CENTRIFUGAL,BIOCHEMICAL, AND ELECTRON MICROSCOPIC ANALYSIS OF CYTOPLASMIC PARTICULATES IN LIVER HOMOGENATES

A combined centrifugal, biochemical, and electron microscopic study of the cytoplasmic particulates present in 0.88 M sucrose homogenates of rat liver has been carried out. Size distribution analyses of particles containing pentose nucleic acid (PNA) and exhibiting several types of enzymatic activity revealed three major size groups within the range of particle radius between 10 and 500 mµ. A different array of biochemical properties was associated with each size group. The largest particles, with an average radius (assuming spherical shape) in the region of 220 to 260 mµ, contained all of the succinic dehydrogenase activity of the cytoplasmic extract, 29 per cent of the diphosphopyridine nucleotide (DPN)-cytochrome c reductase activity, and minor amounts of PNA and acid phosphatase activity. Cytologically, this group of particles was identified with the mitochondria. All of the uricase activity, 58 per cent of the acid phosphatase activity, and 26 per cent of the PNA was apparently associated with a second size group of particles (average radius 120 mµ) which were tentatively identified by electron microscopy with vesicular structures derived from the ergastoplasm of the intact cell. The third particle group demonstrated by centrifugation exhibited a major size distribution peak at 25 mµ and a second smaller peak at 55 mµ. Over 50 per cent of the total cytoplasmic PNA and DPN-cytochrome c reductase activity was associated with particles in this size group. Electron microscopy revealed a morphologically heterogeneous population of particles within this size range.     

CYSTATHIONINE SYNTHESIS AND DEGRADATION IN BRAIN, LIVER AND KIDNEY OF THE DEVELOPING MONKEY

Abstract— The concentration of cystathionine, along with the specific activities of the enzymes involved in its synthesis and degradation, cystathionine synthasc and cystathionase, respectively, have been measured in brain, liver and kidney of the developing Rhesus monkey from mid-gestation, through birth and neonatal life, to maturity. The concentration of cystathionine and the specific activity of cystathionine synthase are low in fetal brain. Both parameters increase slowly after birth and reach values found in adult brain at approx 3 months of postnatal age. The activity of cystathionase in brain is low throughout development.
Liver provides a direct contrast in that the concentration of cystathionine and the specific activity of cystathionine synthase are high in the fetus, decreasing rapidly after birth to values found in the adult by 2 weeks of postnatal age. Cystathionase activity is low in fetal liver and increases slowly after birth reaching values found in adult liver after 2–3 months. Kidney has no more than trace amounts of cystathionine throughout development, higher activity of cystathionine synthase in the fetus than in the adult and high, unchanged activity of cystathionase throughout the period of development studied.
These results indicate that the high concentrations of cystathionine found in primate brain are reached postnatally and suggest that this high concentration of cystathionine may be associated with the functioning of mature brain.     

THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10⁶ cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-¹²⁵I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-¹²⁵I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.     

鸡前、后背阔肌去神经后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用辣根过氧化物酶(HRP)作为大分子标记物,以组织化学和生物化学方法观察了鸡前后背阔肌在去神经后的胞纳现象。结果表明,在去神经后发生肥大的前背阔肌和去神经后发生萎缩的后背阔肌同样都出现胞纳的明显增加。组织化学所观察的结果表明,浸泡在含 HRP 的任氏液中的有完整神经支配的前、后背阔肌只有极少数的肌纤维摄取 HRP,而在去神经的前、后背阔肌中则有不少的肌纤维内部出现 HRP 染色反应。这种反应在有的纤维表现为弥散性染色,有的表现为浓的 HRP 反应颗粒。生物化学的结果显示,去神经后的前、后背阔肌中 HRP 的相对含量分别比有神经支配的对照肌肉明显地增多54%和87%,我们在鸡前背阔肌用组织化学和生物化学所得的实验结果与 Thesleff 等人提出的关于肌肉萎缩机制的假设显然不符。本工作证实了肌肉的胞纳作用的增加并不一定最终导致肌纤维的变性和萎缩。     

