Biochemical polymorphism and the 2,3-diphosphoglycerate in the sheep red blood cells

There was no significant difference in the level of 2,3-DPG in the red blood cells of sheep of different haemoglobin types (Hb A and Hb B) or potassium types (HK and LK). However, low glutathione (GSHL) sheep had significantly higher (p less than 0.01) level of 2,3-DPG in their red blood cells than high glutathione (GSHH) sheep. There was also significant effect of interactions between glutathione, haemoglobin and potassium types (p less than 0.05) and glutathione and haemoglobin types (p less than 0.01) on red cell 2,3-DPG levels.     

Levels of glutathione and 2,3-diphosphoglycerate in the red blood cells of Australian Aborigines

There were no significant differences in packed cell volume (PCV) and red cell 2,3-diphosphoglycerate (2,3-DPG) levels in Australian Aborigines and Caucasians. A highly significant negative correlation was found between PCV and 2,3-DPG in both Aborigines (r = 0.251; n = 231) and Caucasians (r = 0.435; n = 227). Levels of reduced glutathione (GSH) in the red blood cells of Aborigines were significantly lower (P < 0.001) compared to those of Caucasians. There was a significant negative correlation between PCV and GSH in both the groups; (Aborigines r = -0.637, n = 115; Caucasians r = 0.388, n = 111).     

The adaptation of the fetal red cells of newborn lambs to extrauterine life: the role of 2,3-diphosphoglycerate and and adult hemoglobin

The purpose of this study was to determine the interrelationship of the rise and fall of 2,3-diphosphoglycerate (DPG) with the increase in adult hemoglobin and the decrease in red cell oxygen hemoglobin affinity after birth in normal lambs. It was found that the mean maximum DPG level was 26.71 +/- 4.98 mol/g Hb at 7.5 +/- 1.1 days. At the same time the mean P50 and adult hemoglobin level was 27.0 +/- 1.4 mm Hg and 31.1 +/- 11.i%, respectively. In the individual lambs, the level of their maximum DPG correlated inversely with the amount of adult hemoglobin (r-0.77, P less than 0.05). Once the DPG began to decrease, there was an inverse correlation between the DPG and the adult hemoglobin present in the red cell (r = 0.68, P less than 0.001). It appeared that the rise in DPG postanatally is only a compensatory mechanism until an adequate amount of adult hemoglobin is present. This fact was borne out by the second part of the study in which exchange transfusions with adult red cells were performed on five newborn lambs during the first 24 hr after birth and aborted the rise in DPG.     

Tricyclic antidepressant-induced lipidosis in human peripheral monocytes in vitro, as well as in a monocyte-derived cell line, as monitored by spectrofluorimetry and flow cytometry after staining with Nile red

Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.     

Comparison of 5-fluorouracil pharmacokinetics in whole blood, plasma, and red blood cells in patients with colorectal cancer

STUDY OBJECTIVE: To compare 5-fluorouracil (5-FU) pharmacokinetics in whole blood, plasma, and red blood cells in patients with colorectal cancer. DESIGN: Prospective, unblinded observational study in consecutive patients. SETTING: Large regional teaching hospital. PATIENTS: Five patients with colorectal cancer. INTERVENTIONS: Patients received folinic acid 200 mg/m2 intravenously over 2 hours, followed by 5-FU 600 mg/m2 intravenous bolus over 30 minutes, then 5-FU 600 mg/m2 intravenous infusion over 22 hours, administered on days 1 and 2. This 48-hour cycle was repeated every 14 days. MEASUREMENTS AND MAIN RESULTS: Concentrations of 5-FU in whole blood, plasma, and red blood cells were determined by high-performance liquid chromatography. ADAPT II was used for pharmacokinetic computations. The optimum model was determined for each matrix by calculating Akaike's information criteria values. Concentrations of 5-FU in whole blood were 106-115% of simultaneous plasma concentrations (median 112%), and packed red blood cell levels were 5-17% of plasma concentrations (median 11%). The drug's concentration-time profile was similar in the three matrices. The drug is reported to be unstable in whole blood, and red blood cell 5-FU concentrations were near the limit of detection (10 ng/ml), supporting plasma as the preferred matrix for therapeutic drug monitoring studies. Six pharmacokinetic models were fitted to the 5-FU individual data sets to determine the best curve fit. The optimal model for whole blood and plasma data sets was one compartment with both linear and nonlinear elimination models; a one-compartment model with nonlinear elimination provided the best curve fit for 5-FU in red blood cells. A two-compartment model with nonlinear elimination gave a similar degree of curve fit for plasma 5-FU as the one-compartment model with both linear and nonlinear elimination. CONCLUSIONS: These pharmacokinetic results provide the basis for further investigation into the ability to correlate 5-FU systemic exposure with clinical drug activity.     

