THE DEMONSTRATION OF THE SUCCINIC DEHYDROGENASE SYSTEM IN BACILLUS SUBTILIS USING TETRANITRO-BLUE TETRAZOLIUM COMBINED WITH TECHNIQUES OF ELECTRON MICROSCOPY

Activity of the succinic dehydrogenase system was studied in Bacillus subtilis 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 deposition of TNBT-formazan, was found on membranous organelles associated with the cytoplasmic membrane and septal plasma membrane, the nuclear area, and the plasma membrane. Flagella, ~190 A in diameter, with thorn-like projections protruded through the cell wall. Tangential-oblique sections of the cell wall showed many pores ~220 A in diameter with a center-to-center spacing of ~450 A.     

ELECTRON MICROSCOPY OF CELLULAR DIVISION IN ESCHERICHIA COLI.

Conti, S. F. (Brookhaven National Laboratory, Upton, N. Y.) and M. E. Gettner. Electron microscopy of cellular division in Escherichia coli. J. Bacteriol. 83:544-550. 1962.-Exponentially growing cells of Escherichia coli were fixed in formalin, exposed to uranyl nitrate, dehydrated at low temperatures with ethanol, and embedded in methacrylate. Polymerization was carried out at -70 C, by exposure of specimens to radiation from a cobalt(60) source. Electron micrographs revealed that cellular division occurs by the centripetal growth of the cell wall. The fine structure of the cytoplasm, nuclear apparatus, cell wall, and cytoplasmic membrane was also studied.     

THE LOCALIZATION OF ACID PHOSPHATASE IN RAT LIVER CELLS AS REVEALED BY COMBINED CYTOCHEMICAL STAINING AND ELECTRON MICROSCOPY

Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.     

弱氧化醋杆菌阿拉伯糖醇脱氢酶基因克隆及其在大肠杆菌中的表达

The partial genomic library of Acetobacter suboxydans was constructed using Yeast\| E.coli shuttle plasmid YEp352 as vector.Two positive transformants,designated as DH5α(pAD91) and DH5α(pAD98),were obtained by screening the growth of transformants on the agar plate in which D\|arabitol was used as the sole carbon source.The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical.The insert is about 2.3kb.Arabitol dehydrogenase activity assay indic…     

LOCALIZATION OF A BASIC PROTEIN IN THE MYELIN OF VARIOUS SPECIES WITH THE AID OF FLUORESCENCE AND ELECTRON MICROSCOPY

In this study, alanine was shown to be the N-terminal amino acid of a basic protein of low molecular weight that was isolated from either human or guinea pig brain. Antibodies prepared against the guinea pig protein were labeled with either fluorescein or ferritin. Studies with the labeled antibodies showed that an immunohistochemically similar protein is found in the myelin sheaths of central and peripheral nervous tissues of chicken and frog and a variety of mammalian species. Loss of integrity of the myelin during processing was shown to enhance markedly the antigen-antibody reaction.     

THE DISTRIBUTION OF HISTOCHEMICALLY DEMONSTRABLE SUCCINIC DEHYDROGENASE AND OF MITOCHONDRIA IN TONGUE AND SKELETAL MUSCLES

Various skeletal muscles show considerable variations in histochemically demonstrable succinic dehydrogenase activity. In muscles from the lower extremities, the back, and the abdominal wall, only some fibers reveal evidence of enzymatic staining. In the diaphragm and masseter more fibers react positively. In the tongue, however, all fibers show marked activity comparable to that found regularly in the heart. There is a close similarity in the distribution of histochemically demonstrable succinic dehydrogenase and stainable mitochondria in tissue sections.     

LOCALIZATION OF ANTIBODIES BY ELECTRON MICROSCOPY IN DEVELOPING ANTIBODY-PRODUCING CELLS

The development of antibody-producing cells in the early stages of the secondary or hyperimmune response has been studied with the electron microscope in lymph nodes of adult chinchilla rabbits immunized with ferritin or apoferritin. The intracellular distribution of antiferritin antibodies was determined in the lymph node cells at 1 to 5 days after a booster injection, employing the labelling technique previously used by the authors (12) to demonstrate the localization of antibodies in mature plasma cells. Antibodies were first detected at 48 hours in blasts; i.e., cells which have a poorly developed endoplasmic reticulum and a cytoplasm filled with many ribosomes grouped in clusters. The label was subsequently found in forms intermediate between blasts and plasma cells (plasmoblasts, immature plasma cells), in which the endoplasmic reticulum appeared progressively more developed. Antiferritin antibodies were also found in cells in mitosis. In all the above cell types, antigen-antibody precipitates were consistently found in the perinuclear space and in the cisternae of the granular endoplasmic reticulum, from an early stage in the development of the latter. Evidence was also obtained for the presence of antibody in the Golgi area. The results are discussed in relation to the possible cellular sites of antibody synthesis.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : I. DNA and Proteins

If thin sections of Escherichia coli, labeled uniformly with tritium, are radioautographed calculations, based on the distribution of section sizes show that the number of H³ decays per section should be very close to a Poisson distribution. We might, therefore, expect that the distribution of radioautographic grain counts among random cross-sections should follow a Poisson distribution. It can then be inferred that a deviation from a Poisson indicates a high concentration of label in a preferred region. This region can then be identified by analysis of serial section and comparison with electron micrographs. Sections of cells labeled with leucine-H3 gave a Poisson distribution of grain counts, and it was concluded that proteins were distributed fairly uniformly throughout the cell. The situation was not changed if labeled cells were placed in chloramphenicol or if very short pulses of label were used. When Escherichia coli is grown in presence of chloramphenicol a major morphological change concerns the nuclear region: it becomes more regular in outline, nearly spherical, and occupies a smaller proportion of the cell length. The previously described association between DNA labeled with thymidine-H3 and the nuclear region was confirmed by showing that the distribution of the label in the cell followed exactly the morphological changes of the nuclear region. It was also shown that the concentration of DNA in the nuclear region was at least 45 times higher than that of the cytoplasm. Several morphological features of cells grown in chloramphenicol and examined in the electron microscope are discussed.     

