Ureteric obstruction secondary to pelvic actinomycosis.
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Actinomycosis↗
Biomedical subjects
A E Jackson
Publications and source records attributed to A E Jackson.
Calcium channel agonists and antagonists: effects of chronic treatment on pituitary prolactin synthesis and intracellular calcium.
PRL synthesis by GH cells in culture has previously been shown to increase when calcium is added to cultures grown in calcium-depleted medium or when cultures are treated for 18 h or longer with the dihydropyridine calcium channel agonist BAY K8644, whereas the antagonist nimodipine inhibits PRL. The experiments described here were designed to test whether differences in PRL synthesis caused by the dihydropyridines are due to changes in PRL mRNA levels, whether structurally different classes of calcium channel blockers alter PRL production, and whether long term treatment with calcium channel agonists and antagonists alters intracellular free calcium, [Ca2+]i. PRL synthesis and PRL mRNA levels were increased similarly by BAY K8644 and decreased in parallel by the dihydropyridine antagonist nimodipine, while overall protein and RNA synthesis were not changed by either the agonist or antagonist. Two calcium channel blockers which act at different sites on L-type channels than the dihydropyridines also inhibited PRL synthesis without affecting GH; 5 microM verapamil reduced PRL by 64% and 15 microM diltiazem by 89%. Partial depolarization with 5-25 mM KCl increased PRL synthesis up to 2-fold. The intracellular free calcium ion concentration was estimated by Quin 2 and averaged 142 nM for control cultures in normal medium, and 128 and 168 nM for cultures treated 72 h with nimodipine or BAY K8644, respectively. Nimodipine totally prevented the calcium rise obtained upon depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Proximal upstream flanking sequences direct calcium regulation of the rat prolactin gene.
Previous investigations have shown that Ca2+ strongly and specifically stimulates endogenous PRL gene expression by GH3 cells. In this study, addition of Ca2+ to Ca2+-deprived GH3 cells yielded a large (ca. 8-fold) stimulation of transient expression of a transfected PRL-chloramphenical acetyltransferase (CAT) construct containing ca. 1 kilo-base-pair of the PRL promoter region, but only a slight (less than or equal to 2-fold) nonspecific stimulation of CAT activity directed by any of three control promoters: dihydrofolate reductase, Rous sarcoma virus, or thymidine kinase. In GH3 cells never deprived of Ca2+, expression of a PRL-CAT construct was specifically stimulated and inhibited, respectively, by the dihydropyridine voltage-dependent Ca2+ channel modulators Bay K8644 and nimodipine; Ca2+ can thus regulate expression of an exogenous PRL promoter in cells incubated under physiological Ca2+ conditions. By employing a combined protocol, in which Ca2+-deprived cells are exposed to Ca2+ in the presence of Bay K8644, a very large (greater than 35-fold) but still promoter-specific induction of expression of a PRL-CAT construct was obtained. Analysis of 5'-deleted PRL-CAT constructs implied that the PRL gene Ca2+ response element is contained entirely within the first 174 base pairs of upstream flanking DNA sequence.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Association of calmodulin and smooth muscle myosin light chain kinase: application of a label selection technique with trace acetylated calmodulin.
A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-12232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylation↗
PCP-induced alterations in cerebral glucose utilization in rat brain: blockade by metaphit, a PCP-receptor-acylating agent.
The effects of phencyclidine (PCP) on regional cerebral glucose utilization was determined by using quantitative autoradiography with [14C]-2-deoxyglucose. PCP increased brain metabolism in selected areas of cortex, particularly limbic, and in the basal ganglia and thalamus, whereas the drug decreased metabolism in areas related to audition. These results are consistent with the known physiology of central PCP neurons and may help to suggest brain areas involved in PCP-mediated actions. Moreover, based on the behavioral similarities between PCP psychosis and an acute schizophrenic episode, these data may be relevant to the understanding of schizophrenia. The PCP-receptor-acylating agent, metaphit, blocked most of these PCP actions. In addition, metaphit by itself was found to diminish glucose utilization rather uniformly throughout brain. These results indicate an antagonist effect of metaphit on the PCP system and suggest a widespread action of metaphit, putatively at a PCP-related site, possibly in connection with the N-methyl-D-aspartate (NMDA) receptor.
Acylation↗
Effects of ethane dimethane sulphonate (EDS) on seminiferous tubule function in rats.
The effects of a single injection of ethane dimethane sulphonate (EDS) on aspects of seminiferous tubule function were assessed over a period of 49 days. Ethane dimethane sulphonate, which is known to cause destruction of Leydig cells, reduced the levels of testosterone in both serum and testicular interstitial fluid for 21 days, after which recovery occurred. The low testosterone levels were associated with elevated serum levels of LH and FSH. Daily sperm production was decreased from 14 to 42 days post-EDS but returned to control levels at 49 days. The production of seminiferous tubule fluid, measured after unilateral efferent duct ligation, decreased significantly at 7 and 14 days but then recovered. The testicular content of androgen binding protein (ABP) was decreased from 14 to 28 days but returned to normal thereafter. These results demonstrate significant effects on seminiferous tubule function, which may be due to the decrease in testosterone or be associated with a direct effect of EDS.
