Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Asymmetric distribution of anionic sites on rat liver nuclear membranes.

The labelling characteristics of isolated rat liver cell nuclei was studied using polycationized ferritin as an ultrastructural probe for anionic sites. At low concentrations of the marker the nuclear surface was partly labelled eg. at sites of nuclear annuli. At high probe concentrations the entire cytoplasmic surface of the outer nuclear membrane bound ferritin particles. On the other hand, the cisternal surfaces of nuclear membranes could not be labelled although in parallel experiments Concanavalin A-ferritin bound to the cisternal surface of both nuclear membranes indicating free access of ferritin particles to the perinuclear space. The results indicate that nuclear membranes show a distinct vectorial asymmetry in respect to the presence of anionic surface sites.     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

Basement membrane proteoglycans and anionic sites in the fetal rat kidney during late gestation

Proteoglycans were studied in developing rat fetal kidney using cytological and biochemical techniques. These compounds were detected with immunoperoxidase in the nephron basement membranes from the earliest stages of differentiation. In the glomerular basement membrane, immunostaining appeared as both diffuse and granular deposits, as long as this membrane consisted of loose material; however, as soon as a three-layered membrane had formed, staining was confined to the laminae rarae as regularly arranged granules. The same pattern of staining was observed during differentiation of the basement membrane of the proximal tubule. In Bowman's capsule, immunostaining appeared as granules, which were sparsely distributed in the developing glomerulus and then regularly lined the stacked laminae when differentiation was complete. In all basement membranes, anionic sites (disclosed by polyethyleneimine) were colocated with immunostained granular deposits. Total glycosaminoglycan content gradually increased from the beginning of metanephros development to birth. During this period, the relative proportions of glycosaminoglycans changed: heparan sulfate increased and hyaluronic acid decreased as differentiation proceeded. The possible relationship between morphological observations and biochemical changes in glycosaminoglycan content is discussed.     

Genetic systems of red cell blood groups in goats.

Evidence is presented for six genetic systems of goat red cell blood groups. The A system presently consists of one specificity, two alleles, two phenotypes (A1 and no-A1) and appears to be homologous to the A system of sheep. The B system, like its homologue in sheep, is very complex. Fourteen of 21 specificities detected in the present study, i.e. B2, B3, B4, B5, B7, B8, B9, B10, B11, B14, B15, B16, B17 and B20, belong to the B system which involves a large number of phenogroups (31 different B phenogroups identified in 26 sires). Because of their homology with sheep C and R systems, two other genetic systems of goat blood groups are named C and R respectively. Each of the two goat systems is presently a one blood group specificity, two phenotype (C12 and no-C12; R and no-R detectable on the red cells) two allele system. Two specificities, namely E6 and E18, belong to a genetic system called E in which four phenotypes are under the control of two alleles codominant and one recessive at a single locus. The F system involves but a single pair of alleles and two phenotypes (F19 and no-F19). Because of its low frequency in the goats tested, the X13 specificity remains unassigned.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.     

A high red blood cell distribution width predicts failure of arteriovenous fistula

In hemodialysis patients, a native arteriovenous fistula (AVF) is the preferred form of permanent vascular access. Despite recent improvements, vascular access dysfunction remains an important cause of morbidity in these patients. In this prospective observational cohort study, we evaluated potential risk factors for native AVF dysfunction. We included 68 patients with chronic renal disease stage 5 eligible for AVF construction at the Department of General and Vascular Surgery, Central Clinical Hospital Ministry of Internal Affairs, Warsaw, Poland. Patient characteristics and biochemical parameters associated with increased risk for AVF failure were identified using Cox proportional hazards models. Vessel biopsies were analyzed for inflammatory cells and potential associations with biochemical parameters. In multivariable analysis, independent predictors of AVF dysfunction were the number of white blood cells (hazard ratio [HR] 1.67; 95% confidence interval [CI] 1.24 to 2.25; p<0.001), monocyte number (HR 0.02; 95% CI 0.00 to 0.21; p?=?0.001), and red blood cell distribution width (RDW) (HR 1.44; 95% CI 1.17 to 1.78; p<0.001). RDW was the only significant factor in receiver operating characteristic curve analysis (area under the curve 0.644; CI 0.51 to 0.76; p?=?0.046). RDW>16.2% was associated with a significantly reduced AVF patency frequency 24 months after surgery. Immunohistochemical analysis revealed CD45-positive cells in the artery/vein of 39% of patients and CD68-positive cells in 37%. Patients with CD68-positive cells in the vessels had significantly higher white blood cell count. We conclude that RDW, a readily available laboratory value, is a novel prognostic marker for AVF failure. Further studies are warranted to establish the mechanistic link between high RDW and AVF failure.     

A simple method for determination of red blood cell mechanical fragility in the rat

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.     

Control of red blood cell mass during spaceflight

Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.     

High red blood cell distribution width and preclinical carotid atherosclerosis

Abstract

Background: Preclinical carotid atherosclerosis is associated with future risk of stroke. Data regarding the correlation between carotid atherosclerosis and biomarkers, which might predict the risk for the disease has been inconsistent and conflicting. Red blood cell distribution width (RDW) is also related to adverse clinical outcomes. Studies examining the relationship between RDW and preclinical and clinical carotid atherosclerosis were non-conclusive.

Objective: To study the association between RDW and preclinical carotid atherosclerosis in a large heterogeneous cohort.

Methods: Patients underwent Doppler ultrasound of the common carotid artery and Carotid Intima Media Thickness (CIMT). Advanced CIMT software analyzed over 100 samples in each exam. Blood samples for RDW were obtained on the same day. Logistic regression was used to evaluate the correlation between RDW and preclinical carotid atherosclerosis.

