Dexamethasone Inhibits Mitogen-dependent Synthesis of Histamine Due to Histidine Decarboxylase Induced in Cultured Mouse Spleen Cells

Schizosaccharomyces pombe has four α-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Δ was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Δ, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Δ had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.     

Lipopolysaccharide and Interleukin-1β Augmented Histidine Decarboxylase Activity in Cultured Cells of the Rat Embryonic Brain

Abstract: We investigated the effect of lipopolysaccharide (LPS) and various inflammatory cytokines on the histidine decarboxylase (HDC) activity in cultured cells of the rat embryonic brain. Histaminergic neuronal cell bodies were supposed to exist in cultured cells of the diencephalon but not in those of the cortex. The HDC activity was elevated by adding LPS and interleukin-1 β (IL-1β) but not by tumor necrosis factor-α (TNF-α) and IL-6 to the mixed primary cultures of diencephalon. In the adherent cell fraction of the cultured diencephalon cells, HDC activity was also enhanced by LPS and IL-1β. In a similar manner, LPS augmented HDC activity in the mixed primary culture of cerebral cortical cells and in its adherent cell fraction. The effects of IL-1β but not LPS in the mixed primary culture of diencephalon were canceled by a prior exposure to cytosine-β- d -arabinofuranoside. The changes in HDC activity after exposure to LPS for 12 h were not accompanied by increased mRNA levels. In these cell cultures, mast cells were not detected by Alcian Blue staining. These results indicated the presence of the third type of HDC-bearing cell besides neurons and mast cells in the brain. The increase of HDC activity by IL-1β might be due to cell proliferation.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Detection of Ornithine Decarboxylase Antizyme in Mouse Brain

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, is known to be regulated by a macromolecular inhibitor, termed antizyme, in a number of cellular systems. The present results show that the antizyme is also a functional component of polyamine metabolism in the brain. It could be demonstrated both in normal randomly selected mice and in animals which had been subjected either to intracerebroventricular injection of saline, which is known to cause a transient activation of ornithine decarboxylase, or to 1,3-diamino-2-propanol, an antizyme-inducing agent. When compared to tissues or cell systems studied so far, the cytosol fraction from mouse brain homogenate appeared to contain an exceptionally high amount of antizyme, that was bound to some material other than active ornithine decarboxylase. This feature was seen in all the animal groups studied, being most prominent after saline injection, when the amount of dissociable antizyme exceeded 14-fold the corresponding released ornithine decarboxylase activity. In untreated animals the excess was about eightfold and after 1,3-diamino-2-propanol about fivefold.     

Effect of Muramyldipeptide,a Synthetic Bacterial Adjuvant,on Enzyme Release from Cultured Mouse Macrophages

Monolayer cultures of macrophages obtained by peritoneal lavage of normal or thioglycollate-stimulated mice spontaneously secreted lysosomal enzymes into the culture medium. When the elicited macrophages were cultured in the presence of muramyldipeptide (MDP), a 20–30% increase in the release of β-glucuro-nidase was consistently observed and the intracellular activity decreased to about 45% of that of control cells after 6–8 days' culture. A stimulatory effect of MDP on lysozyme secretion, though less profound, was also observed. In contrast, release of neither enzyme was stimulated in resident macrophages by the addition of MDP. A neutral α-glucosidase, which has recently been found to localize also in granules of macrophages, remained inside the cells and neither its activity nor its release was affected by the addition of MDP to either type of macrophages. A large amount of lactic dehydrogenase was released only when the resident, not the elicited, macrophages were cultured for 3–4 days and then phagocytosed zymosan.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

Antisera Against Rat Recombinant Histidine Decarboxylase: Immunocytochemical Studies in Different Species

Histamine is involved in many important biological processes such as allergic reactions, gastric acid secretion and neurotransmission. The formation of histamine is catalysed by the enzyme histidine decarboxylase. In order to understand the role of histamine in different tissues, information about the cellular localisation of the decarboxylase is important. However, the availability of antisera against the enzyme, which can be used in immunocytochemical techniques, has so far been limited, mainly due to the difficulties in purifying sufficient amounts of histidine decarboxylase from various tissues. In the present study we describe the use of antisera raised against rat recombinant histidine decarboxylase to localise the enzyme immunocytochemically in the gastric mucosa of different mammals and submammalian vertebrates. The antisera specifically stained histidine decarboxylase-immunoreactive cells in the gastric mucosa of not only rat, but also of species like frog, chicken, mouse and dog. This is the first report describing the immunocytochemical distribution of the decarboxylase in the gastric mucosa of species other than rat. These antisera are likely to become valuable tools for further studies of the immunocytochemical localisation of histidine decarboxylase.     

Two-Step Purification of Mouse Kidney Ornithine Decarboxylase

Abstract

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4. 1. 1. 17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1. 0 × 10⁶?1. 4 × 10⁶ nmol/h. mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54, 000 and a minor band of Mr 51, 000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [¹⁴C)difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Calcium Effects on the Solubilization of Membrane-Bound Histidine Decarboxylase in the Rat Brain

In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane-bound form. A major part (82%) of the membrane-bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane-containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of approximately 50% of membrane-bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.     