THE INTERACTION OF CATIONIC LIPOSOMES CONTAINING ENTRAPPED HORSERADISH PEROXIDASE WITH CELLS IN CULTURE

Cationic liposomes composed of sphingomyelin, cholesterol, and stearylamine were prepared with horseradish peroxidase trapped inside. Stable particles were formed in which 10–12% of the enzymic activity appeared to be located at, or near, the outer surface of the liposome. Adsorption and uptake of liposomes by HeLa cells were followed cytochemically by electron microscopy and quantitated by enzyme assay and by the distribution and fate of particles labeled with [¹⁴C]cholesterol and [¹²⁵I]horseradish peroxidase. The particles were adsorbed by HeLa cells at least 300 times as efficiently as was free horseradish peroxidase. Many of the particles remained at the cell surface, but numerous membrane-bound cytoplasmic inclusions were observed to contain peroxidase-staining material. In addition, many areas of the cell membrane gave a positive staining reaction. It was concluded that many particles (presumably the larger ones) did not gain access to the interior of the cells, many were phagocytized, and some enzyme was transferred to the cell membrane, perhaps as a result of fusion of the liposomal membrane with the cell membrane.     

ACTIVITIES OF SOME ENZYMES INVOLVED IN HOMOCYSTEINE METHYLATION IN BRAIN, LIVER AND KIDNEY OF THE DEVELOPING RHESUS MONKEY

Abstract— The specific activities of the enzymes responsible for remethylation of homocysteine to methionine. N⁵-methyltetrahydrofolate-homocysteine methyltransferase and betaine-homocysteine methyl-transferase, and of the enzyme responsible for transferring the β-carbon atom of serine to tetrahydrofolate. serine tetrahydrofolate 5,10-hydroxymethyltransferase, have been measured in brain, liver and kidney of the developing Rhesus monkey from mid-gestation, from birth and neonatal life to maturity. The specific activity of N⁵-methyltetrahydrofolate-homocysteine methyltransferase in all tissues is higher during late gestation and shortly after birth than it is in the adult, and in brain and liver it shows a positive correlation with increasing gestational age. Betaine-homocysteine methyltransferase activity is not measurable in brain. In liver it increases linearly during fetal and neonatal development until values found in the adult are reached. In kidney there is a sharp linear increase during the period of gestation studied. The drop at birth is followed by a sharp increase back to values noted at the end of gestation and thereafter a slow increase to values found in the adult. The specific activity of serine-tetrahydrofolate 5,10-hydroxymethyltransferase tends to be higher in fetal and early neonatal brain than it is in adult brain, whereas in liver and kidney it is low in the fetus and rises during development to reach values found in the adult some months after birth.     

DEMONSTRATION OF ACID PHOSPHATASE-CONTAINING GRANULES AND CYTOPLASMIC BODIES IN THE EPITHELIUM OF FOETAL RAT DUODENUM DURING CERTAIN STAGES OF DIFFERENTIATION

Dense cytoplasmic bodies surrounded by one or two unit membranes and containing mitochondria, vesicles, ribosomes, rough and smooth surfaced endoplasmic reticulum, and lamellated membranes (myelin figures) have been observed in the differentiating mucosa of the duodenum of rat foetuses by electron microscopy. Generally, the cytoplasmic components in the bodies seem to be in varying stages of disintegration. The bodies are found in greatest number on the 17th and 18th day of gestation, i.e. at the onset of differentiation. At this period of development the epithelium is stratified, and the villus formation is initiated by invagination of the epithelium by buds of mesenchyme followed by a splitting of the epithelium along the sides of the invaginations. When the villi have formed, the stratified epithelium has changed to the simple columnar type and the dense bodies have largely disappeared. Simultaneously, the lumen has widened considerably. In a parallel study with the light microscope, frozen sections incubated for the demonstration of acid phosphatase activity revealed the reaction product to be localized in bodies of the same size and distribution as the dense bodies found by electron microscopy. Hence, it seems that the bodies are altered and enlarged lysosomes (cytolysomes) active during the intensive differentiative events in the small intestine during the last part of intra-uterine life.     