Progressive transmural electrographic, myocardial potassium ion/sodium ion ratio and ultrastructural changes as a function of time after acute coronary occlusion

The progressive transmural electrographic, biochemical and ultrastructural changes as a function of time after acute coronary occlusion were systematically assessed in eight dogs. Transmural plunge electrodes with poles 1 mm apart were placed in the ischemic and nonischemic zones, and coronary occlusion was maintained for 4 hours. Transmural full thickness biopsy specimens were obtained from each zone for electron microscopy before, and 1 and 4 hours after occlusion. Endocardial and epicardial layers were also obtained for assessment of myocardial potassium ion (K+) and sodium ion (Na+) concentrations. Before coronary occlusion, local Q waves were recorded an average depth of 1.0 +/- 0.34 mm from the endocardial surface. After 1 hour of occlusion, Q waves appeared at an average depth of 3.8 +/- 0.67 mm and progressed to a depth of 5.2 +/- 0.7 mm at 2 hours, 6.2 +/- 0.5 mm at 3 hours and 7.0 +/- 0.5 mm at 4 hours. After 1 hour, ultrastructural changes of early ischemia, including a decrease in glycogen and mild mitochondrial swelling, were seen in the endocardial layer; the epicardial layer showed normal morphologic features. After 4 hours, the endocardial layer showed well developed ischemic changes marked by the loss of mitochondrial cristae, vacuolization, the appearance of amorhopous mitochondrial cristae, vacuolization, the appearance of amorphous mitochondrial densities, an increase in interfibrillary space and the appearance of I bands. In contrast, the epicardial layer at this time showed only early ischemic changes. At the end of 4 hours, the endocardial layer showed a marked decrease in myocardial K+ concentration and an increase in Na+ concentration leading to complete reversal of K+/Na+ ratio (0.7 +/- 1.0; P less than 0.001). In the epicardial layer, a smaller decrease in K+ concentration and an increase in Na+ concentration occurred, resulting in a diminution but not a reversal of K+/Na+ ratio (1.4 +/- 0.2; P less than 0.005). Thus, the dynamic evolution of an acute myocardal infarction involves a sequential progression from endocardium to epicardium as a function of time, resulting in an epicardial "border zone" in the early stages after acute coronary occlusion.     

Distribution of sulfonamides and sulfonamide potentiators between red blood cells, proteins and aqueous phases of the blood of different species

The uptake of sulfonamides and sulfonamide potentiators by plasma albumin, red blood cells and hemoglobin of man, ox, rabbit and mouse has been determined. From the figures obtained the distribution of these compounds between cells, macromolecules and aqueous phases of blood has been calculated. In most cases more than 50% of a total sulfonamide in blood is bound to plasma albumin. The time necessary for establishment of concentration equilibrium of the drugs investigated between erythrocytes and surrounding medium varies between a few seconds and several minutes, differences between the species being encountered. Hemoglobin binding of the drugs is much smaller than albumin binding. Nevertheless, drug concentration within the erythrocytes is generally higher than in the surrounding medium.     

Contents of D-lactate and its related metabolites as well as enzyme activities in the liver, muscle and blood plasma of aging rats

Regulatory elements within the promoter of the pollen-specific NTP303 gene from tobacco were analysed by transient and stable expression analyses. Analysis of precisely targeted mutations showed that the NTP303 promoter is not regulated by any of the previously described pollen-specific cis-regulatory elements. However, two adjacent regions from -103 to -86 bp and from -86 to -59 bp were shown to contain sequences which positively regulated the NTP303 promoter. Both of these regions were capable of driving pollen-specific expression from a heterologous promoter, independent of orientation and in an additive manner. The boundaries of the minimal, functional NTP303 promoter were determined to lie within the region -86 to -51 bp. The sequence AAATGA localized from -94 to -89 bp was identified as a novel cis-acting element, of which the TGA triplet was shown to comprise an active part. This element was shown to be completely conserved in the similarly regulated promoter of the Bp 10 gene from Brassica napus encoding a homologue of the NTP303 gene.     

Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr-loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.     

Activities of superoxide dismutase and glutathione peroxidase in the red cells of Japanese acatalasemia blood

Trichophyton violaceum was cultured on a Sabouraud's dextrose agar slants for freeze-fracture replication. It was cryoprotected with glycerol and sucrose. The surface structure of the cell wall showed a rodlet pattern. Invagination-like craters were observed in the plasmalemma. The nuclear envelope with pores, mitochondria, and vacuoles were clearly shown. Three-dimensional views of the fine structure were presented.     

