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Biomedical subjects

A Jaworowski

Publications and source records attributed to A Jaworowski.

At least 37 records · Page 2Linked to original sources

Detection of jun but not fos protein during developmental cell death in sympathetic neurons.

A large proportion of neurons die during normal development of the nervous system via an active process known as apoptosis. We counted the total number of neurons and apoptotic neurons in the superior cervical ganglion of the GH Wistar rat strain, which possesses a neurotrophic deficit leading to excessive perinatal cell death, and in its normal counterpart (N) by using the optical disector method to quantify the extent of apoptosis during postnatal development. Total neuron numbers fell between postnatal days 3 and 14 by 10 and 40% in N and GH, respectively. In GH ganglia, 1.5% of neurons were apoptotic at any given time, as determined by the presence of condensed chromatin clumps. Some types of cell death have been associated with expression of the immediate-early genes c-fos and c-jun. Therefore, we used histological and immunocytochemical techniques to characterise individual neurons and to detect the products of these immediate-early genes during developmental cell death. All apoptotic cells were immunopositive for c-jun protein, whereas no c-jun protein was detected in nonapoptotic cells. Conversely, members of the fos family of transcription factors were detected in the nucleus of 60% of nonapoptotic cells but in only a minor proportion of cells undergoing apoptosis. These results indicate that c-jun occurs in neurons that are committed to die. This is the first situation in which the presence of jun protein has been correlated with normal programmed cell death in individual apoptotic neurons.

Aging↗

Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts.

Colony-stimulating factor 1 (CSF-1) causes the activation of STAT1 and STAT3 transcription factors in bone marrow macrophages (BMM), in the macrophage cell line BAC1.2F5, and in fibroblasts that express the wild-type receptor for CSF-1. Fibroblasts expressing a mutant receptor in which the tyrosine 809 is replaced with phenylalanine do not activate STAT proteins in response to CSF-1. The activation of the STAT proteins in BMM is accompanied by tyrosine phosphorylation of Tyk2. In fibroblasts, the activation of the STAT proteins is accompanied by tyrosine phosphorylation of Tyk2 and JAK1. We propose that these JAK kinases are subjected to very rapid phosphorylation in response to CSF-1, followed by rapid dephosphorylation. Furthermore, we propose that kinases other than JAK kinase may be involved in the phosphorylation of the STAT proteins in response to CSF-1.

3T3 Cells↗

Protein synthesis and secretion by cultured retinal pigment epithelia.

Protein synthesis and secretion by post-natal sheep and calf retinal pigment epithelial (RPE) cells was investigated following labelling of choroidal pieces, isolated RPE cells and RPE cells in tissue culture with L-[U-14C] leucine. We show that RPE cells secrete a specific set of proteins that includes retinol binding protein (RBP) and transthyretin (TTR), which are both involved in retinol transport in blood. Using a two-chambered culture system we show that protein secretion by the post-natal RPE cells occurs predominantly across the apical pole of the cells, i.e., across the surface of the cells which, in vivo, faces the retina. In agreement with results of others using foetal RPE cells (Ong, D.E., Davis, J.T., O'Day, W.T. and Bok, D. (1994) Biochemistry 33, 1835-1842) we show that RBP and, to a lesser extent, TTR are also secreted predominantly across the apical pole of the cell. We have developed a cell culture model for the RPE that may be used as an in vitro model for studying transport across the blood-retinal barrier.

Animals↗

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres.

Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.

Actins↗

Phospholipase D is activated by phorbol ester but not CSF-1 in murine bone marrow-derived macrophages.

Phospholipase D activity was measured in murine bone marrow-derived macrophages (BMM) treated with either colony stimulating factor-1 (CSF-1) or phorbol myristyl acetate (PMA) by measuring formation of phosphatidylbutanol (PtBut) in cells preloaded with n-butanol. Addition of 10(-7) M PMA for 15 min stimulated the amount of PtBut formed in growth arrested cells by 3-4 fold whereas no stimulation was observed with 5000 units mL-1 CSF-1 for 0.5, 2 or 15 min. Protein kinase C activity was determined in growth-arrested BMM by phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS). PMA stimulation for 5 min increased protein kinase C activity 5-6 fold whereas CSF-1 treatment for 5 min or 15 min did not. Contrary to earlier reports, CSF-1 did not stimulate diradyl glycerol formation in BMM. These results show that stimulation of protein kinase C and the activation of phospholipase D are not involved in the early events of CSF-1-stimulated signal transduction pathways in BMM.

