Biomedical subjects
Publications and source records attributed to R M Johnson.
The uptake of exogenous 32Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[32P]ATP, and suggests a possible reinterpretation of those experiments.
The growth temperature of Bacillus stearothermophilus was previously reported to affect the esterase band pattern obtained after gel electrophoresis. In this study gel electrophoresis of esterases was done on a group of Antarctic bacterial strains cultivated at different temperatures to investigate whether band shift due to a change in growth temperature was a general phenomenon or limited only to select groups. Most strains studied were in the Bacillus genus. Standard strains of known Bacillus species and other Gram-positive genera were examined for comparative purposes. A change in the esterase band pattern was observed as a result of variation in growth temperature. Two major esterase band were dominant in this diverse group of species.
The uptake of 32P from exogenous 32Pi into membrane proteins of sickle erythrocytes has been analyzed. The phosphorylation of spectrin is normal in sickle cells. There is, however, a substantial increase in 32P in the sialoproteins of the membrane, which can be demonstrated after fractionation or selective proteolysis. Normal and sickle erythrocytes were separated on Stractan gradients and average cell age was determined using the remaining pyruvate kinase activity as a marker. The altered phosphorylation of sickle cells was not seen in young normal cells, suggesting that it was not related to cell age. The altered phosphorylation was also not correlated with the level of reticulocytes in these fractions. This result is further evidence for abnormalities in the sialoproteins of sickle erythrocytes and is the first demonstration of altered sialoprotein phosphorylation in the red cell.
Epidermal growth factor (EGF) causes rapid increases in free intracellular Ca2+ and stimulates the phosphorylation of 11 cytosolic proteins in hepatocytes. Ten of the 11 cytosolic proteins altered by EGF are identical to those affected by angiotensin II, a hormone that stimulates the breakdown of phosphatidylinositol 4,5-bisphosphate. An increase in the phosphorylation of the other protein, spot c (Mr = 36,000, pI = 5.5), is observed only with EGF. Treatment of intact rats with pertussis toxin to ADP-ribosylate Ni, the inhibitory GTP-binding protein of the adenylate cyclase complex, abolished the effect of EGF on Ca2+ mobilization and on the phosphorylation of the 10 proteins affected in common with angiotensin II. This treatment had minimal effects on the ability of EGF to stimulate the phosphorylation of its unique substrate, spot c. In marked contrast, modification of Ni did not block the ability of angiotensin II to stimulate Ca2+ mobilization or protein phosphorylation. Pretreatment of normal hepatocytes with 4 beta-phorbol 12-myristate 13-acetate blocked all responses to EGF, including the increased phosphorylation of spot c, but had no effect on the responses to angiotensin II. These results imply that Ni or a similar pertussis toxin substrate may mediate the apparent effects of EGF on phosphatidylinositol breakdown and that protein kinase C may regulate a site in the transduction pathway. Angiotensin II appears to use a different signal transduction mechanism to stimulate phosphatidylinositol metabolism in hepatocytes.
An antibiotic factor obtained from Alteromonas luteoviolacea was shown to kill Paramecium caudatum. This substance was shown to be destroyed by boiling, sonication, or freezing. It passed through a 0.45-micron-pore-size filter and was not precipitated from a culture supernatant with ethyl alcohol. Its production varied with A. luteoviolacea strains and medium constituents. It was also shown to be distinct from the A. luteoviolacea antibiotic that affects bacterial cells.
Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at greater than 1500S; (b) in ribonucleoprotein particles that sediment at 20-120S and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120-360S. We have compared the RNA and protein components of the first two of these RNP fractions. The polyadenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril-associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibril-associated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre-mRNP). RNA-binding experiments demonstrate that the polypeptide most tightly bound in pre-mRNP is a 54-kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60-kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre-mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre-mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA-associated protein in blocking translation are discussed.
