 to 31 hours (mean 16.2 +/- 2.6 hours, p less than 0.001). In the study group aortic systolic pressure ranged from 64 +/- 5 to 92 +/- 7 mm Hg, arterial oxygen tension from 180 +/- 35 to 285 +/- 66 mm Hg. Urine output ranged from 15 to 70 ml/hour. Blood urea nitrogen declined from 15.6 +/- 2.5 to 6.6 +/- 1.3 mg/dl (p less than 0.01); creatinine declined from 0.8 +/- 0.03 to 0.3 +/- 0.01 mg/dl (p less than 0.01). Severe liver congestion and premature renal failure occurred in the control group but did not occur in the study group. In the study group one lung was transplanted after 33 hours of preservation with simultaneous contralateral pulmonary artery ligation. Good lung function was maintained after transplantation. Although the exact mechanism by which hibernation induction trigger extends tissue survival time is still not clear, its effect on organ preservation is profound. This study also produced one of the longest average survival times for organ preservation.

Animals↗