β-Nerve Growth Factor Measurements in Mouse Serum

Mouse serum beta-nerve growth factor (NGF) levels were measured using a newly developed competitive beta-NGF radioimmunoassay. The basal serum beta-NGF levels in male and female mice were consistently less than 2 ng/ml when these animals were maintained in individual cages for at least 7 days before they were killed. However, in male mice, serum beta-NGF levels were significantly elevated when they were housed 5 per cage. The rise in serum beta-NGF levels, presumably mediated by intermale aggression, was confirmed by grouping previously isolated mice together in one cage for 20 min before they were killed. In all aggressive male mice, serum beta-NGF levels were elevated by two orders of magnitude. The beta-NGF radioimmunoassay values were also validated by a neurite outgrowth bioassay system using the serum of aggressive male mice. In summary, both measurement techniques confirm that mouse serum beta-NGF levels undergo marked changes depending on animal handling conditions.     

Temporal Profiles of Nerve Growth Factor β-Subunit Level in Rat Brain Regions After Transient Ischemia

To determine the role of nerve growth factor (NGF) in ischemic brain damage, we measured the temporal and regional changes in the level of NGF in the hippocampal subfields, the cerebral cortex, the striatum, and the septum at 1, 2, 7, and 30 days after transient forebrain ischemia using a highly sensitive sandwich-type enzyme immunoassay system for the beta-subunit of mouse 7S NGF (beta-NGF). We also analyzed glial fibrillary acidic protein immunoreactivity in the hippocampus to ascertain the contribution of reactive astrocytes to NGF production after an ischemic insult. In the CA1 subfield of the hippocampus, the level of beta-NGF decreased slightly 2 days after ischemia (not significant), at which time CA1 pyramidal cell loss began to occur, and increased by 40% 30 days after ischemia (p less than 0.05). A marked increase in glial fibrillary acidic protein-positive astrocytes in the CA1 subfield 2-30 days after ischemia suggests that the reactive astrocytes participated in a gradual increase in the level of beta-NGF after recirculation. The level of beta-NGF in the dentate gyrus decreased transiently 2 days (p less than 0.05) and 7 days (p less than 0.01) after ischemia, followed by recovery to the level of control animals 30 days after ischemia. The level of beta-NGF in the septum gradually decreased 7 days (-27%, p less than 0.05) and 30 days (-43%, p less than 0.01) after ischemia. The levels of beta-NGF in the cerebral cortex and striatum remained unaltered throughout the observation period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental Changes in Distribution of Acidic Fibroblast Growth Factor in Rat Brain Evaluated by a Sensitive Two-Site Enzyme Immunoassay

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.     

Effects of Transforming Growth Factor β1 on Astroglial Cells in Culture

The effects of transforming growth factor beta 1 (TGF beta 1) on DNA synthesis and functional differentiation of astroglial cells cultured in serum-free medium were investigated. TGF beta 1 diminished and delayed the peak of DNA synthesis induced by serum. TGF beta 1-treated cells were larger than control cells. This factor delayed the appearance of process-bearing cells induced by acidic fibroblast growth factor treatment and also affected the astrocyte-specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGF beta 1 inhibited the induction of GS activity by HC in a dose- and time-dependent manner. Moreover, pretreatment with TGF beta 1 for 4 h maintained the inhibition of GS activity for approximately 16 h after removal of this factor from culture medium. These results suggest that TGF beta 1 may be an important regulator of astrocyte growth and differentiation.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

β,β''-Iminodipropionitrile Impairs Retrograde Axonal Transport

beta,beta'-Iminodipropionitrile (IDPN), a neurotoxin, causes redistribution of neurofilaments in axons followed by the development of proximal axonal swellings and, in chronic intoxication, a distal decrease in axonal caliber. The latter changes are caused by a selective impairment in the slow anterograde axonal transport of neurofilament proteins. To assess the role of retrograde axonal transport in IDPN toxicity, we used [3H]N-succinimidyl propionate ([3H]NSP) to label covalently endogenous axonal proteins in sciatic nerve of the rat and measured the accumulation of radioactively labeled proteins in the cell bodies of motor and sensory neurons over time. IDPN was injected intraneurally 6 h or intraperitoneally 1 day before subepineurial injection of [3H]NSP into the sciatic nerve, and the animals were killed 1, 2, and 7 days after [3H]NSP injection. Neurotoxicity was assessed by electron microscopic observation of the nerves of similarly treated animals. Both intraneural and intraperitoneal injection of IDPN caused an acute reduction in the amount of labeled proteins transported back to the cell bodies. The early appearance of these changes suggests that alterations in retrograde transport may play a role in the production of the neuropathic changes.     

