LOCALIZATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN ESCHERICHIA COLI USING COMBINED TECHNIQUES OF CYTOCHEMISTRY AND ELECTRON MICROSCOPY

The activity of the succinic dehydrogenase system was studied in Escherichia coli utilizing combined techniques of cytochemistry and electron microscopy. Organisms were incubated in a medium containing tetranitro-blue tetrazolium (TNBT) which served as an electron acceptor. Enzymatic activity, as evidenced by the deposition of aggregates of TNBT-formazan, was found associated with the site of the plasma membrane of the bacterium.     

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q₁₀, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.     

THE USE OF TETRANITRO-BLUE TETRAZOLIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE : Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

轮枝菌形态的低温扫描电镜观察

本文报道寄生在昆虫、锈菌、蜘蛛及植物上的7株轮枝菌(Verticillium)和虫草菌(Cordyceps)的轮枝菌无性型的低温扫描电镜观察结果。试验表明该方法可以清晰地观察到保持完好而未变形的轮枝菌头状分生孢子球及分生孢子链,蜡蚧轮枝菌寄生在昆虫与锈菌号的形态具有较大差异。     

THE DEMONSTRATION OF ENZYMATIC ACTIVITY IN PINOCYTIC VESICLES OF BLOOD CAPILLARIES WITH THE ELECTRON MICROSCOPE

In aldehyde-fixed myocardium, enzymatic activity to nucleotide substrates was localized to the pinocytic vesicles of blood capillaries with the electron microscope. No other structures of the endothelial cell showed activity under the conditions employed. These findings are discussed in relation to recent concepts of transport across the capillary wall.     

ELECTRON MICROSCOPY OF THE UTERINE EPITHELIUM IN THE RABBIT

The ultrastructure of the uterine epithelium has been studied in estrous, ovariectomized, pregnant, and pseudopregnant rabbits. Tissue for light microscopy was fixed in Bouin''s solution and stained with hematoxylin and eosin, by the periodic acid-Schiff (PAS) method, and with methylene blue. Tissue for electron microscopy was fixed in 1 per cent osmium tetroxide in White''s saline and embedded in Araldite. The uterine epithelium in estrus is comprised of ciliated and non-ciliated cells. After ovariectomy the epithelium becomes reduced in height and PAS-positive material disappears. Multinucleated cells are formed in the epithelium in pregnancy, pseudopregnancy, and in the non-pregnant horn in unilateral pregnancy. They degenerate during the 3rd week of pseudopregnancy and during the 4th week of pregnancy in the non-pregnant horn. The formation of multinucleated cells is believed to be under hormonal control. The uterine epithelium in contact with the blastocyst changes into a "symplasma," presumably under the influence of a local (chemical?) effect produced by the blastocyst. This change is not seen in pseudopregnancy nor in the non-pregnant horn in unilateral pregnancy. A complex infolding of the basal cell membrane of the epithelium accompanies the "symplasmic" change. The remaining uterine epithelium in pregnancy shows a well developed ergastoplasm suggesting a production of secretion materials, some of which may be available for absorption by the fetus through the yolk sac or paraplacental chorion.     

THE INACTIVATION OF BACILLUS SUBTILIS SPORES IN PENICILLIN BY GAMMA RADIATION

SUMMARY: Sodium benzylpenicillin, contaminated with Bacillus subtilis spores by freeze-drying a suspension of spores in an aqueous penicillin solution ( c . 50% w/v), was exposed to gamma radiation and a 70-tube dilution method was used to determine the surviving spores after various doses. The correlation coefficient between log₁₀ percentage survival and dose was −0.9523. The regression of the former on the latter was calculated and the decimal reduction dose found to be 20.2 × 10⁴ rads. The regression and the decimal reduction dose were similar to those obtained when suspensions of spores in distilled water were irradiated.     

pNK289衍生质粒在牙孢杆菌中的分离稳定性

报道关于一系列pNK289衍生质粒分离稳定性研究结果。这些起源相同的质粒在Bacillus,subtilis AS1.1176中的分离稳定性存在差异,这种差异与质粒的大小和复制方式无关,而与质粒的考贝数有一定的关系。由于不稳定质粒pNK219在B.subtilis BD224宿主中能稳定遗传,所以推测宿主的遗传背景可能影响质粒的分离稳定性,这些研究不仅为进一步寻找与pNK289衍生质粒稳定性相关的基因奠定了基础,而且为在芽孢杆中构建稳定的重组质粒提供了理论依据。     

pNK289衍生质粒在芽孢杆菌中的分离稳定性*

报道关于一系列ｐＮＫ２８９衍生质粒分离稳定性研究结果。这些起源相同的质粒在Ｂａｃｉ－ｌｌｕｓ．ｓｕｂｔｉｌｉｓＡＳＩ．１１７６中的分离稳定性存在差异,这种差异与质粒的大小和复制方式无关,而与质粒的拷贝数有一定的关系。由于不稳定质粒ｐＮＫ２１９在Ｂ．Ｓｕｂｔｉｌｉｓ ＢＤ２２４宿主中能稳定遗传,所以推测宿主的遗传背景可能影响质粒的分离稳定性。这些研究不仅为进一步寻找与ＰＮＫ２８９衍生质粒稳定性相关的基因奠定了基础,而且为在芽孢杆中构建稳定的重组质粒提供了理论依据。     

耳蜗琥珀酸脱氢酶显示方法的改进

在采用耳蜗顶部灌流方法测定琥珀酸脱氢酶的活性时,本文提出了两点改进,实验动物为豚鼠:(1)孵育前用2%戊二醛磷酸盐缓冲液(pH7.4,4℃)将耳蜗预固定30min,再冰凉(—20——40℃)约3min,以便消除血管纹屏障,使该部位和柯蒂氏器官的琥珀酸脱氢酶活性能同时被显示出来。(2)在孵育液中加入细胞呼吸抑制剂(阿密妥),可使耳蜗背景色变淡,从而减少假阳性现象。通过对一些豚鼠的实验观察,所得结果基本一致。     

STUDIES ON THE MECHANISM OF HYDROGEN TRANSPORT IN ANIMAL TISSUES : VI. INHIBITOR STUDIES WITH SUCCINIC DEHYDROGENASE

1. The mechanism of succinic dehydrogenase action was studied by means of inhibitors. 2. The enzyme is inhibited by a large number of diverse compounds whose only common denominator appears to be their ability to react with SH groups. These compounds include quinonoid structures, sulfhydryl reagents, sulfhydryl compounds, copper, zinc, selenite, and arsenite. 3. In contrast to the above inhibitors, the action of malonate does not appear to involve sulfhydryl groups and is explained on the basis of its affinity for the enzyme groups which react with the carboxyl groups of succinate. 4. The action of malonate and the sulfhydryl reactants is mutually exclusive, and this fact suggests the conclusion that the sulfhydryl group of the enzyme is located between the carboxyl affinity points. 5. On the basis of the deduced structure of the succinate-activating center of the enzyme, it is suggested that the enzyme may function by oscillating between the EnSH and EnS· forms, rather than by a thiol-disulfide equilibrium.