Results: Five hundred and twenty-two consecutive patients were included, with a mean age of 6.6?±?11. A cut-off value of 14.1% was used to differentiate between high and low RDW groups. The higher RDW group (RDW above 14.1%) was significantly older and with more cardiovascular risk factors. In a multivariate analysis, in all the patients including those treated by lipid modifying therapies, high RDW was significantly associated with advanced CIMT (OR?=?2.35, CI 95% 1.28–4.30, p?=?0.006). This association remained significant in subgroups of non-diabetic patients as well as patients not treated by lipid modifying drugs. RDW was also associated with significant carotid artery stenosis (OR?=?1.77, CI 95% 1.12–2.82, p?=?0.015).

Conclusions: High RDW correlates with increased risk for preclinical and clinical carotid atherosclerosis.     

Velocity distribution on the membrane of a tank-treading red blood cell

The kinematics of an area-conserving tank-treading disk-shaped red blood cell membrane is studied using the stream function method suggested by Secomb and Skalak (Q. Jl Mech. appl. Math. 35, Pt 2, 233–247, 1982). Two simple area-conserving velocity fields are superimposed to satisfy the continuity condition at the curved edges of the disk. A differential equation for the trajectory of any material point of the membrane is derived. The requirement of synchrony of the cycle for all membrane points leads to an integral equation which determines a magnitude function. An approximate solution is made possible by assuming small trajectory deflections.     

Classification and localization of hemoglobin binding sites on the red blood cell membrane.

The binding of hemoglobin to the red cell membrane was characterized over a wide range of free hemoglobin concentrations by measurement of membrane bound and supernatant hemoglobin. Scatchard analysis of the binding data revealed two classes of sites: high affinity sites with a binding constant of 1 X 10(8) M-1 and 1.2 X 10(6) sites per cell, and a second, low affinity class of sites with a binding constant of 6 X 10(6)M-1 and 6 X 10(6) sites per cell. The low affinity sites are shown to be nonspecific and appear to be a result of the ghost preparation. The high affinity sites are shown to be specific to the inner surface of the red cell membrane. The competition of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase suggests band III proteins as a potential binding site for hemoglobin.     

Studies of the sheep red blood cell receptor using anti-lymphocyte antibodies

Human thymus derived lymphocytes (T cells) interact with sheep red blood cells (SRBC) to form rosettes. We wanted to determine whether the lymphocyte's receptor for SRBC is associated with serologically detectable cell surface antigens. Antisera were prepared by immunizing horses with either fresh human thymus (ATG) or with B lymphocytes from an established lymphoid cell line in culture (ALG). ATG, ALG or Concanavalin A (Con A) were added to lymphocyte preparations to determine their effect on rosetting. The results showed that ATG inhibited rosettes in a dose dependent manner. In contrast, both the Con A and ALG had no effect. By immunofluorescence, Con A and ALG staining cells were able to form rosettes. ATG staining cells were unable to form rosettes. Removal of the ATG receptor by capping could not restore the rosette forming capacity suggesting that inhibition was not due to steric hindrance. We conclude that antibody directed against T cells but not B cells binds to surface antigens which appear to be identical with or in close proximity to the specific SRBC receptor.     

Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes

Summary Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid), SITS (4-acetamido-4-isothiocyano-stilbene-2-2-disulfonic acid), and Zn⁺⁺, and SH reactive Hg⁺⁺ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na⁺ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifier decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na⁺ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg⁺⁺ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn⁺⁺ modify different amino sites. First, simultaneous modification with TNBS and Zn⁺⁺ results in an increase in Na⁺ permeability equal to the sum of their individual effects. Secondly, Zn⁺⁺ causes an increase in Na⁺ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn⁺⁺ modification is opposite that for TNBS modification. These pH experiments suggest that Zn⁺⁺ enhances Na⁺ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper sulfhydryl sites in the sodium channels.     

Basal lamina anionic sites in the embryonic submandibular salivary gland: resolution and distribution using ruthenium red and polyethyleneimine as cationic probes

The basal lamina of the embryonic submandibular epithelium is a dynamic compartment of the extracellular matrix required for branching morphogenesis. A transmission electron microscopy (TEM) structural analysis of the basal lamina, at a time of intense branching activity, was conducted, comparing standard glutaraldehyde-fixed preparations with ones that included tannic acid in the primary fixative, and comparing anionic site resolution and distribution with two cationic probes, ruthenium red (RR) and polyethyleneimine (PEI). Standard TEM revealed a conventional basal lamina structure, with a lamina densa, a lamina lucida interna and a lamina lucida externa. Fine filaments emanated from the lamina densa, traversing both lamina lucidae. Tannic acid revealed approximately 35 nm diameter electron-dense particles in the lamina densa with a spacing repeat of approximately 45 nm. Basal lamina anionic sites were resolved as approximately 26 nm diameter RR-particles and approximately 50 nm diameter PEI-particles, present in the lamina lucida interna and associated with the lamina lucida externa. RR-particle linear spacing was 70 nm in the externa and 50 nm in the interna, while the PEI-particle spacing repeat was 90 nm in both compartments. Binding of both probes was blocked by testicular hyaluronidase or chondroitinase treatment, a result suggesting that the anionic sites were chondroitin sulfate proteoglycan, hyaluronic acid, or both. The greater particle spacing observed with PEI was not simply a physical limitation resulting from the average PEI particle diameter being almost twice that of RR particles, since PEI-resolved anionic sites on interstitial collagen were much more closely spaced (approximately 60 nm) than RR-resolved sites (approximately 105 nm).(ABSTRACT TRUNCATED AT 250 WORDS)