Androgen-Binding Protein↗
Binding of trifluoperazine and fluorene-containing compounds to calmodulin and adducts.
Calmodulin can be specifically acylated with a fluorene-containing hydrophobic spin-labeling reagent at just Lys 75 or at Lys 75 and Lys 148. The binding of trifluoperazine to calmodulin and the two adducts was determined using a Hummel-Dreyer procedure, and binding of the phenothiazine was found to be characterized by apparent positive cooperativity and an apparent limiting stoichiometry of about seven binding sites per protein molecule. Two non-reactive fluorene-containing compounds were synthesized, and both reagents exhibited far less binding to calmodulin than did trifluoperazine. One of these was also assayed for binding to the monolabeled adduct, and this binding was about half that observed with calmodulin and was non-cooperative. Thus, the qualitative and quantitative binding parameters of hydrophobic groups to calmodulin can be quite different.
Calmodulin↗
Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines.
The effects of the binding of smooth muscle myosin light chain (MLC) kinase on the microenvironments of different regions of calmodulin (CaM) were investigated by comparing the acylation rate constants of the seven lysine amino groups of free CaM with those of CaM complexed with MLC kinase. Equimolar amounts of CaM and CaM-MLC kinase complex were trace labeled with [3H]acetic anhydride in the presence of phenylalanine as a standard nucleophile. After completion of the reaction, equal amounts of a trace 14C-acetylated CaM sample, together with [14C]acetylphenylalanine, were added to each reaction mixture. The 3H/14C-labeled CaM and acetylphenylalanine were then isolated from each solution. After complete reaction with nonradioactive acetylating reagent, 3H/14C ratios (r) were determined for each epsilon-N-acetyllysine in the two CaM samples. These values were obtained either from isolated peptide fragments containing one lysine or from epsilon-N-acetyl phenylthiohydantoin lysine obtained by Edman degradation of peptide fragments containing two lysines. From the ratios, protection factors (= rfree/rcomplex) were determined as a measure of the perturbation produced by MLC kinase binding. These protection factors were corrected, using the isotope ratios of the internal standard, for differences in the degree of competition for labeling reagent between the two mixtures. In two separate labeling experiments employing different levels of trace labeling, very little change was observed in the reactivities of four lysines on MLC kinase binding (lysines 13, 30, 77, and 94). Small but reproducible decreases (about 2-fold) were observed in the reactivities of lysines 21 and 148, while lysine 75 underwent a major (more then 7-fold) decrease in labeling. In conjunction with previously published data, these results are interpreted as suggesting that the major perturbation in lysine 75 is a direct effect of MLC kinase contact with CaM and that a region in the central helix containing this residue, but not lysine 77, represents or is near the CaM-binding site for MLC kinase. The smaller changes in reactivities at lysines 21 and 148 may reflect a conformational change that occurs in CaM as a result of binding to MLC kinase.
Acetylation↗
The effects of ethane dimethane sulphonate (EDS) on bilaterally cryptorchid rat testes.
A single dose of ethane dimethane sulphonate (EDS) given to adult male rats has a specific destructive effects on Leydig cells, which are removed from the intertubular area by macrophages. This is associated with a decrease in testosterone (T) and a rise in serum FSH and LH for 21-28 days after EDS. Recovery of Leydig cells occurs from connective tissue precursors. This study uses the bilaterally cryptorchid rat to investigate the influence of local factors from the seminiferous tubules on the restoration of Leydig cell morphology and function. Morphometric data of intertubular tissue, hCG binding, serum T, FSH and LH levels all indicate that the Leydig cell population in cryptorchid testes is more rapidly restored from connective tissue cells when compared to the normal situation. The precise mechanism for this faster recovery will require further study.
Animals↗
Evaluation of testicular hCG binding in unilaterally cryptorchid rats following administration of ethane dimethane sulphonate (EDS).
Administration of ethane dimethane sulphonate (EDS) (75 mg/kg) to unilaterally cryptorchid rats, results in a rapid decline in serum testosterone levels and a marked reduction in hCG binding to homogenates of scrotal and cryptorchid testes, consistent with the destruction of Leydig cells within the testes as shown by morphological and morphometric analysis. Between 3 and 7 days after EDS, serum LH and FSH levels rise, LH to levels found in castrate rats. hCG binding begins to recover in scrotal testes, reaching 32% of control values 28 days after EDS. However, the recovery of hCG binding to cryptorchid testes is more rapid, reaching 51% of control levels by 21 days which coincides with the re-establishment of serum testosterone to normal levels. Since a different rate of recovery is observed for hCG binding to scrotal and cryptorchid testes, and both testes are exposed to the same circulating levels of LH and FSH, the likely stimulus for recovery of hCG binding and regeneration of Leydig cells is intratesticular in origin. This supports the concept that local factors, released following damage to the seminiferous epithelium by cryptorchidism, play a role in the differentiation and growth of Leydig cells within the testis.