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.     

ELECTRON MICROSCOPY OF THE VOLVOCACEAE AND ASTREPHOMENACEAE

Lang, Norma J. (U. Texas, Austin.) Electron microscopy of the Volvocaceae and Astrephomenaceae. Amer. Jour. Bot. 50(3): 280-300. Illus. 1963.—Clonal cultures of Gonium sociale, G. pectorale, Pandorina morum, Eudorina elegans, Eudorina sp., Volvulina steinii, V. pringsheimii, Platydorina caudata, Pleodorina illinoisensis, P. californica, Volvox aureus, V. tertius, V. globator, V. barberi, and Astrephomene gubernaculifera representing the Volvocaceae and Astrephomenaceae in the Volvocales were examined with the electron microscope and their ultrastructure compared. The ultrastructure of the various organelles is basically similar in the species studied and no increase in cellular complexity is found to accompany the evolutionary trends evidenced in the Volvocaceae. The ultrastructure of a colonial cell is basically that of Chlamydotnonas. A cytoplasmic membrane having a unit membrane structure encompasses a cell and is continuous with the double-membraned flagellar sheaths. The flagella contain the typical 9 + 2 fibril arrangement with the 2 axial fibrils terminating in a cylinder at the flagellar base and the 9 peripheral pairs continuing into the cytoplasm as a basal body. The organelles comprising the cytoplasm are: mitochondria with plate-like cristae; dictyosomes composed of stacks of agranular cisternae; small, rough or smooth-surfaced vesicles; an endoplasmic reticulum of granule-bearing and agranular tubules, lamellae and broad cisternae; vacuoles which are either contractile, contain fine granular and fibrillar material, or have dense contents probably representing polyphosphate; lipid bodies; and dense granules 100–150 A which have been called ribosomes. The finely granular nucleoplasm is surrounded by a porous, double-membraned nuclear envelope and contains a centric nucleolus composed of dense, spherical granules. The outer membrane of the nuclear envelope bears granules and may have granular extensions into the perinuclear cytoplasm. Each extension appears to encompass one or several dictyosomes and has been termed an “amplexus.” The amplexi are agranular on the surface contiguous to a dictyosome. A double-membraned chloroplast envelope is continuous around the single, cup-shaped chloroplast. The basic chloroplast units are discs closed at each end, occurring in stacks of varying number parallel to the envelope. The presumed proteinaceous matrix of the basal pyrenoid is penetrated by elongated, tubular elements which connect with the lamellar discs. Multiple rows of granules, associated with individual discs, form the anterior stigma within the chloroplast envelope. The colonial matrix is not a structureless, mucilaginous material uniting the cells in colonies, but it has rather a highly complex structure especially around the periphery of the colony and the flagellar channels. The apparent substitution of a fibrillar layer of the colonial matrix for the discrete compact cell wall, such as is found in Chlamydomonas, implies a greater degree of complexity in the evolution of these colonial genera than is generally assumed.     

THE LOCALIZATION OF MITOCHONDRIAL NADP-DEPENDENT ISOCITRATE DEHYDROGENASE IN NORMAL AND HYPOXIC CONDITIONS

NADP-dependent isocitrate dehydrogenase is localized in both external and internal membranes and in the mitochondrial matrix from brains of adult and newborn animals. Electrophoretic studies indicate that the mitochondrial enzyme occurs in the form of two isoenzymes; the isoenzyme of the external membranes behaves similarly to that of the cytoplasm. Ischaemia in adult animals results in a decrease in enzyme specific activity, particularly in external membranes, while in the mitochondrial fraction of the newborn the activity of the enzyme increased.     

LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE RAT SPERM TAIL AS REVEALED BY ELECTRON MICROSCOPY

The epididymides of rat testis were fixed in glutaraldehyde and cut as frozen sections. The sections were incubated in lead nitrate solution containing as a substrate either ATP, AMP, creatinine phosphate, beta glycerophosphate, or phenyl phosphate. Then they were postfixed in osmium tetroxide, embedded, sectioned, and examined with the electron microscope. In the sperm tail, when ATP is used as a substrate the reaction product (lead phosphate) is observed both in the tail filament complex and on the surface membrane of the mitochondrial helix of the middle piece. In the tail filament complex, this product is seen near the nine paired peripheral and two central filaments, and in the matrix between the outer coarse fibers. But the product is not observed within these filaments and fibers. In longitudinal sections, no periodicity of the deposits in the complex is observed. When the other phosphate compounds are used as substrates the reaction products appear on the surface membrane of the mitochondrial helix, and are not found in the tail filament complex. No distinctly different localization of the reaction products is observed when substrates other than ATP are used. Possible relationships between the structure and the function of the sperm tail are discussed in the light of these findings.