Measurement of hemoglobin in long-term fixed erythroid cells: application development for cell science experiments in microgravity

Studying the effects of microgravity on cell differentiation will enhance our understanding of fundamental biology and is indispensable for a sustained space program. Rauscher murine erythroleukemic cells were chosen as a model system to study erythroid cell differentiation aboard the International Space Station because these cells undergo differentiation in response to the natural inducer, erythropoietin, as well as various chemical inducers. We have now developed a method to quantify hemoglobin in Rauscher cells after weeks of fixation and storage required for such space biology experiments. By exploiting the pseudoperoxidase activity of hemoglobin and by using reagents that yield a soluble chromophore that freely passes out of fixed cells, we developed a highly specific and sensitive assay applicable to cells fixed as long as 4 months.     

A lectin horseradish peroxidase study of the origin of ascending fibers in the medial forebrain bundle of the rat. The upper brainstem

The origins of projections within the medial forebrain bundle from the upper brainstem were examined with the horseradish peroxidase technique. Labeled cells were found in approximately 15 upper brainstem nuclei following injections of a conjugate of horseradish peroxidase and wheat germ agglutinin at various levels of the medial forebrain bundle. Labeled nuclei included (from caudal to rostral): dorsal and ventral parabrachial nuclei; Kolliker-Fuse nucleus; dorsolateral tegmental nucleus; A7 (lateral pontine tegmentum medial to lateral lemniscus); median and dorsal raphe nuclei; distinct group of cells oriented mediolaterally in the dorsal pontine tegmentum below the central gray; B9 (ventral midbrain tegmentum dorsal to medial lemniscus); retrorubral nucleus; nucleus of Darkschewitsch, interfascicular nucleus; rostral and caudal linear nuclei; ventral tegmental area; medial part of substantia nigra, pars compacta; and the supramammillary nucleus. With the exception of the ventral parabrachial nucleus, Kolliker-Fuse, A7, B9 and substantia nigra, pars compacta, each of the nuclei mentioned above sent strong projections along the medial forebrain bundle to the rostral forebrain. Sparse labeling was observed throughout the pontine and midbrain reticular formation. With the exception of the dorsal raphe nucleus, projections to the most anterior regions of the medial forebrain bundle (level of the anterior commissure) essentially only arose from presumed dopamine-containing nuclei-retrorubral nucleus (A8 area), interfascicular nucleus, rostral and caudal linear nuclei, substantia nigra pars compacta, and ventral tegmental area. Evidence was reviewed indicating that major forebrain sites of termination for these dopaminergic nuclei are structures that have been collectively referred to as the 'ventral striatum'. It is concluded from the present findings that several pontine and mesencephalic cell groups are in a position to exert a strong, direct effect on structures in the anterior forebrain and that the medial forebrain bundle is the main communication route between the upper brainstem and the forebrain.     

Development of the retinal pathway to the pretectum of the cat

The development of retinal projections to the pretectal complex of prenatal and early postnatal cats has been examined using the anterograde transport of horseradish peroxidase and tritiated amino acids. As early as embryonic day 38, the entire dorsal pretectum is penetrated by retinal ganglion cell axons. At this stage the bilateral complement of retinal efferents appears to be dispersed uniformly within the pretectal anlage. A week later, on embryonic day 46, indistinct foci of peroxidase reaction product can be discerned within 2 of the primordial nuclei: the nucleus of the optic tract and the olivary nucleus. By embryonic day 56, five distinct bilateral fields of retinal fiber termination are apparent within the following regions:

(i) the nucleus of the optic tract;

(ii) the pretectal olivary nucleus;

(iii) the posterior pretectal nucleus;

(iv) the anterior pretectal nucleus; and

(v) the medial pretectal nucleus. Four days before birth, on embryonic day 61, crossed and uncrossed retinal arbors are partially segregated within the nucleus of the optic tract and the pretectal olivary nucleus.

The early postnatal retinal connection to the pretectum has an overall pattern virtually indistinguishable from that of the mature cat. The ontogeny of the retinal influx to the pretectum is similar to that of the retinocollicular projection.⁶¹ However, the development of retinal projections to the pretectum and superior colliculus appears to lag behind those to the dorsal lateral geniculate nucleus.⁴⁹ These differences may reflect temporal and spatial gradients in the maturation of three major classes of retinal ganglion cells.     

一种灵敏稳定的酶联免疫吸附试验底物的研制及评价

目的：研制一种灵敏、稳定的酶联免疫吸附试验底物并进行方法学评价。方法制备了以四甲基联苯胺和过氧化氢脲为主要成分的底物体系,并对其灵敏度、精密性和存放稳定性进行了方法学鉴定试验,且与其他3种底物进行了比较。结果本研究自制底物与3种商品化底物相比,灵敏度高于其中2种,精密性试验平均变异系数为3．50％,4℃存放自制底物第1天、7天、1月、6月、12月测定其酶联物反应所得吸光度值分别为2．268、2．403、2．358、2．278、2．330,标准差为0．056185,变异系数为2．40％。结论本研究自制底物灵敏度和精密度高、稳定性强,配制方法简便、重复性好。既能满足实验室科研需要,作为检验试剂盒的一个重要组分也达到了研发商品化试剂盒的要求,为科研实验及科研转化提供了一种经济、有效的选择。     

A study of the origin of brain stem projections to monkey spinal cord using the retrograde transport method.