Animals↗
The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells.
Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.
Animals↗
Detection of chloramphenicol acetyl transferase activity in transfected cells: a rapid and sensitive HPLC-based method.
An assay based on high-performance liquid chromatography (HPLC) has been developed for determining the expression of the acetyl coenzyme A: chloramphenicol 3-O-acetyltransferase gene in transfected cells. The assay is based on the separation of extracted, nonradioactive chloramphenicol and its acetylated derivatives by reverse-phase HPLC, using UV absorbance for detection. In addition to eliminating the need for costly [14C]chloramphenicol, this assay is as sensitive and much faster than the commonly used method involving thin-layer chromatography.
Acetyltransferases↗
Specific acylation of calmodulin. Synthesis and adduct formation with a fluorenyl-based spin label.
The spin-labeling reagent, N4-(9'-fluorenylmethyloxycarbonyl)-4-amino-1-oxyl-4-succinimidyloxyca rbonyl- 2,2,6,6-tetramethylpiperidine, and the same enriched in 14C at the 4-formyl group, were synthesized as new acylating compounds for protein amino groups that can preserve charge. Porcine testicular calmodulin was modified with this reagent at pH 7.8 in the presence of Ca2+ under conditions that yielded a fairly homogeneous derivative as judged by electrophoretic analysis and tryptic digestion patterns. The tryptic peptides were separated by gel filtration and reverse-phase high-performance liquid chromatography, and the resulting, highly purified 14C-labeled peptides were hydrolyzed and their amino acid compositions determined. The results indicate that at least 87% of the modifications occur at lysyl residues 75 and 148, and the former appears to be the most reactive. This bilabeled calmodulin adduct does not activate a bovine brain cyclic nucleotide phosphodiesterase preparation. The fluorenylmethyloxycarbonyl portion of this inactive calmodulin derivative can, however, be removed by conditions that do not diminish native calmodulin activity in the phosphodiesterase assay. The resulting calmodulin adduct is active in the enzymic assay, although with diminished potency compared to calmodulin. The specificity of the reaction of this acylating reagent with calmodulin may be due to recognition of the tricyclic fluorene ring by the phenothiazine-binding sites since it was found that trifluoperazine inhibited the labeling reaction. Also, calmodulin was far more reactive to this reagent than were several other proteins. This is the first report of a specific, characterized lysine modification on calmodulin, and it is possible that other phenothiazine-binding proteins may also exhibit similar selectivity for acylation. Electron paramagnetic resonance spectra of the calmodulin adducts suggest a high degree of spin immobilization in both the Ca2+-free and Ca2+-saturated states.
Acylation↗
Mouse glomerular culture.
A method is described for the culture of isolated intact mouse glomeruli. Cultures showed adherent glomeruli after 2 days and from 2-4 days a monolayer of cells began to develop around the points of adherence. The predominant cell (Type I) was 80-200 micrometer in diameter with cytoplasmic extensions, later becoming polyhedral and often multinucleated. Type II spindle cells, 70-100 micrometer in length, were less prominent in the earlier stages and later formed sheaves between Type I cells. Small phagocytic Type III cells, 10-20 micrometer in diameter, were not prominent in cultures from normal kidneys and tended to form small clusters on top of the Type I cell monolayer.
Animals↗
Phagocytic cells in glomerular cultures from NZB/W mice.
Comparative studies of glomerular cultures from NZB/W and normal control mice aged 2, 4, 6, 8, 10 and 12 months revealed a significant increase in the number of phagocytic cells in cultures from NZB/W mice at all ages. These cells on electron microscopy had the appearance of macrophages and on immunofluorescence showed Fc receptors and reacted with rabbit anti-mouse macrophage serum. The cells appear to be of blood monocyte origin and it is likely that they play a role in the pathogenesis in NZB/W mice of the glomerulopathy associated with clearance of immune complexes.
Animals↗
Macrophages in NZB/NZW mouse glomerular disease.
Glomerular cultures from mice have been established and the various cells associated with them observed microscopically by phase contrast time-lapse cinematography, after histological staining and ultrastructurally with the transmission and scanning electron microscope. The most common cell types were large 80-200 microns diameter irregular polyhedral cells appearing as flattened plate-like structures morphologically, possibly of epithelial origin. Smaller 70-100 microns diameter fusiform cells possibly derived from either endothelium or mesangium were also often seen. A third cell type, small 10-20 microns diameter circular, motile and actively phagocytic was especially noticeable in cultures of hybrid New Zealand Black/New Zealand White mice but was not present in appreciable numbers in control cultures from strains without renal disease. This third cell type appeared to have Fc receptors, stained positively with non-specific esterase and by indirect immunofluorescence with specific rabbit anti-mouse macrophage serum and had the morphological appearance of a macrophage by scanning and transmission electron microscopy. It is concluded that cells of macrophage origin are associated with the immune complex glomerulonephritis in these hybrid mice.
Age Factors↗
Congenital arthrogryposis in Charolais calves.
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Animals↗
Recrudescent melioidosis associated with diabetic ketoacidosis.
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Adult↗