Mechanism of endotoxin-induced reduction in the number of beta-adrenergic receptors in dog livers: role of phospholipase A

The role of phospholipase A on the endotoxin-induced reduction in the number of beta-adrenergic receptors in dog liver plasma membranes was investigated. The results show that digestion of control liver plasma membranes with exogenous phospholipase A2 (0.2 unit/200 micrograms protein) decreased the specific binding of (-)-[3H]dihydroalprenolol by 37.3% (P less than 0.01) and reduced the number of receptor sites by 31.7% (P less than 0.05). These decreases in the specific binding and the number of beta-adrenergic receptors were completely reversible by the addition of phosphatidylcholine (0.2 mM). Endotoxin administration (2 hr postendotoxin) decreased the specific binding by 36% (P less than 0.05) and reduced the number of beta-adrenergic receptors by 33% (P less than 0.05), and these decreases were completely reversible by the addition of 0.2 mM phosphatidylcholine. Digestion of control liver membranes with exogenous phospholipase A2 decreased phosphatidylcholine and phosphatidylethanolamine levels by 50.6 and 51.2%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine levels by 12- and 8.4-fold, respectively. Endotoxin administration decreased phosphatidylcholine and phosphatidylethanolamine contents by 21.4 and 23.8%, respectively, but increased lysophosphatidylcholine and lysophosphatidylethanolamine contents by 2.1- and 1.4-fold, respectively. In addition, endotoxin administration increased endogenous phospholipase A activity by 73.5%. Based on these results, it is suggested that the decreases in the specific binding and the number of beta-adrenergic receptors in dog livers during endotoxic shock are a result of phospholipase A activation.     

大鼠脊髓中的α受体在减压反射和失血性休克中的作用

大鼠脊髓蛛网膜下腔注射α激动剂可乐宁1μg,引起血压降低、心率减慢及腹腔神经节后交感神经干放电抑制。应用α阻断剂酚妥拉明阻断脊髓内源性 NE的作用,可部分抑制血压升高时反射性的心率减慢和交感神经放电抑制反应,使压力感受器反射的敏感性降低。在颈动脉放血造成不可逆性失血性休克的动物,脊髓蛛网膜下腔注射酚妥拉明可使动脉血压有一定程度的回升。以上结果表明,由脊髓α受体调制的心血管抑制效应参与减压反射以及失血性休克的发病机制。     

Kinase Signaling in the Spindle Checkpoint

The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the “wait anaphase” signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.     

Expression of mosquitocidal crystal protein genes in non-insecticidal Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis NTB-1 isolated from soil samples in Korea produces ovoidal parasporal inclusions with proteins of approximately 24–40 kDa in size. Although serological study indicated that the isolate has a flagella (H) antigen identical with subsp. israelensis , it seemed to be non-insecticidal against Lepidoptera and Coleoptera as well as Diptera. To investigate the activity of non-insecticidal B. thuringiensis transformed with insecticidal crystal protein genes, cryIVD and cytA genes of B. thuringiensis subsp. morrisoni PG-14, highly toxic to mosquito larvae, were introduced into the isolate NTB-1. The expression of mosquitocidal crystal protein genes in NTB-1 was characterized by SDS–PAGE analysis and electron microscopy. The results showed that crystalline inclusions of host, CryIVD and CytA were stably expressed in the transformant. However, the mosquitocidal activity of transformant was similar to that of B. thuringiensis subsp. kurstaki Cry⁻ B harbouring cryIVD and cytA genes, demonstrating that a synergistic effect by an interaction of both introduced insecticidal and resident non-insecticidal crystal proteins was not observed.     

Component proteins in crude extract of adult Paragonimus westermani purified by immunoaffinity chromatography using monoclonal antibodies.

The nature of 2 component proteins in crude saline extract of adult Paragonimus westermani was investigated. By immunoaffinity chromatography using monoclonal antibodies (MAb) as ligands, the proteins were purified from the crude extract. Band 1 protein in disc-polyacrylamide gel electrophoresis (PAGE) was purified by PFCK-136 MAb. The protein, known to have molecular mass of 440 kDa, was composed of 23, 46 and 92 kDa subunits when observed by reducing SDS-PAGE and SDS-PAGE/immunoblot. This protein was originated from eggs of the worm as revealed by immunohistochemical staining with PFCK-136 Mab. Another affinity purified protein utilizing PFCK-44 MAb was the band 4 protein of 17 kDa in disc-PAGE. This was a monomer protein in reducing SDS-PAGE and SDS-PAGE/immunoblot. The protein was produced at intestinal epithelium of the worm.     

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.     

INHIBITION OF CATALASE IN MESENCEPHALIC CULTURES BY l-DOPA AND DOPAMINE

Catalase activity in cell cultures of fetal rat mesencephalon was decreased by 42 and 50%, respectively, after exposure to l-3,4-dihydroxyphenylalanine (l-DOPA, 100 μM) or dopamine (100 μM) for 48 h. Catalase activity was also decreased 21% by 10 μM hydroquinone. Ascorbic acid (200 μM), an agent that suppresses the autoxidation of l-DOPA and dopamine, blocked the anti-catalase effect of l-DOPA, but not that of dopamine. Inhibitors of the A and B forms of monoamine oxidase (20 μM clorgyline plus 20 μM pargyline) had no effect on the anti-catalase action of either l-DOPA or dopamine. The latter results suggest that products of the oxidative deamination of dopamine by monoamine oxidase are not involved in the suppression of catalase activity. However, autoxidation reactions of l-DOPA may play a role since ascorbate suppressed the anti-catalase effect of l-DOPA. On the contrary, the basis for the failure of ascorbate to similarly block the anti-catalase effect of dopamine is uncertain. l-DOPA and dopamine (25 μM) also inhibited crystalline catalase in solution after incubation for 1 h at neutral pH (40–50% inhibition). Inhibition was blocked by 0.45 M ethanol, indicating a need for autoxidation and the formation of compound II, which is an enzymatically inactive form of catalase. The ability to model the enzyme inhibition in purely chemical experiments indicates a probable mechanism for loss of enzymatic activity in cell cultures. Inhibition of catalase may contribute to cell damage during incubation of cultures with l-DOPA, dopamine, or other autoxidizable compounds. Copyright © 1996 Elsevier Science Ltd     

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.