We identified brain stem neurons projecting to cervical and lumbar levels of the spinal cord in young rhesus monkeys using the retrograde transport method. The somatotopic organizations of the red nucleus and lateral vestibular nucleus were clarified. In addition, the presence of bulbospinal neurons in the medial vestibular nucleus; the nucleus of the tractus solitarius; the medial and lateral reticular formations; the raphe nuclei magnus, obscurus, and pallidus; the hypothalamus; and the nuclei of the locus ceruleus and subceruleus was confirmed.     

Cancer mortality and incidence among a cohort of benzidine and dichlorobenzidine dye manufacturing workers

BACKGROUND: Benzidine is classified as a definite human carcinogen and dichlorobenzidine as a probable human carcinogen. METHODS: A cohort study of 538 workers potentially exposed to benzidine and/or dichlorobenzidine from a single chemical manufacturing facility was conducted. Social Security records were used to identify all employees who worked at the facility from 1960 to 1977. Vital status was determined through 2001 and cancer incidence through 2002. RESULTS: A total of 22 bladder cancer cases were identified. For three individuals, bladder cancer was the underlying cause of death. Increased standardized mortality ratios (SMRs) were found for all cancer 1.54 (95% CI 1.04-2.19), bladder cancer 8.34 (95% CI 1.72-24.78), and lymphohematopoietic cancer 2.84 (95% CI 1.04-6.18). The standardized incidence ratio (SIR) for bladder cancer was 6.85 (95% CI 4.30-10.4). Only one case of bladder cancer was identified among the workers who were exposed to dichlorobenzidine only. However, an increased risk for lymphohematopoietic cancer was found among these dichlorobenzidine only workers (SMR 6.62 (95% CI 1.37-19.36)). CONCLUSIONS: This study confirms the high risk of bladder cancer among benzidine exposed workers even years after exposure has ceased, and raises suggestive evidence of increased risk to lymphohematopoietic cancer from exposure to benzidine or dichlorobenzidine.     

改进血清铜蓝蛋白凝胶电泳——氧化酶活性盐酸联苯胺显示法

目的：改进血清铜蓝蛋白（ＣＰ）的检测方法。方法：不连续淀粉－琼脂糖凝胶电泳分离血清ＣＰ，利用其氧化酶活性使 苯胺氧化，形成的有色产物沉积物凝胶板上ＣＰＲ 相应位置，可直接观察ＣＰ量的变化。结果：通过比较各种联苯胺的显色时间及显色效果。从而筛选出一种更为有效的血清ＣＰ测定方法。结论：氧化酶活性盐酸联苯胺显示法是最有效的血清ＣＰ检测方法。     

中药川芎嗪对慢性压力超负荷大鼠心肌纤维化的干预作用

目的为验证中药川芎嗪对心肌肥大心功能不全时改善心肌生物力学方面的功能。观察中药川芎嗪对压力超负荷大鼠心肌纤维化动态改变的影响。方法采用缩窄腹主动脉造成大鼠压力负荷模型。将63只大鼠SPSS软件采用随机分为3组:假手术组(shamoperatedgroups,SOG)、手术组(operatedgroups,OG)和手术+川芎嗪干预组(OG+川芎嗪)。每组分为术后1,2,4,7,14,21,30d7个时间点。采用组织病理学和计算机图像分析技术对在各时间点大鼠心肌纤维化进行形态学和形态计量法上的比较。结果(1)OG组光镜下显示出反应性纤维化和修复性纤维化阶段,OG+川芎嗪组心肌纤维化程度明显比OG组减轻,未见修复性纤维化出现。(2)OG组心肌血管周围纤维化(perivascularcollagenarea,PVCA)在术后1d(2.09±0.45)就已显著高于SOG组(0.83±0.06),以后平稳上升,而与此同时OG+川芎嗪组PVCA(1.16±0.06)显著低于OG组;OG组心肌胶原容积分数(collagenvol-umefraction,CVF)在术后2d(3.08±0.56)已显著高于SOG组(2.78±0.64),从术后21d始呈现迅速上升的趋势,而OG+川芎嗪组CVF从术后21d(4.69±0.85)开始显著低于OG组(7.56±0.88),P均<0.05。结论川芎嗪可减缓压力超负荷大鼠心肌胶原的沉积,具有心脏保护作用。     

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFNγ, IL-1α, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1α, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.     

中药制剂中川芎嗪含量的高效液相色谱法测定

用高效液相色谱法(HPLC)建立了制剂中川芎嗪含量的测定方法。实验结果表明,其回收率(97.93±0.5220%)、精密度(CV为同日内1.39%、日间1.51%)都能满足定量检测要求,最低检出量可达0.005μg。