鼠神经生长因子对电烧伤患者神经修复的作用及其机制研究

目的:探讨鼠神经生长因子对电烧伤患者神经修复的作用及其机制。方法:选取2013年2月至2014年11月期间我院确诊治疗的四肢电烧伤患者128例,依据随机分配原则分为对照组和神经组,对照组患者给予常规皮瓣修复术治疗,神经组患者在此基础上给予鼠神经生长因子治疗,且依据给药方式分为全身亚组和局部亚组。统计分析所有患者创面愈合时间、感染和出血发生情况,采用BMRC感觉、运动功能评级法评估患者感觉、运动功能恢复情况,应用Spearman分析法分析二者之间的关系。结果:神经组患者创面愈合时间、感染和出血发生率明显低于对照组,差异有统计学意义(P0.05);局部亚组患者感觉功能优良率为90.63%,全身亚组为84.38%,对照组为71.88%,局部亚组全身亚组对照组,差异有统计学意义(P0.05);局部亚组患者运动功能优良率为93.75%,全身亚组为84.38%,对照组为76.56%,局部亚组全身亚组对照组,差异有统计学意义(P0.05);Spearman分析法结果显示,感觉功能与运动功能呈正相关(r=0.812,P0.05)。结论:鼠神经生长因子可有效提高电烧伤患者神经修复的作用,有利于改善患者术后创面愈合、感染、出血情况,促进患者感觉、运动功能恢复,且通过局部给药方式具有更为良好的神经修复作用,值得临床作进一步推广。     

Glycosidases of apple fruit: A multi-functional β-galactosidase

Extraction of Spartan apple ( Malus domestica ) fruit acetone powder and fractionation of the extract on DEAE-agarose allowed detection and quantification of 10 glycosidases active toward 4-methylumbelliferyl glycosides. Hydrolysis was measured fluorimetrically. The predominant activity, a β- d -galactosidase (EC 3.2.1.23), labile upon purification, was stabilized by soluble PVP. Molecular weights, measured by gel permeation HPLC, pH optima and K_m values were obtained for most glycosidase activities. Multiple forms of several activities were found. The major α- d - and β- d -galactosidases were resolved on phosphocellulose. The β- d -galactosidase so obtained had associated α- l -arabinopyranosidase and β- d -fucosidase activities which were retained upon GP-HPLC. Mixed substrate kinetic analysis and inhibition analysis of this fraction indicated that the enzyme has 3 catalytic sites, 1 for each substrate, whose substrates mutually influence each other's activity positively.     

利用SEC—HPLC测定鼠神经生长因子的纯度

目的：建立检测鼠神经生长因子（mNGF）纯度的分子排阻高效液相色谱法（SEC-HPLC）。方法：利用TSK G3000 SWXL分析柱和Waters 2695/2487高效液相色谱系统检测mNGF标准品纯度,并对方法的专属性、线性、重复性、中间精密度、检测限及耐用性等指标进行评估。结果：空白溶剂在mNGF积分范围内无特异峰出现;样品浓度与吸收峰之间线性回归系数R2=0.9998;6次进样峰面积相对标准偏差（RSD）为1.18%;中间精密度分析RSD为0.45%;检测限为0.2μg;耐用性实验表明磷酸盐浓度在0.2~0.3 mol/L之间变动对检测结果无影响。结论：各项指标符合规定,SEC-HPLC法可用于检测mNGF的纯度。     

