Evaluation of the ability of intact platelets to accumulate acridine orange

A new approach to the evaluation of the uptake of fluorescent probes by intact cells is described. Acridine orange (AO) was used because it can be selectively accumulated by serotonin-containing granules of platelets. Analysis of the fluorescence signal allows the estimation of the relative volume of the granules and the equilibrium coefficients for AO transport across the cytoplasm and granule membranes. The following results were obtained for human and rabbit platelets: the relative volumes of the granules were 14 +/- 1% and 29 +/- 2%, the ratios of intragranular-extracellular probe concentration were 2260 +/- 382 and 30,000 +/- 5550, and the cytoplasm-extracellular medium concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.     

Lipid characterization and14C-acetate metabolism in catfish taste epithelium

Summary The catfish,Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62±1% of the total, neutrals for 38±1%. Phosphatidyl-cholines, serines and ethanolamines are the major constitutents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [¹⁴C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37°C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.This work was supported in part by NIH Research Grant No. NS-23622, NS-22620, and by the Veterans Administration.     

Satellite cells in the regenerated and regrafted skeletal muscles of rats

Summary Soleus (SOL) muscles were grafted into extensor digitorum longus (EDL) muscle beds (EDL-first-graft). Sixty days later, some mature EDL-first-grafts were regrafted into their own beds (EDL-second-grafts). Fully regenerated muscle fibers and satellite cells were observed in both types of mature grafts. The ratios of satellite cell nuclei per total nuclei (myonuclei and satellite cell nuclei) were 4.81±0.47% for EDL-2nd graft, 4.26±0.51% for EDL-1st-graft, 4.30±0.33% for control SOL, and 3.30±0.18% for control EDL. It is thought that satellite cells are required for the repeated activity of muscle fiber regeneration. The persistance of satellite cells in EDL-second-grafts suggests that satellite cells are not depleted during the first grafting, making second-grafts possible.     

Änderungen des Elektrolytgehaltes von Erythrozyten und Plasma bei nephrektomierten Ratten

Summary After two-stage nephrectomy in rats the potassium concentration in the red blood corpuscles (RBC) decreases from 10.6 ± 0.3 to 5.5 ± 0.3 mEq per 100 ml of RBC within 48 h. The decrease is accompanied by a much smaller increase in the plasma potassium concentration; the hyperkaliemia in nephrectomized rats is less pronounced than in nephrectomized dogs or anuric humans. Na⁺ in RBC increases by about 44% after nephrectomy; while there is only a very slight decrease of Na⁺ in plasma. Plasma chlorides drop from 10.92 ± 0.08 mEq/100 ml of plasma to 6.00 ± 0.81 mEq % within 48 h after nephrectomy. RBC chlorides tend to increase again after an initial drop from 5.22 ± 0.07 to 3.82 ± 0.90 mEq% within the first 8 h.     

Reduction of bovine pulmonary hypertension by normoxia,verapamil and hexaprenaline

Summary In calves with hypoxic pulmonary hypertension, resistance was reduced by 40±3% with normoxia, 19±4% with verapamil, and 60±1% with hexoprenaline. It is possible that the increased resistance during normoxia is due partly to vasoconstriction rather than solely to vascular thickening, and that the vasoconstriction is due to an abnormality in calcium metabolism by the hypertensive vasculature.Acknowledgments. We thank Rosann Glas, Mary Munroe, Diann Smith and Linda Panepinto for technical assistance. This work was supported by the Colorado Heart Association and by the National Institutes of Health Grants HL-05973 and HL-29237. Verapamil (Isoptin) was supplied by Knoll Pharmaceutical Co. Hexoprenaline (ST 1512 HCl, Ipradol) was supplied by Österreichische Stickstoffwerke Linz.     

Serum cholesterol levels in a nigerian population sample

Summary Serum cholesterol was determined in 104 healthy Nigerians (57 males and 47 females). The average cholesterol concentration was 187±72.8 mg/100 ml (mean±SD) with a range of 60–480 mg/100 ml. Cholesterol levels were higher in males than in females and increased with age. Distribution of cholesterol level was generally skew to the left.Acknowledgment. I am grateful to Mr S. O. Oguike an Mr O. Uzoechi for technical assistance. I also thank Dr. E. O. Anosike for his help in arranging for the subjects of study.     

Ultradian and other rhythms in human respiration rate

Summary Twelve male subjects had their respiration rate (RR) measured at 3 min intervals for 6 h. Although there were substantial individual differences, most subjects demonstrated a 90±15 min rhythm of RR, with several subjects also showing 60±10 min and 30±3 min rhythms.     