In hereditary pyropoikilocytosis (HPP) and one type of hereditary elliptocytosis (HE), spectrin self-association is abnormal [5,7]. Spectrin extracted from normal erythrocyte membranes at 0 degree C is nearly all tetrameric, while in HPP and HE (type 1) a substantial amount of the extracted spectrin is dimeric. Abnormal reassociation of spectrin dimers to tetramers can also be demonstrated. We here report the case of a family in which the child has moderately severe hemolysis, with extreme microcytosis and poikilocytosis. The spectrin extracted at 0 degree C was predominately dimer. Parents had levels of dimer intermediate between patient and control values. The temperature dependence was normal for erythrocyte fragmentation; spectrin extractability; and circular dichroism of purified spectrin. Neither the patient nor either parent had elliptocytic red cells as judged from smears and scanning electron microscopy. The presence of substantial amounts of dimeric spectrin in the parents is consistent with a model in which each parent is heterozygous for a different nonassociating mutant spectrin, while the child has inherited a nonassociating molecule from each parent. In each individual, the degree of mechanical stability of the erythrocyte membrane, determined by ektacytometry, was proportional to the amount of tetramer found in the membrane. The description of this case is consistent with either HPP or a form of homozygous HE which is asymptomatic in the carrier state.
The transfer of attributes from a test stimulus (TS) to a masking stimulus (MS) during backward masking was investigated. Results indicated that a gap inserted on a TS transferred to a MS and that the degree of transfer varied as an inverted U-shaped function of ISI. Two explanations of the findings were offered. One was based on preservation of distinctive features and the second was based on "filling in" theory and the decrease in masking which occurs with increased spatial separation. The suggestion was made that an explanation of the transposition effect should be consistent with theories of backward masking and apparent motion.
The effects of nitroprusside (NP), glyceryl trinitrate (GTN), and the 8-bromo analog of cyclic GMP (8-Br-cGMP) on norepinephrine (NE)-stimulated phosphorylase a formation and myosin light chain (MLC) phosphorylation were examined in the rat aorta. NE produced a time-dependent increase in tension, phosphorylase a formation, and MLC phosphorylation. The formation of phosphorylase a and phosphorylation of MLC were transient, since both processes declined to basal levels within 30 min after the addition of NE even though tension remained elevated. NP and GTN inhibited tension, phosphorylase a formation, and MLC phosphorylation although inhibition of phosphorylase was greater when strips were treated with submaximal (i.e., 0.01 microM) NE concentrations. GTN was a more effective inhibitor of phosphorylase a formation than NP in NE-treated strips, although both agents and 8-Br-cGMP inhibited MLC phosphorylation. The guanylate cyclase inhibitor methylene blue (10 microM) effectively prevented the effects of NP and GTN. The results suggest that NP, GTN, and 8-Br-cGMP inhibit phosphorylase kinase and MLC kinase activation by lowering Ca2+ in the cell. This hypothesis is supported by the observations that 8-Br-cGMP inhibited the Ca2+-dependent, KCl-induced phosphorylase a formation most markedly at reduced concentrations of extra-cellular Ca2+. In addition, neither NP, GTN, nor 8-Br-cGMP inhibited phosphorylase a formation in forskolin-treated tissues, which occurred in response to cAMP-dependent phosphorylation of phosphorylase b kinase.
The effect of pH on erythrocyte deformability was determined in the ektacytometer. In isotonic solutions, cell deformability was essentially unaltered at pH values from 6.44 to 7.7, despite the known cell volume changes that occur over this pH range. Analysis by osmotic scan ektacytometry showed that erythrocyte deformability is homeostatically maintained constant during acidosis and alkalosis because of a balance between the countervailing effects of changes in intracellular viscosity and in the surface to volume ratio. This balance is perturbed in the sickle erythrocyte, so that acidosis improves deformability.
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Interleukin-3 (IL-3) (multipotential colony-stimulating factor [multi-CSF] ) is an important regulator of hemopoiesis. We recently isolated a cDNA clone of this gene and describe in this manuscript the biologic properties of the expressed gene product. An SV40 expression vector carrying cDNA encoding murine interleukin-3 was constructed so that expression of the IL-3 gene was placed under the control of the SV40 early promoter. When the expression vector was transfected to COS-1 monkey cells, IL-3 activity was secreted into the medium, reaching maximal levels 72 hours after transfection. The IL-3 produced by the COS-1 cells was partially purified using diethylaminoethyl Sephacel and phenyl-Sepharose, and its chromatographic properties were the same as IL-3 produced by the WEHI-3 cell line. The biologic activities of the "expressed" IL-3 include the "induction" of 20-alpha-hydroxysteroid dehydrogenase (20-alpha-SDH) in splenic lymphocytes from nu/nu mice, proliferative activity for 32D cl-23 and FDC-P1 cell lines, and colony-stimulating activity for granulocyte-macrophage, eosinophil, megakaryocyte, natural killer-like, erythroid, and multipotential colony-forming cells from murine fetal liver and adult bone marrow.