Determination of tripamide and its metabolites in plasma, red blood cells and urine by high-performance liquid chromatography

A procedure for the determination of tripamide and its hydroxylated metabolites in plasma, red blood cells and urine by reversed-phase high-performance liquid chromatography is described. The concentrations in red blood cells showed a monophasic decline and the half-life was 9.5 h. The concentration in red blood cells was markedly higher than that in plasma, showing that 95-98% of the drug is present in whole blood, after a dose of tripamide (90 mg) in man. The specificity and sensitivity of this procedure appear to be satisfactory for pharmacokinetic studies.     

Uptake of chromate in human red blood cells and isolated rat liver cells: the role of the anion carrier

The transport of [51Cr]chromate into human erythrocytes and isolated rat hepatocytes has been investigated. It was found that uptake in both cell types could be inhibited by the established anion carrier inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The uptake was very fast, and in kinetic studies a very low Km was found for both cell types, indicating either a high affinity of chromate for the carrier, and/or, more probably, an efficient intracellular reduction and trapping of 51Cr. The transport capacity, however, was of the same magnitude as for physiological substrates, such as lactate and sulfate. The uptake was temperature dependent and the activation energy was of the same magnitude as that for the physiological substrates. The uptake could be partly inhibited by high levels (mmol l-1) of lactate, pyruvate or sulfate. The uptake rate was greatly increased at lower pH (6.0 versus 7.4) which could indicate transport of the HCrO4- form or an increased intracellular rate of CrVI reduction. The results showed efficient uptake of 51CrO4(2-) by erythrocytes and hepatocytes. They were consistent with a mechanism of uptake which involved the cell membrane anion-exchange carrier in the transport and trapping of 51Cr within the cell.     

Prenatal dexamethasone causes oligonephronia, sodium retention, and higher blood pressure in the offspring

OBJECTIVES: It is not known whether changes in the biomechanics of elderly gait are related to aging per se, or to reduced walking speed in this population. The goals of the present study were to identify specific biomechanical changes, independent of speed, that might impair gait performance in healthy older people by identifying age-associated changes in the biomechanics of gait, and to determine which of these changes persist at increased walking speed. DESIGN: Stereophotogrammetric and force platform data were collected. Differences in peak joint motion (kinematic) and joint moment and power (kinetic) values between healthy young and elderly subjects at comfortable and increased walking speed were measured. SETTING: A gait laboratory. SUBJECTS: Thirty-one healthy elderly (age 65 to 84 years) and 31 healthy young adult subjects (age 18 to 36 years), all without known neurologic, musculoskeletal, cardiac, or pulmonary problems. MAIN OUTCOME MEASURES: All major peak kinematic and kinetic variables during the gait cycle. RESULTS: Several kinematic and kinetic differences between young and elderly adults were found that did not persist when walking speed was increased. Differences that persisted at both comfortable and fast walking speeds were reduced peak hip extension, increased anterior pelvic tilt, and reduced ankle plantarflexion and ankle power generation. CONCLUSION: Gait performance in the elderly may be limited by both subtle hip flexion contracture and ankle plantarflexor concentric weakness. Results of the current study should motivate future experimental trials of specific hip flexor stretching and ankle plantarflexor concentric strengthening exercises to preserve and potentially improve walking performance in the elderly.     

Relation between the osmolality trend and ornithynedecarboxylase activity in red blood cells of uremic patients during hemodialytic treatment

We validated the Hypomania Interview Guide-Seasonal Affective Disorder version (HIGH-SAD) in patients with rapid cycling bipolar disorder (RCBD). Fourteen outpatients were rated on six separate occasions (total = 84 visits). On each visit the patients were rated with the HIGH-SAD and the Young Mania Rating Scale (YMRS) in a counterbalanced order. Clinical assessment was completed at the end of the visit by the treating psychiatrist. Patients were assessed as hypomanic/manic on 22 of the visits. Pearson correlation coefficient between the YMRS total scores and the HIGH-SAD total scores for those 22 visits in which patients were hypomanic/manic was r = 0.629 (P < 0.05) and for all visits was r = 0.769 (P < 0.0001). Analysis with only one rating per patient yielded a Pearson correlation coefficient of r = 0.792 (P < 0.0004). We found that the HIGH-SAD was a valid scale for the measurement of hypomania in patients with RCBD. However, the scale does not differentiate hypomania from mania in this group of patients.     

Two receptor tyrosine phosphatases of the LAR family are expressed in the developing leech by specific central neurons as well as select peripheral neurons, muscles, and other cells

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. We have cloned and sequenced two Hirudo medicinalis rPTPs that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. In addition, HmLAR2 is expressed on the cell body, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, we hypothesize that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. Furthermore, we hypothesize that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. Lastly, we propose that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2.     

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.