Animals↗

Immunohistochemical localization of transthyretin in glomerular peripolar cells of newborn sheep.

Purified transthyretin has been isolated from sheep serum. Antiserum raised against this protein has been used with an indirect immunoperoxidase histochemical technique to identify transthyretin in newborn lamb kidney tissue. Transthyretin was found in proximal tubule cells and in glomerular peripolar cells. Preabsorption studies using purified transthyretin protein indicate that the immunoreactivity of the antiserum is specific to transthyretin.

Animals↗

Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates.

A chicken liver cDNA library was constructed in bacteriophage lambda gt10. A full-length transthyretin cDNA clone was identified by screening with rat transthyretin cDNA and was sequenced. A three-dimensional model of chicken transthyretin was obtained by computer-graphics-based prediction from the derived amino acid sequence for chicken transthyretin and from the structure of human transthyretin determined by X-ray diffraction analysis [Blake, C.C.F., Geisow, M.J., Oatley, S.J., Rérat, B. & Rérat, C. (1978) J. Mol. Biol. 121, 339-356]. The similarity of the amino acid sequences of chicken and human transthyretins was 75% overall and 100% for the central channel containing the thyroxine-binding site. Also, the organization of the transthyretin gene into exons and introns and the tissue specificity of expression of the transthyretin gene were similar in chicken and mammals, despite an evolutionary distance of about 3 x 10(8) years from their common ancestor, the Cotylosaurus. By far the highest levels of transthyretin mRNA were found in choroid plexus. The data suggest a fundamental role for the cerebral expression of transthyretin in all vertebrates. It has been proposed that this role is the transport of thyroxine from the bloodstream to the brain [Schreiber, G., Aldred, A.R., Jaworowski, A., Nilsson, C., Achen, M.G. & Segal, M.B. (1990) Am. J. Physiol. 258, R338-R345].

Amino Acid Sequence↗

Enolpyruvate: chemical determination as a pyruvate kinase intermediate.

Despite many studies suggesting the role of enolpyruvate as a bound intermediate in the pyruvate kinase reaction, direct evidence for it has been lacking. By use of a combination of chemical trapping and isolation of a derivative, significant amounts of enzyme-bound enolpyruvate have now been demonstrated. The method distinguishes enolpyruvate. It is based on reaction of bromine with enolpyruvate in acid, derivatization of formed bromopyruvate with thionitrobenzoate, and resolution by reversed-phase HPLC of the thioether derivative. As little as 10 pmol of the thioether derivative could be quantitated reliably. With this method, the internal equilibria, including the E.ATP.enolpyruvate intermediate, have been determined. Enzyme-enolpyruvate concentration was shown to be pH-dependent. Phosphoenolpyruvate also reacts with bromine to form bromopyruvate. To quantitate enolpyruvate specifically in a background of phosphoenolpyruvate, advantage was taken of phosphoenolpyruvate's much greater stability in acid. When bromide/was added 10 min after the acid quench, ketonization of enolpyruvate was complete, and only phosphoenolpyruvate was measured. Enolpyruvate is thus determined by difference between the bromopyruvate measured with and without delayed bromine addition.

Adenosine Triphosphate↗

Transthyretin (prealbumin) gene expression in choroid plexus is strongly conserved during evolution of vertebrates.

1. The major protein synthesized and secreted by the choroid plexus from mammals, birds, reptiles and probably amphibians is similar in subunit structure to transthyretin. 2. In mammals and birds the proportion of transthyretin mRNA is much higher in choroid plexus RNA than in liver RNA. No transthyretin mRNA is found in brain outside the choroid plexus. 3. Transthyretin-like protein, such as that secreted by the choroid plexus, was not detected in amphibian serum and was present in very low levels in reptile serum. 4. It is proposed that transthyretin synthesis and secretion arose earlier in evolution in the choroid plexus than in the liver.

Animals↗

Thyroxine transport from blood to brain via transthyretin synthesis in choroid plexus.

