CYTOCHEMICAL OBSERVATIONS ON THE RELATIONSHIP BETWEEN LYSOSOMES AND PHAGOSOMES IN KIDNEY AND LIVER BY COMBINED STAINING FOR ACID PHOSPHATASE AND INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE

After incubation of formalin-fixed, frozen sections of kidney and liver from peroxidase-treated rats in an azo dye medium for acid phosphatase, and after subsequent incubation of the same sections with benzidine, phagosomes were stained blue and lysosomes were stained red in the same cells. It was observed that newly formed phagosomes were separate from preexisting lysosomes in the tubule cells of the kidney and in the Kupffer cells of the liver at early periods after treatment with peroxidase. At later periods, the color reactions for acid phosphatase and peroxidase occurred in the same granules. The reaction of peroxidase decreased gradually and disappeared from the phago-lysosomes after 2 to 3 days, whereas the reaction for acid phosphatase persisted. In the liver, most of the injected protein was concentrated in large phagosomes located at the periphery of the cells lining the sinusoids. The peribiliary lysosomes showed a relatively weak reaction for peroxidase in the proximity of the portal veins. After pathological changes of permeability, phagosomes and lysosomes lost their normal location and fused, in the interior of many liver cells, to form large vacuoles or spheres. The effects of a reduced load of peroxidase and the effects of the pretreatment with another protein (egg white) on the phago-lysosomes of the kidney were tested. The relationship of the fusion of phagosomes with lysosomes to the size of normal and pathological phago-lysosomes was discussed.     

COLORIMETRIC INVESTIGATION OF THE UPTAKE OF AN INTRAVENOUSLY INJECTED PROTEIN (HORSERADISH PEROXIDASE) BY RAT KIDNEY AND EFFECTS OF COMPETITION BY EGG WHITE

After intravenous injection of horseradish peroxidase into rats, the foreign protein appeared in the kidney first in the small phagosomes and its concentration there decreased quickly; it then was concentrated and "stored" for several days in the large phagosomes. After injection of 10 mg of peroxidase per 100 gm of body weight, the concentration of peroxidase in blood and urine decreased exponentially during the first 6 hours; small amounts of peroxidase were excreted in the urine for several days. When 0.05 to 1.0 mg of peroxidase per 100 gm were administered, most of the peroxidase was taken up by the liver and little by the kidney, and a portion was excreted in the urine even at the lowest dose. At doses above 1.5 mg per 100 gm, the liver cells were saturated, and large reabsorption droplets appeared in the tubule cells of the kidney. With further dosage increase, the concentration of peroxidase in the phagosomes of the kidney increased rapidly until saturation was reached at doses of 13 mg per 100 gm. After intraperitoneal injection of egg white 18 hours prior to the administration of peroxidase, the concentration of peroxidase in all kidney fractions was only 10 to 25 per cent of the values for the untreated animals, the disappearance of peroxidase from the blood was delayed, and 81 percent more peroxidase was excreted in the urine. The treatment with egg white had no effect on the uptake of peroxidase by the liver. The ability of kidney tissue to degrade and adsorb peroxidase in vitro was tested.     

OCCURRENCE OF PHAGOSOMES AND PHAGO-LYSOSOMES IN DIFFERENT SEGMENTS OF THE NEPHRON IN RELATION TO THE REABSORPTION, TRANSPORT, DIGESTION, AND EXTRUSION OF INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE

The size, number, and location of lysosomes, phagosomes, and phago-lysosomes in different segments of the proximal and distal tubules, in the collecting tubules, and in invading macrophages of the kidneys of rats were compared by staining lysosomes (acid phosphatase) red, and phagosomes (injected horseradish peroxidase) blue in separate sections, and by staining phago-lysosomes purple by successive application of the reactions for the two enzymes in the same sections. It was concluded from these observations that the absorption of the foreign protein from the lumen and its gradual digestion in large phago-lysosomes took place mainly in the cells of the proximal convoluted tubules of the outer cortex. Several segments of the proximal convoluted tubules were distinguished on the basis of differences in the size and location of the phago-lysosomes and the amounts of peroxidase ingested. The distal tubules showed, in addition to moderate numbers of phago-lysosomes, many small phagosomes in the apical and basal zones of the cells. Moderate numbers of phagosomes and phago-lysosomes were observed in the cells of the collecting tubules. Macrophages showing very large phago-lysosomes were seen in the peritubular capillaries of the medulla, after injection of peroxidase. When high doses of peroxidase were administered, enlarged phago-lysosomes, parts of which seemed to be extruded into the lumen, were formed in the terminal segments of the proximal convoluted tubules.     

