The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Bradykinin-induced transient accumulation of inositol trisphosphate in neuron-like cell line NG108-15 cells

Studies were undertaken to further elucidate the mechanism(s) by which bradykinin-dependent phosphoinositide metabolism takes place in neuroblastoma X glioma hybrid NG108-15 cells [(1984) J. Biol. Chem. 259, 10201-10207] using [3H]inositol-labelled cells. Bradykinin produced net increases in the level of [3H]inositol phosphates, especially of [3H]inositol trisphosphate which is formed transiently and most rapidly. The results indicate that bradykinin activates a phosphodiesterase to break down phosphatidylinositol 4,5-bisphosphate, generating two recently recognized intracellular messengers, 1,2-diacylglycerol and inositol trisphosphate.     

Paratropomyosin: a new myofibrillar protein that modifies the actin-myosin interaction in postrigor skeletal muscle. I. Preparation and characterization

A protein component which is released from skeletal-muscle myofibrils on the treatment with Ca2+ at concentrations above 10(-5) M and modifies the actin-myosin interaction was purified by a method involving column chromatography on Sephadex G-200 and DEAE-cellulose in succession. Although this protein resembles tropomyosin in some physicochemical properties, it has the same chain weight of 34,000 as the alpha-component of tropomyosin on SDS-polyacrylamide gel electrophoresis, and differed from tropomyosin not only in the amino acid composition but also in prolonging the clearing phase of superprecipitation of reconstituted actomyosin. We therefore concluded that this protein is a new myofibrillar one, and termed it "paratropomyosin." In postrigor muscle, it seems likely that paratropomyosin is released from its original locus with an increased concentration of Ca2+, and that it weakens rigor linkages formed between actin and myosin.     

Fine structural changes in the rat brain after local injections of gliotoxin, alpha-aminoadipic acid

Toxic effects of locally injected gliotoxin, L-alpha-aminoadipic acid, were examined in the adult rat brain at the fine structural level. Astrocytes were the only cell type in the corpus striatum that showed clear structural degeneration 1 to 3 days after the treatment. With a 7 day survival period, two major structural changes were detectable, including tremendous invasion of astrocytes and their processes, and an appearance of a large number of microglia around injection sites. Both the stereospecificity and cellular specificity were firmly established on the toxic effects of this chemical.     

Diazepam action onγ-aminobutyric acid-activated chloride currents in internally perfused frog sensory neurons

The Cl- current (ICl) in gamma-aminobutyric acid (GABA)-sensitive frog sensory neuron was separated from other Na+, Ca2+, and K+ currents using a suction pipette technique which allows internal perfusion under a single-electrode voltage clamp. Diazepam (DZP) itself evoked no response but facilitated the dose- and time-dependently GABA-induced ICl without changing the GABA equilibrium potential (EGABA) at concentrations ranging widely, from 3 X 10(-9) to 10(-4) M. In the presence of DZP, the GABA dose-response curve shifted to the left without changing the maximum current, indicating that DZP modifies the interaction between GABA and its receptor rather than affecting directly the channel activation step. The enhancement of the GABA-induced ICl by DZP depended neither on the membrane voltage nor on the inward or outward direction of the ICl. DZP also potentiated the ICl elicited by GABA agonists such as beta-alanine, taurine, homotaurine, 5-aminovaleric acid, l-GABOB, d-GABOB, glycine, and muscimol. The GABA response enhanced by pentobarbital (PB) was further enhanced by adding DZP, indicating that DZP and PB do not act in the same way. Ro5-3663, a diazepam analogue, enhanced the GABA-induced ICl only in a narrow range of the concentrations but inhibited the current at concentrations higher than 2 X 10(-6) M.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

Kinetics of microcolony formation of a soil oligotrophic bacterium, Agromonas sp.

Abstract The kinetics of budding/dividing of parent cells at different culture ages, spread on a fresh medium, was formulated by the following model N _t= N _∞ [1 − exp (− λ ( t − t _r)] where N _t is the number of budding/dividing cells in the parent population at time t , N _∞ is the expected number of budding/dividing cells at infinite time, λ is the rate of budding/dividing of parent cells, and t _r is the retardation time. The rate of budding/dividing λ decreased with the increase in the culture age of the parent cell population.