The transport of thyroxine from the bloodstream to the brain and the synthesis and secretion of transthyretin (formerly called prealbumin) were studied in rats and in sheep choroid plexus perfused in vitro. Rat choroid plexus contained 4.4 micrograms and rat liver 0.39 micrograms transthyretin mRNA per gram wet tissue. The specific radioactivity of transthyretin isolated from cerebrospinal fluid of rats 60 min after intravenous injection of [14C]leucine was greater than 50 times that of transthyretin from serum. After adding [14C]leucine to the perfusion medium of an in vitro perfused sheep choroid plexus, highly radioactive transthyretin was isolated from freshly secreted cerebrospinal fluid collected from the exposed choroid plexus surface. Secretion of newly synthesized transthyretin into the perfusion medium could not be demonstrated. After intravenous injection of [125I]-thyroxine into rats, a maximum in the curve of radioactivity in tissue plotted against time after injection was observed first for choroid plexus, thereafter for cerebrospinal fluid, and still later for cortex and striatum. Based on the obtained data, a hypothesis is derived for the mechanism of the transport of thyroid hormones from the bloodstream to the brain involving transthyretin synthesized in choroid plexus and secreted into the cerebrospinal fluid.

Animals↗

Developmental patterns of gene expression of secreted proteins in brain and choroid plexus.

The proteins secreted by the choroid plexus throughout rat brain development were analyzed by two-dimensional polyacrylamide gel electrophoresis following biosynthetic labeling of choroid plexus pieces with [14C]leucine in vitro. Approximately 20 major protein species were resolved which, with the exception of transferrin, transthyretin, and alpha 2-macroglobulin, appear to be unrelated to proteins found in high concentrations in plasma. Several patterns of developmental regulation were observed. At least two of the proteins were synthesized and secreted at high levels only by fetal choroid plexus, whereas the secretion of several other proteins including transferrin and proteins comigrating with cystatin C and alpha 2-macroglobulin increased only after birth. The levels of mRNA coding for transferrin, ceruloplasmin, cystatin C, alpha 2-macroglobulin, beta 2-microglobulin, and transthyretin were measured in the brain during development by dot hybridization and northern gel analysis. No mRNA was detected coding for the proteins alpha-fetoprotein, alpha 1-antitrypsin, haptoglobin, and thiostatin in the brain at any stage. For those proteins, which are produced in other parts of the brain as well as by the choroid plexus, the changes in their corresponding mRNA levels measured in whole brain paralleled the changes in their secretion by the choroid plexus. The results presented in this paper show that the choroid plexus is active in protein secretion at all stages studied. The changing pattern of protein secretion by the choroid plexus, combined with its early development compared with other tissues in the brain, suggests that it is active in providing the appropriate extracellular environment for the growth and differentiation of the brain.

Aging↗

Internalization of IL-2 by an IL-2-dependent murine T cell lymphoma expressing no detectable cell surface receptors for IL-2.

A murine Radiation leukemia virus-induced T cell lymphoma, 5C2, which is dependent on interleukin-2 (IL-2) for proliferation has been analyzed for interleukin-2 receptor (IL-2R) expression. Using fluorescence-activated cell sorter and electron microscopic analysis together with antibody specific for the known p55 chain of the murine IL-2R, no evidence has been obtained to suggest that these cells express detectable numbers of receptors with high affinity for IL-2. However, two different antibodies with specificity for the p55 chain of the IL-2R have been shown to inhibit 5C2 proliferation. An analysis of 125I-IL-2 binding has precluded a cell surface receptor density of greater than 80 molecules per cell. A temperature-dependent, nonspecific uptake of 125I-IL-2 has been described for 5C2. Uptake is saturated at 8.5 nM 125I-IL-2 with equilibrium being established within 60 min. When incubated at 37 degrees C in the presence of 400 pM 125I-IL-2, a maximum of approximately 2,000 molecules are internalized within 40 min. Uptake of other iodinated proteins by 5C2 was not observed. This property is unique to 5C2 and not to the control C6VL/1 cell line. Intracellular vesicles have also been found in 5C2 cells by electron microscopy which stain positively with gold-conjugated antibody specific for the p55 chain of the IL-2R. 5C2 appears to exhibit unique IL-2 regulatory characteristics.

Animals↗

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum.

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).

Glyceric Acids↗

Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction.

At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.

Borohydrides↗

Cloning of the gene for the respiratory D-lactate dehydrogenase of Escherichia coli.

