Estimation of uncertainties in X-ray refinement results by use of perturbed structures

The uncertainties in the refined parameters for a 1.5-A X-ray structure of carbon-monoxy (FeII) myoglobin are estimated by combining energy minimization with least-squares refinement against the X-ray data. The energy minimizations, done without reference to the X-ray data, provide perturbed structures which are used to restart conventional X-ray refinement. The resulting refined structures have the same, or better, R-factor and stereochemical parameters as the original X-ray structure, but deviate from it by 0.13 A rms for the backbone atoms and 0.31 A rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B-factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X-ray structures are more consistent with the energy parameters used in simulations.     

Molecular biology of Philadelphia chromosome in chronic granulocytic leukaemia and acute lymphoblastic leukaemia

In classical t(9;22) translocation, as observed in chronic granulocytic leukemia (CGL), a hybrid DNA unit is produced, including a rearranged PHL gene, previously known as bcr (breakpoint cluster region) plus the translocated c-abl gene from chromosome 9: a hybrid bcr-abl protein, p210 is formed, with increased tyrosine kinase activity. Such DNA rearrangement, with a p210 protein synthesis, is also found in cases of Philadelphia-positive acute lymphoblastic leukemia (ALL), but in apparently similar cases the bcr gene is not rearranged, and a novel p190 abl-related protein can be found; c-abl rearrangement has also been observed.It is thus established that correlations between cytogenetic and molecular events can be found in CGL and ALL, as in other haemopoietic malignancies: translocation and possible rearrangement of the c-abl oncogene seem of particular importance in this case.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Phosphorylation of cytosolic proteins by casein kinases in human erythrocytes. Response to ionic strength and to 2,3-bisphosphoglycerate

The endogenous phosphorylation of human erythrocyte cytosolic proteins is markedly increased when the crude cytosol, prior to incubation in the presence of [y-³²P] ATP, is submitted to DEAE-cellulose chromatography. Some proteins, including 22 and 23 kDa proteins, are preferentially phosphorylated by cytosolic casein kinase CS, whereas other proteins, including 42 kDa protein, are preferentially phosphorylated by casein kinase CTS. The CS-catalyzed phosphorylation is strongly inhibited by physiological ionic strength (150 mM KCl or NaCl) and by physiological levels (3 mM) of 2,3-bisphosphoglycerate, while CTS-catalyzed phosphorylation is unaffected. The very poor endogenous phosphorylation of these proteins in the crude cytosol may be due to the presence of other cytosolic inhibitors which are removed by DEAE-cellulose chromatography.     

Radioactive labeling techniques for the identification of G antigen in the human Rh blood group system

The G antigen is one of the erythrocyte membrane Rh antigens. The amount of Rh antigen present on the red blood cell is about 10(-15) g and radioactive labeling of membrane proteins is a useful method for its identification and characterization. In this paper, we compare 4 labeling techniques. Using a human monoclonal anti-Rh(G) antibody and an immunofixation technique, we located the G antigen on a polypeptide of an average molecular weight of 28,000 Da.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation

Summary The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of preand postganglionic NF-positive nerves in the ganglion. Cuts in both preand postganglionic nerves result in a marked increase in GFAP immunoreactivity in SCG, whereas NF immunoreactivity increases in nerve cell bodies after preganglionic cuts. Only a few ganglion cells show NF immunoreactivity in the normal SCG of guinea pig. All intraganglionic NF-positive nerves are of preganglionic origin; decentralization abolishes NF immunoreactivity in these nerve fibers. The inferior mesenteric ganglion, the hypogastric nerves and colonic nerves in guinea pigs contain large numbers of strongly NF-immunoreactive nerve fibers.When the SCG of adult rat is grafted to the anterior eye chamber of adult rat recipients, both ganglionic cell bodies and nerve fibers, forming on the host iris from the grafted ganglion, are NF-positive. As only the perikarya of these neurons normally exhibit NF immunoreactivity, and the terminal iris arborizations are NF-negative, it appears that the grafting procedure causes NF immunoreactivity to become more widespread in growing SCG neurons.     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council