血锌与肝锌,骨骼锌及肌肉锌的相关性研究

用~(65)Zn同位素示踪法研究了不同年龄LACA小鼠体内血锌与肝脏锌、骨骼锌及肌肉锌的相关性。实验结果表明,血锌的代谢与骨骼内锌及肌肉内锌的代谢有明显的相关关系,而与肝脏内锌代谢无相关关系;骨骼及肌肉中锌含量占全身锌含量的90％,上述结果提示血锌的监测可反映体内锌的水平。     

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.     

不同年龄喜马拉雅旱獭大脑皮质结构与血液气体测定的比较研究

本文报道饲养于海拔3300米地区室内(室温5-20℃)非冬眠的幼年、成年与老年喜马拉雅旱獭大脑的某些生理参数、血液气体分析、大脑皮质神经元数量与锥体细胞树突棘棘器的数量变化以及大脑皮质超微结构特点。结果表明:成年组与幼年组相比,脑重占体重百分比有所下降,血红蛋白浓度、红细胞压积和氧利用率有所升高,锥体细胞数量增加9.7％(P>0.05),锥体细胞树突棘的棘器数目增加22.4％(P<0.05);老年组与成年组相比,脑重占体重百分比进一步下降,血红蛋白浓度、红细胞压积进一步升高,氧利用率明显下降(P<0.01),锥体细胞数减少29.8％(P<0.01),锥体细胞树突棘的棘器数目减少33.8％(P<0.01),锥体细胞浆中的粗面内质网、游离核糖体及突触前膜中的突触小泡数量也明显减少,脂褐素含量升高。     

体感皮层细胞放电的非时锁反应和时锁反应的比较分析

刺激腓浅神经诱发皮层单位放电的非时锁反应(N-TLR)可分为兴奋、抑制、无关型,时锁反应(TLR)可分为Ⅰ、Ⅱ、Ⅲ型。N-TLR的兴奋型单位可有Ⅰ、Ⅱ或Ⅲ型TLR,无关型单位也可有Ⅰ、Ⅱ或Ⅲ型TLR,抑制型单位有Ⅰ或Ⅲ型TLR。表明TLR和N-TLR各型的关系比较复杂,两者可能反映不同信息,TLR可能反映更多信息。提示研究体感皮层单位放电对外周刺激的反应时,不仅要分析N-TLR,还要分析TLR,后者可能更为重要。只分析一种就会遗漏许多信息。     

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.     

LIPOPROTEIN GRANULES IN THE CORTICAL COLLECTING TUBULES OF MOUSE KIDNEY

The light and, to a lesser extent, the dark cells of the cortical collecting tubules in mouse kidney contain a great number of granules which according to histochemical tests are composed of phospholipids and proteins. These granules are bounded by a triple-layered membrane measuring approximately 75 A across, and contain one or several crystals with a hexagonal or square lattice. These crystals are built up of rod-shaped units, which appear dense after osmium fixation, measure about 48 A in diameter, and are separated by a light interspace of similar dimensions. The mean center-to-center distance of the rods is about 96 A. The structure is explained as a lipoprotein crystallized within a membrane-bounded vacuole. No relationship between these granules and mitochondria was found. The physiological significance of the granules remains unknown.     

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.     

草鱼肾脏和脾脏血细胞发育过程的观察

鱼类肾脏、脾脏是机体造血的主要器官。印迹涂片显示:草鱼肾脏和脾脏内血细胞的发育过程大致经历了三个阶段,即原始阶段、幼稚阶段、成熟阶段。本文着重描述了各阶段细胞的形态特征井对草鱼血细胞的发育及命名等问题作了初步的探讨。       

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.     

几种植物组织及其原生质体过氧化物酶同工酶的比较研究

对马尾松幼苗子叶和胚轴、甘蔗、小麦、烟草、黄花菜等幼叶及其原生质体的过氧化物酶同工酶(POI)分别作比较研究。观察到凡经纤维素酶处理的各植物组织POI酶带数均多于未经纤维素酶处理的组织的酶带数;除个别例外,后者一般又比无壁原生质体的酶带数多。此种差异随植株生长年龄而增大,表明植物组织内大部分POI主要存于质外体中。