Modulation of Leishmania (L.) amazonensis Growth in Cultured Mouse Macrophages by Prostaglandins and Platelet Activating Factor

The role of endogenously synthesized PAF and prostaglandins on the infection of mouse macrophages by Letsbmanta (L.) amazonensis was investigated, as well as the possible correlation between the effects of these inflammatory mediators with nitric oxide production. It was found that pretreatment of macrophages with 10(-5) M of the PAF antagonists, BN-52021 or WEB-2086, increased macrophage infection by 17 and 59%, respectively. The cyclooxygenase inhibitor, indomethacin (10 mug/ml), induced a significant inhibition which was reversed by addition of PGE (10-3 M) to the culture medium. These results suggested that the infection of macrophages by leisbmanla is inhibited by PAF and enhanced by prostaglandins and that these mediators are produced by macrophages during this infection. This was confirmed by addition of these mediators to the culture medium before infection; PAF (10(-6), 10(-9) and 10(-12)M) reduced significantly the infection whereas PGE(2) (10(-5) M) induced a marked enhancement. This effect of exogenous PAF on macrophage infection was reversed by the two PAF antagonists used in this study as well as by the inhibitor of nitric oxide synthesis, L-arginine methyl ester (100 mM). Taken together the data suggest that endogenous production of PAF and PGE(2) exert opposing effects on Lesbmana-macrophage interaction and that nitric oxide may be involved in the augmented destruction of parasites induced by PAF.     

Histamine Receptor Expression, Hippocampal Plasticity and Ammonia in Histidine Decarboxylase Knockout Mice

Genetic ablation of the histamine producing enzyme histidine decarboxylase (HDC) leads to alteration in exploratory behaviour and hippocampus-dependent learning. We investigated how brain histamine deficiency in HDC knockout mice (HDC KO) affects hippocampal excitability, synaptic plasticity, and the expression of histamine receptors. No significant alterations in: basal synaptic transmission, long-term potentiation (LTP) in the Schaffer collateral synapses, histamine-induced transient changes in the CA1 pyramidal cell excitability, and the expression of H1 and H2 receptor mRNAs were found in hippocampal slices from HDC KO mice. However, when compared to WT mice, HDC KO mice demonstrated: 1. a stronger enhancement of LTP by histamine, 2. a stronger impairment of LTP by ammonia, 3. no long-lasting potentiation of population spikes by histamine, 4. a decreased expression of H3 receptor mRNA, and 5. less potentiation of population spikes by H3 receptor agonism. Parallel measurements in the hypothalamic tuberomamillary nucleus, the origin of neuronal histamine, demonstrated an increased expression of H3 receptors in HDC KO mice without any changes in the spontaneous firing of “histaminergic” neurons without histamine and their responses to the H3 receptor agonist (R)-α-methylhistamine. We conclude that the absence of neuronal histamine results in subtle changes in hippocampal synaptic transmission and plasticity associated with alteration in the expression of H3 receptors.     

乳鼠成骨细胞体外培养

目的建立乳鼠成骨细胞体外培养方法,探讨该方法的可行性和应用价值。方法用出生1~3 d乳鼠颅骨,采用多次胶原酶消化法进行细胞体外培养。倒置显微镜观察细胞形态,对其碱性磷酸酶(ALP)活性及矿化能力进行鉴定,并测定细胞生长曲线。结果原代培养24 h后,大量细胞贴壁生长,细胞呈圆形,48 h后,贴壁细胞呈长梭形、三角形或不规则多边形,并且贴壁细胞伸出2~3个突起,胞质透亮、饱满,7 d后细胞铺满整个平皿底面。经鉴定,培养细胞具有体内成骨细胞的生物学特性。细胞接种后第1与第2个24 h为细胞的潜伏适应期,第3与第7个24 h生长曲线基本为线性曲线,是细胞的对数生长期。结论采用胶原酶消化法分离培养成骨细胞的方法切实可行。     

Transcription-Dependent and -Independent Induction of Cerebral Ornithine Decarboxylase

Ornithine decarboxylase (ODC; EC 4.1.1.17) is a highly inducible, rate-limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonal changes and seizures on these regional differences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK-801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression.     

Partial Purification and Characterization of A Novel Histidine Decarboxylase from Enterobacter aerogenes DL-1

Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca²⁺ and Mn²⁺ showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver–Burk plot showed that K _m and V _max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.     

Fas-Mediated Apoptosis in Primary Cultured Mouse Hepatocytes

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.     

小鼠腹腔巨噬细胞的分离与培养

目的:建立一种小鼠腹腔巨噬细胞分离的简便方法,为低强度激光照射对巨噬细胞功能的影响研究提供实验细胞.方法:以无血清的RPMI-1640培养液灌洗小鼠腹腔,分离获取小鼠腹腔巨噬细胞,在含10%小牛血清的RPMI-1640培养液中培养.采用倒置显微镜观察细胞形态,台盼蓝染色计算存活率,瑞氏染色计算纯度.结果:获得高纯度的巨噬细胞,具备巨噬细胞的形态特征.结论:本法是一种简便实用的分离小鼠腹腔巨噬细胞的方法.