Action of β-N-Oxalylamino-l-Alanine on Mouse Brain NADH-Dehydrogenase Activity

Abstract: β- N -Oxalylamino- l -alanine ( l -BOAA), a non-protein neuroexcitatory amino acid present in the seeds of Lathyrus sativus (chickling or grass pea), is known to produce its neurotoxic effects by overstimulation of non- N -methyl- d -aspartate receptors, especially α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, at micromolar concentrations. It has recently been reported that l -BOAA selectively inhibits mitochondrial enzyme NADH-dehydrogenase (NADH-DH) in brain slices at subpicomolar concentrations. The present study finds that up to 4 m M concentrations of pure l -BOAA fail to inhibit NADH-DH activity in mouse brain homogenate and isolated brain mitochondria. Two known inhibitors (rotenone and 1-methyl-4-phenylpyridinium ion, MPP⁺) of this mitochondrial enzyme produced significant inhibition under identical conditions. NADH-DH inhibition was also not observed in the homogenate or mitochondria from the brains of animals systemically treated with convulsive doses of l -BOAA. Some inhibition (20–37%) of NADH-DH activity was observed in mouse brain slices incubated with 100–1,000 µ M concentrations of l -BOAA for 1 h at 37°C in an atmosphere of 95% O₂ and 5% CO₂, but the inhibition was nonselective, because the activity of another mitochondrial enzyme, succinic dehydrogenase, was similarly inhibited by l -BOAA. These results are in contrast with the report that l -BOAA inhibits mitochondrial NADH-DH selectively at subpicomolar concentrations. We suggest the observed nonselective NADH-DH inhibition in mouse brain slices treated with l -BOAA is caused by neuronal damage through an excitotoxic mechanism.     

小鼠神经生长因子直接竞争酶联免疫检测方法的建立

目的:利用小鼠神经生长因子(mNGF)和纯化的兔抗mNGF IgG,建立mNGF直接竞争酶联免疫检测方法。方法:以亲和纯化兔抗mNGF IgG作为包被抗体,封闭、洗涤后加入用稀释液稀释的不同浓度的标准mNGF或样品液和生物素标记的mNGF混合液,于37℃孵育80 min或室温孵育120 min,洗涤后加入辣根过氧化物酶标记的链酶亲和素,洗涤后加入显色液显色,测定D450nm值,通过标准曲线计算待测样品中mNGF含量。结果与结论:建立了mNGF直接竞争酶联免疫检测方法;该方法的灵敏度约20 ng,变异系数为批内≤10%、批间≤15%,回收率为85%～115%;利用该方法检测了3批样品中mNGF的含量。     

石杉碱甲联合注射用鼠神经生长因子对老年痴呆MMSE评分及日常生活能力的影响

目的:研究石杉碱甲联合注射用鼠神经生长因子对老年痴呆简易智力状态检查量表(MMSE)及日常生活能力量表(ADL)评分的影响。方法:选择2015年6月～2018年6月我院收治的98例老年痴呆患者,随机分为两组。对照组口服石杉碱甲,起始给药剂量为每次100μg,每天2次,第2周给药剂量调整为每次150μg,每天2次,维持用量。观察组联合静脉滴注鼠神经生长因子,每次20μg,每天1次。比较两组治疗前后ADL和MMSE评分、血清白细胞介素(IL)-1β、C-反应蛋白(CRP)、IL-6、肿瘤坏死因子-α(TNF-α)水平的变化。结果:治疗后,观察组的总有效率为87.75%(43/49),明显高于对照组[69.39%(34/49)](P0.05);两组治疗后的ADL和MMSE评分及血清IL-1β、CRP、IL-6和TNF-α水平均较治疗前明显降低(P0.05),且观察组以上指标均明显低于对照组(P0.05)。结论:与单用石杉碱甲相比,注射用鼠神经生长因子联合石杉碱甲可更有效改善其日常生活能力和认知功能,且能显著降低血清炎症因子水平。     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β-NGF levels disappeared. Average levels of β-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions. The concentrations of mRNA for β-NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain-derived neurotrophic factor and neurotrophin-3 between the two sexes. Thus, in some regions of the brain, the production of β-NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β-NGF in these region.     

Three Distinct Types of Monoclonal Antibodies After Long-Term Immunization of Rats with Mouse Nerve Growth Factor

This article reports the results of a systematic investigation of the different types of antibodies produced in the course of a long-term immunization of rats with mouse nerve growth factor (NGF). We have characterized three types of monoclonal antibodies, namely: (1) antibodies that bind to NGF and inhibit its binding to target cells and its biological activity in culture (type A); (2) antibodies that bind to and precipitate NGF but do not inhibit its binding to target cells or its biological activity (type B); (3) antibodies that fail to recognize NGF itself, but inhibit nonetheless its binding to target cells (type C). These antibodies bind to an antigen present on NGF target cells and not on rat fibroblasts lacking NGF receptor. They appear thus to be antiidiotypic antibodies directed against the NGF receptor, developed as a consequence of the long-term immunization with NGF.