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The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.
Helical strips of rat aorta contracted with norepinephrine (10 nM) respond to ultraviolet (UV) radiation (340-360 nm) with a transient decrease in tension. The UV radiation-dependent relaxation is completely reversible and endothelial cell-independent. Although decreased tension is associated with a rise in tissue cGMP content, the cAMP level is unchanged after UV radiation. A significant inhibition of phosphorylase a formation which occurs coincidently with the rise in cGMP and decline in tension is observed with UV radiation. The effects of UV radiation on cGMP, phosphorylase a formation and tension were blocked by methylene blue. Relaxation and inhibition of phosphorylase a formation in response to UV radiation were also partially reversed with higher concentrations of norepinephrine (100 nM). Our results suggest that cGMP may mediate UV radiation-dependent reactions by reducing cytoplasmic Ca2+.
At early stages of oogenesis in Xenopus laevis most of the ribosomal 5S RNA is complexed with three proteins to form two types of cytoplasmic RNP storage particle. A particle sedimenting at 42S contains 5S RNA and tRNA together with two proteins of Mr 48000 (P48) and Mr 43000 (P43) and a second particle sedimenting at 7S contains 5S RNA plus a protein of Mr 40000 (P40, also known as the transcription factor, TFIIIA). In this report we use antibodies monospecific for each protein to follow the movement of 5S RNA from nucleus to cytoplasm to nucleolus to cytoplasm and to determine the fate of each of the proteins that associate with 5S RNA during these transitions. Both P48 and P43 have roles additional to the formation of the 42S RNP storage particle; P48 is detected in the nucleus during early oogenesis and is cleaved to yield an Mr-33000 fragment that remains associated with 5S RNA that is excess to ribosome requirement during late oogenesis; P43 appears to be cleaved to yield fragments of Mr 28000 and 17000, the latter being present in ribosomal fractions. Apparently, there is no function for P40 in addition to those already described in transcription of 5S RNA genes and in storage of 5S RNA as a 7S RNP particle.
Membrane protein phosphorylation has been measured in both normal and dystrophic (BIO 53.58) hamsters. After radiolabeling intact erythrocytes with exogenous 32Pi, band 3 polypeptides from dystrophic hamsters had increased incorporation of 32P, compared to age-matched controls. This was verified by membrane protein fractionation. The use of intact cell phosphorylation avoids many of the sources of experimental variability noted in experiments with isolated membranes. This work, using an animal model for the disease, supports the hypothesis of a generalized membrane defect in the muscular dystrophies.
In small oocytes of Xenopus laevis, the three most abundant proteins are isolated as basic polypeptides with molecular weights of 48 kD (P48), 43 kD (P43) and 40 kD (P40, also known as transcription factor IIIA). All three proteins share common properties in being able to bind specifically ribosomal 5S RNA molecules and influence, in different ways, their rates of production and utilization. It has been shown by biochemical analysis and immunological characterization that the three proteins are structurally distinct and are most probably the products of different genes. Immunostaining and radio-immunoassays indicate that both P48 and P43 have diverged considerably in structure between the amphibian genera Xenopus and Triturus. Antibodies raised against the transcription factor for Xenopus laevis 5S RNA genes (P40/TFIIIA) do not cross-react with the transcription factor isolated from oocytes of the closely related species Xenopus borealis. A protein equivalent of TFIIIA is not found in 5S RNA-containing RNP storage particles of Triturus oocytes. The functions of the three Xenopus oocyte proteins in transporting 5S RNA between different cellular compartments are considered in the light of these variations.