饲喂泰和乌骨鸡及其黑素对小鼠肝、肠、肾酶组织化学活性的影响

应用酶组织化学方法观察了老年小鼠经饲喂泰和乌骨鸡及其黑素一个半月后,肝、肠、肾SDH、Mg^2 －ATPase、G－6－Pase、5′－Nase和MAO的酶活性变化。结果显示;肝、肠、肾SDH、Mg^2 -ATPase、G－6－Pase、5′-Nase的酶活性增强。MAO的酶活性减弱。结果提示：泰和乌骨鸡及其黑素具有促进机体代谢、维持内环境稳定,延缓衰老的作用。     

血糖浓度变化与消化间期综合肌电周期的关系

在狗的十二指肠浆膜面埋植银-氯化银双极电极,记录空腹时的肌电活动,观察血糖浓度变化与消化间期综合肌电(IDMEC)周期的关系。实验结果表明:(1)血糖浓度随 IDMEC周期的不同时相而变动,Ⅰ相最高,Ⅱ相次之,Ⅲ相最低(P<0.01)。(2)静脉注射酚妥拉明或心得安后。血糖浓度随 IDMEC 周期的波动均消失。(3)切除双侧迷走神经干后,血糖浓度略有升高,随 IDMEC 的周期性波动依然存在。(4)分别阻断肾上腺素α、β受体及切除迷走神经后,不影响 IDMEC 的周期性活动,但可使周期延长,各相所占时程百分比发生变化。以上结果证明,空腹情况下血糖浓度随 IDMEC 周期的不同时相而变动,交感神经和迷走神经具有一定的调节作用。     

A COMPARATIVE STUDY OF THE ISOLATION OF THE CORTEX AND THE ROLE OF THE CALCIUM-INSOLUBLE PROTEIN IN SEVERAL SPECIES OF SEA URCHIN EGG

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.     

泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

为了探讨谷胱甘肽过氧化物酶（Glutathione peroxidase,GPx）基因在泥蚶应激反应中的作用,研究采用RACE技术克隆了泥蚶GPx基因（TgGPx）cDNA全长,其cDNA全长1195 bp,包含45 bp 5’-UTR,639 bp开放阅读框（ORF）和511 bp 3’-UTR。ORF编码212个氨基酸残基,预测蛋白分子量为24.3 kD,理论等电点为8.33,其中,第53个氨基酸U是由密码子202UGA204编码的硒代半胱氨酸（Se-Cys）。在3’-UTR上存在一段序列,形成一种独特的茎环结构,即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析,发现软体动物中也存在不同种类的GPx基因,TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析,结果表明,TgGPx在泥蚶的5个组织中都有表达,但存在组织特异性,在外套膜中的表达量最高,在血细胞中的表达量最低。用重金属铅、铜、镉刺激后,TgGPx在肝胰脏中的表达量显著升高,表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。     

大鼠活体注射台盼蓝后肝和肾的组织学与组织化学变化的观察

大鼠连续4天腹腔注射1%台盼蓝后,观察隔天、隔周、隔二周后肝和肾的组织结构及PAS、AlP、AcP、G-6-Pase、Mg~(2+)-ATPase和SDH的活性变化。结果发现:肝细胞和肾小管的上皮细胞中有台盼蓝颗粒;肝PAS反应阴性;隔天后肝AlP、AcP、G-6-Pase和SDH活性增强,Mg~(2+)-ATPase活性减弱;肾的上述组化反应活性都减弱;隔二周后肝和肾的上述组化反应接近对照。实验结果提示:活体注射台盼蓝对肝和肾未构成实质性损伤,隔天后的组化变化可能是一种生理适应性反应。     

小鼠骨胳肌在切腱、协同肌切腱和肉毒杆菌毒素中毒后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用生物化学方法测定离体小鼠比目鱼肌对 HRP 的胞纳作用。结果表明,在切断神经或切腱后引起萎缩的肌肉侧或在协同肌切腱后引起代偿性肥大的肌肉侧,与它们各自对照的正常肌肉侧相比,都可发生对 HRP 胞纳的明显增加。而在肉毒杆菌毒素中毒后引起萎缩的肌肉侧,和它正常的对照肌肉侧相比,却意外地不发生这种胞纳摄取的明显增加。本文的实验结果进一步证实。肌肉的胞纳增加并不一定导致肌纤维的变性和萎缩(例如代偿性肥大的肌肉);而肌纤维的变性和萎缩亦不一定需要肌肉的胞纳增加为前提(例如肉毒杆菌毒素中毒的肌肉)。本工作结果还提示:肌肉的胞纳增加有其神经原性和肌原性因素。本文还对肌肉胞纳增加的可能机制进行了讨论。