Distribution of calmodulin in insects as determined by radioimmunoassay

The distribution of calmodulin in 11 separate tissues of the cockroach Leucophaea maderae was determined by radioimmunoassay. The highest levels of this protein were found in Malpighian tubules, visceral muscle and the central nervous system. Distribution of calmodulin was also determined for the following insect species: Apis mellifera, Canthon imitator, Heliothis zea (larvae) and Periplaneta americana. Abdominal tissues contained the highest levels of the calcium binding protein, while lesser amounts were found in the thorax and head. The detected levels of calmodulin suggest that this protein serves as an important regulator of biochemical functions in the viscera and nervous system of insects.     

Characteristics of the granules formed in nucleated erythrocytes as affected by cardiotrast

Numerous granules are formed in frog erythrocytes under the influence of cardiotrast (C) (diethanolamine-3,5-diiodo-4-pyridone-1-acetic acid). However, as revealed by cytospectrophotometric investigation and X-ray microanalysis, no C was accumulated in these granules. It is known that C can dissociate into diethanolamine and 3,5-diiodo-4-pyridone-1-acetic acid. It was assumed that under the influence of C granule formation may occur at the expense of an uncharged diethanolamine form penetrating into lysosome-like structures to be accumulated by protonation process. Diethanolamine was found to provoke granule formation in frog erythrocytes. It is impossible to reveal substance in granules because it is colourless and has no ultraviolet absorbtion band. Under the influence of some inhibitors of energy metabolism on granule formation and the granules formed, their inhibitory effect is exerted on the process of granule formation. In granules isolated by differential centrifugation activity of some marker lysosomal enzymes was found which enabled us to attribute these granules to lysosome-like structures.     

H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA.

The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones.     

Endotoxin stimulates endothelin-release in vivo and in vitro as determined by radioimmunoassay

A marked increase in immunoreactive endothelin was observed in rat serum collected within 10-15 min after infusion of endotoxin. Endothelin level was 117 +/- 11.5 pg/ml (mean +/- S.E., N = 4) in rats exposed to endotoxin as compared with undetectable levels (less than 2 pg/ml, N = 4) in controls. We have also observed a significant stimulation of endothelin-release by endotoxin from cultured bovine transformed thoractic aortic endothelial cells at concentrations of endotoxin ranging between 0.1 and 10.0 micrograms/ml. Serum was indispensable for the stimulating effect of endotoxin, although serum itself did not show any effect at the concentration used (1%). These results suggest that endothelin plays an important role in mediation of pathophysiological responses caused by endotoxin. The levels of endothelin were measured by radioimmunoassay with high sensitivity.     

Enzyme loading of nucleated chicken erythrocytes

Exogenous enzymes can be loaded into nucleated chicken erythrocytes by hypotonic hemolysis. The hemolysed and resealed cells retain the ability to activate transport ions and to survive while circulating in the blood of chickens. It is proposed that these cells may be useful for the investigation of several aspects of cellular biochemistry.     

A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [(32)P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35mu(3) and are isopycnic with sucrose of density 1.2871 (measured at 20 degrees ). Maturation of the nuclei causes them to shrink to a volume of 25mu(3) and the density to increase to 1.2944.     

Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay

We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an ED50 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, beta-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 ng/mg tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem.     

Labelling of Histone H5 and Its Interaction with DNA. 1. Histone H5 Labelling with Fluorescein Isothiocyanate

Abstract

Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific α-NH₂ terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA.

FTTC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4–8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound UTC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.     

The metabolism of ribavirin in erythrocytes and nucleated cells

1. The metabolism of the broad-spectrum antiviral drug ribavirin was examined in intact human erythrocytes, cultured skin fibroblasts and EBV-transformed lymphoblasts. At an extracellular ribavirin concentration of 35 microM all the cell types produced ribavirin mono-, di- and triphosphate, with the nucleotide concentration reaching half-maximum in 210, 245 and 267 min for fibroblasts, lymphoblasts and erythrocytes, respectively. The ratio of mono-, di- and triphosphates was ca 4:1:40 in fibroblasts, 3:1:8 in lymphoblasts and 1:5:17 in erythrocytes. 2. When ribavirin was removed from the medium, the half-life of ribavirin nucleotides was less than 2 hr in fibroblasts and lymphoblasts, but greater than 24 hr in erythrocytes. 3. In energy-starved erythrocytes, the catabolism of ribavirin nucleotides closely followed the catabolism of adenine nucleotides. 4. None of these cell types excreted appreciable amounts of the ribavirin catabolites triazole carboxamide, triazole carboxylate or triazole carboxylate ribonucleoside. 5. Cells deficient in adenosine kinase produced greater than 4% of the ribavirin nucleotides of normal controls. 6. The nucleated cells quickly hydrolyzed ribavirin 5'monophosphate to ribavirin, whereas this activity was negligible in erythrocytes.