During attempts to clone the gene coding for the respiratory NADH dehydrogenase of Escherichia coli, two hybrid plasmids were constructed from E. coli chromosomal DNA [Young, I. G., Jaworowski, A., & Poulis, M. I. (1978) Gene 4, 25-36]. One of these plasmids, pIY1, derived from EcoRI-digested chromosomal DNA, was studied in detail and shown to possess the gene coding for the NADH dehydrogenase of the aerobic respiratory chain of E. coli. We now report the characterization of the other hybrid plasmid, pIY2, derived from HindIII-digested chromosomal DNA, and shown that it complements ndh mutants not by virtue of carrying the ndh gene but because it carries the gene coding for the respiratory D-lactate dehydrogenase. Cells carrying this hybrid plasmid overproduce the respiratory D-lactate dehydrogenase in their cell membranes by 15-20-fold with negligible activity appearing in the cytoplasm. This results in an amplification of the levels of the D-lactate oxidase. The amplified D-lactate oxidase activity, coupled with the pyridine nucleotide linked D-lactate dehydrogenase, apparently provides a new pathway for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) in the cell, independent of the respiratory NADH oxidase.

Cloning, Molecular↗

Characterization of the respiratory NADH dehydrogenase of Escherichia coli and reconstitution of NADH oxidase in ndh mutant membrane vesicles.

Highly purified preparations of the cholate-solubilized respiratory NADH dehydrogenase, isolated from genetically amplified Escherichia coli strains [Jaworowski, A., Campbell, H. D., Poulis, M. I., & Young, I. G. (1981) Biochemistry 20, 2041-2047], have been characterized. Enzyme preparations were shown to contain 70% (w/w) lipid, predominantly phosphatidylethanolamine. One mol of noncovalently bound FAD and approximately 1 mol of ubiquinone/mol of enzyme subunit were detected. The purified enzyme was shown to contain only low levels of Fe and acid-labile S, indicating the absence of iron-sulfur clusters. No Cu, Mo, W, or covalently bound P was detected, and no evidence for other chromophores was obtained from visible and ultraviolet absorption spectra of the purified enzyme or of the delipidated polypeptide prepared by gel filtration in sodium dodecyl sulfate. Protein chemical studies verified that the enzyme consists of a single polypeptide species of Mr 47 000, and the N- and C-terminal cyanogen bromide peptides were identified. The pure enzyme was shown to reconstitute membrane-bound, cyanide-sensitive NADH oxidase activity in membrane vesicles prepared from ndh mutant strains.

Amino Acids↗

Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli.

Escherichia coli membrane particles were solubilized with potassium cholate. An NADH:ubiquinone oxidoreductase was resolved by hydroxylapatite chromatography of the solubilized material. This enzyme has been identified as the respiratory NADH dehydrogenase since it is absent in chromatograms of solubilized material from an ndh mutant strain. Such mutants lack membrane-bound NADH oxidase activity and have previously been shown to have an inactive NADH dehydrogenase complex [Young, I. G., & Wallace, B. J. (1976) Biochim. Biophys. Acta 449, 376-385]. The respiratory NADH dehydrogenase was amplified 50- to 100-fold in vivo by using multicopy plasmid vectors carrying the ndh gene and then purified to homogeneity on hydroxylapatite. Hydroxylapatite chromatography of cholate-solubilized material from genetically amplified strains purified the enzyme approximately 800- to 100-fold relatively to the activity in wild-type membranes. By use of a large-scale purification procedure, 50-100 mg of protein with a specific activity of 500-600 mumol of reduced nicotinamide adenine dinucleotide oxidized min-1 mg-1 at pH 7.5, 30 degrees C, was obtained. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme showed that the enzyme consists of a single polypeptide with an apparent Mr of 45 000.

Cell Membrane↗

Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli. UUG initiation codon.

The nucleotide sequence of the structural gene coding for the respiratory NADH dehydrogenase of Escherichia coli has been determined by the chain-termination method. The reading frame for the protein starts with the unusual initiation codon UUG and predicts an amino acid sequence of 434 residues (Mr = 47 304). The reading frame was confirmed by protein chemical studies including determination of the N-terminal sequence of the protein. The product made in vivo was found to have threonine as its N-terminal residue, indicating that the initiating N-formylmethionine had been removed by post-translational processing.

Amino Acid Sequence↗