Effects of blood sampling,anesthesia and surgery on plasma vasopressin concentration in rats

The influence of blood sampling, anesthesia and surgery on plasma vasopressin concentration was assessed in rats. Mean plasma concentration in conscious, chronically catheterized rats was 1.4±0.1 pg/ml (n=6). This value remained constant over repeated plasma samplings in the same animals. On the other hand, decapitation increased the plasma vasopressin concentration to 6.0±2.4 (in pg/ml) (n=6), inactin anesthesia to 2.9±0.6 (n=6), anesthesia and femoral cannulation to 13.3±5.8 (n=6) and surgery for renal micropuncture to 81.3±35.0 (n=6). It is concluded that the level of circulating plasma vasopressin is highly dependent on the sampling technique and is closely related to the extent of surgery.     

Electro-osmotic and iontophoretic release of noradrenaline from micropipettes

Summary The relative contributions of electro-osmosis and iontophoresis to the electrophoretic release of noradrenaline from micropipettes were examined. Electro-osmosis was responsible for 23.1% (±5.0%) of the total rate of release.This work was supported by the Wellcome Trust. We are grateful to Dr M.N. Jones for his help. Some of the results have been communicated to the British Pharmacological Society. Dublin, July 1979.     

The regulatory peptide system of the large bowel in equine grass sickness

Summary In recent years, distinct changes in regulatory peptides have been found in a number of gastrointestinal diseases. Grass sickness is a fatal disease of horses for which the etiology has yet to be fully ascertained. In this study, the peptide-containing nerves and ganglionic and mucosal endocrine cells of the ileum, colon and rectum were investigated in horses with sub-acute or chronic grass sickness and compared with normal controls using immunocytochemistry, at both the light and electron microscopical levels, and radioimmunoassay. A substantial loss of both peptide-containing cells and nerves was found in all of the sick horses, particularly in the ileum. Electron microscopy revealed marked degeneration of nerves in the gut wall. fibers containing granules immunostained for substance P or VIP, using the immunogold staining technique, underwent extensive degranulation in grass sickness, with the formation of multiple vacuoles.Radioimmunoassay of peptide content also showed that the most drastic changes occurred in the ileum. For example, VIP content was significantly reduced from 109±19.8 (mean±SEM) pmoles/g in controls to 6.8±1.4 pmoles/g in grass sickness (p<0.001) and substance P from 65.9±8.1 to 31.3±9.5 (p<0.02). These results may have applications in the diagnosis and treatment of grass sickness.The authors gratefully acknowledge the financial support of the Wellcome Trust and the Grass Sickness Fund.     

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64±0.18 (m±SE, n=11) and 3.12±0.24 (n=11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14±3% (n=34) and 22±13% (n=10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12±0.04 (n=11) and 0.07±0.01 nmol/min/mg cell protein (n=11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.     

Synthesis and contractile activity of the C-terminal heptapeptide of substance P with N5-dimethyl glutamine in the 6-position. Active site studies

Summary The synthesis and testing of [N⁵-dimethyl-Gln⁶]-SP_5–11 showed 37±12% contractile activity relative to SP, and intrinsic efficacy 98±4%. This finding indicates that the carboxamide groups of the dual Gln⁵-Gln⁶ moiety are not equally related with the contractile response of the C-terminal heptapeptide of SP.The authors wish to express their deep appreciation to Professor H. Niedrich and Dr J. Bergmann of the Institute of Drug Research, Academy of Sciences of DDR, Berlin, for their valuable help in providing the biological data.     

Études des variations quantitatives du passage de la ferritine au niveau de la membrane basale glomérulaire en fonction du temps et sous l'influence de la théophylline

Summary A fine quantitative evaluation of ferritin aggregates in rat GBM permits to counts 5.08±1.51 and 18.30±3.21 g/cm², respectively 30 and 60 min after ferritin injection; likewise, 30 min after ferritin administration, 5.08±1.51 and 17.11±3.9 g/cm², respectively in normal and theophylline-treated animals.     

Le potassium et le sodium de l'humeur aqueuse du lapin et leurs variations sous l'effet de l'acétazolamide (Diamox)

Summary In 21 rabbits, the average concentration of sodium and potassium in the aqueous humor was found to be respectively 149±4.3 mEq/l and 5.34±mEq/l.Eleven of these animals were injected with Diamox (100 mg/kg of body weight); a significant drop of potassium concentration in the aqueous humor was observed three hours later, as compared with the ten control animals. No change in sodium concentration occurred.Our findings are considered in correlation with the fall of intraocular pressure produced by the carbonic anhydrase inhibitor, and the role of this enzyme is discussed.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

A simple,sensitive, non-stimulated photon counting system for detection of superoxide anion in whole blood

A simple, sensitive, non-stimulated assay was developed to measure the superoxide anion concentration in whole blood, using an ultra-sensitive chemiluminescence (CL) analyzer, and lucignin amplification. The assay system can be performed without leukocyte isolation or stimulant administration. The blood CL levels of healthy males (362.8±337.7 counts/10 sec) were not different from those of females (335±308.7 counts/10 sec) (p=0.64), whereas the CL levels in whole blood in patients with acute pancreatitis (2522±2014 counts/10 sec) were significantly higher than those of healthy controls (p<0.001). This assay system may be valuable in the future for quantitative measurement of reactive oxygen species in various disorders.     

Transmural pressure as a determinant of basic intrinsic heart rate

Summary It was observed the heart rate was minimum at zero transmural pressure. The mean heart rate at zero transmural pressure was 23±5/min. This mean heart rate increased from 23±5/min to a peak value of 40±6/min (74% acceleratin) when the transmural pressure was raised from 0 to +4 mm Hg and to a similar peak value of 36±8/min (56% acceleration) when the transmural pressure was lowered from 0 to –4 mm Hg. The peak values attained at ±4 mm Hg were higly significant (p<0.001). It is concluded that the heart rate at zero transmural pressure represents the basic intrinsic pacemaker frequency independent of neural, humoral, thermal and haemodynamically induced mechanical influences.     

Acid phosphatase in eggs of the zebrafish,Brachydanio rerio

Summary 2 isozymes of acid phosphatase have been identified by polyacrylamide disc gel electrophoresis in the ovary and mature, unfertilized eggs ofB. rerio. Histochemically, the enzyme appears to be localized in preyolk bodies of previtellogenic oocytes and in yolk platelets of vitellogenic and postvitellogenic oocytes. The contents of the cortical granules at all stages of oocyte differentiation were acid phosphatase negative.     

Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects

Phenylketonuric (PKU) subjects have a limited supply of selenium (Se) in their phenylalanine-restricted diet. A Se repletion (1 g Se/kg/day)/depletion study was conducted in PKU children to determine the effect of Se on thyroid function parameters.The initial plasma Se concentration (mean±SD: 0.26±0.12 mol/L, p<0.00003, n=10) and glutathione peroxidase (GSH-Px) activity (140±58 U/L, p<0.00003, n=10) were significantly lower compared to agematched controls. After 14 weeks of supplementation, the plasma Se concentration (mean ±SD: 0.74±0.20 mol/L) normalized (normal range: 0.57–1.15 mol/L, mean ±SD: 0.76±0.13 mol/L, n=32) and remained stable thereafter during repletion. Plasma GSH-Px activity reached normal values after 18 weeks of supplementation (312±57 U/L; normal range: 238–492 U/L, mean ±SD: 345±54 U/L, n=32) and increased significantly for up to eight weeks thereafter (332±52 U/L). Individual and mean thyroid parameters were initially normal in all cases. The mean concentrations of plasma thyroxine (T₄: p<0.025), free T₄ (FT₄: p<0.01) and reverse triiodothyronine (rT₃: p<0.005) decreased to 75% of their initial value within three weeks of Se supplementation and remained stable thereafter, within a normal physiological range during selenium supplementation. They increased back to their initial values three weeks (T₄: p<0.05, FT₄: p<0.05) and six weeks (rT₃: p<0.025) respectively, after the end of the supplementation. In conclusion, Se supplementation modifies thyroid function parameters in Se-deficient PKU subjects most likely by an increase in activity of type I 5-deiodinase (5-DIase I).Preliminary results of this study were presented as posters on the 4th International Congress on Trace Elements in Medicine and Biology, Chamonix, France (1993) and the 4th Joint Meeting of the Lawson Wilkens Pediatric Endocrine Society and the European Society for Paediatric Endocrinology, San Francisco, California, Ped Res (1993) 33: S93 (abstract 537). Support in part by Research Grants of Nutricia BV, (The Netherlands).     

Differentiation of human megakaryocytes in culture starting from the primordial circulating cells in the newborn]

Human neonatal blood mononuclear cells were seeded in plasma clot containing high dose of a crude erythropietin. Pure megakaryocyte colonies were observed rarely and most of the colonies were mixed, megakaryocytes being located between subcolonies of erythrocytic bursts. The megakaryocytic nature of large cells could be clearly confirmed by the presence of platelet peroxidase, demarcation membranes, and alpha granules detected by electron microscopy; in addition mature micromegakaryocytes were recognized, shedding platelets.