A CYTOCHEMICAL LOCALIZATION OF REDUCTIVE SITES IN A GRAM-NEGATIVE BACTERIUM : Tellurite Reduction in Proteus vulgaris

In order to obtain information on the exact location of the respiratory enzyme chain in Gram-negative bacteria, an electron microscopic study was made of the sites of reducing activity of cells that had, in the living state, incorporated tellurite. In the test object Proteus vulgaris, the reduced tellurite was found to be deposited in bodies contiguous with the plasma membrane but different in structure from those described in the Gram-positive Bacillus subtilis (2). In fact, the bodies proved to consist of a conglomerate of elements which contained the strongly electron-scattering reduced tellurite and a delicately granular "matrix." A limiting membrane was not observed around these complexes. In serial sections details of the complexes are illustrated. Reduced tellurite was not deposited in the plasma membrane to any important degree. Since no other sites of deposition of the reduced product were revealed, it is assumed that the complexes represent the mitochondrial equivalents in the investigated organism. In addition, the bodies might function as the basal granules of the flagella.     

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASES IN EUGLENA GRACILIS

The localization of induced and constitutive acid phosphatase activity in Euglena was studied by light and electron microscopy, using two different cytochemical methods. Cells grown in high phosphate medium have constitutive acid phosphatase activity in three regions: in the Golgi complex, around the paramylum bodies, and in the peri-reservoir vesicles. Cells that have formed an induced acid phosphatase by exposure to a phosphate-deficient medium have, in addition to the constitutive activity localized exactly as in the uninduced cell, a strong activity in the pellicle. The induced activity is not uniformly distributed over the pellicle, but is localized at the notch of each pellicle complex, near a group of about four fibrils and near a characteristic vesicle of the endoplasmic reticulum. In the cytostome, where fission begins during division, there is an alternation of large and small pellicle complexes, both of which have induced phosphatase activity. A similar alternation is seen over the entire pellicle of dividing cells.     

小鼠胸腺皮质外位腺苷三磷酸酶的细胞化学定位

对探索细胞之间相互作用的机制,对ＢＡＬＢ／ｃ小鼠胸腺皮质内腺苷三磷酸酶进行了细胞化学定位。结果表明该酶活性主要位于毛细血管基膜,内此细胞皮膜和吞饮小泡;上皮性网状细胞和胸腺细胞的质膜外层,特别是二者的相邻面。巨噬细胞及上皮性网状细胞的溶酶和囊泡也具有此酶活性。本文对外位腺苷三磷酸酶有抑制腺苷三磷酸诱发胸腺细胞凋落死亡的可能性进行了讨论。     

THE CYTOCHEMICAL LOCALIZATION OF ASCORBIC ACID IN ROOT TIP CELLS

The intracellular distribution of ascorbic acid was studied in frozen-dried root tips of Allium cepa and Vicia faba by the silver nitrate procedure. The sites of the ascorbic acid as indicated by the deposited silver appear as spherical (0.2 to 0.6 µ in diameter) cytoplasmic particles. The site appears to have small amounts of lipides and to be rich in ribonucleic acid. These particles are concluded to be submicroscopic in size and associated, in the elongating cell, with the cell surface. In the meristematic cells they appear fewer in number and are distributed throughout the cytoplasm.     

RADIOAUTOGRAPHIC LOCALIZATION OF SODIUM PUMP SITES IN RABBIT INTESTINE

Direct demonstration of the cellular location of sodium pumping constitutes a key problem in the solution of intestinal sodium absorption. Utilizing silicone-impregnated epoxy sections of freeze-dried, osmium-fixed tissue, ouabain-³H and inulin-³H light microscope radioautographs have been produced which show that: lateral but not brush border membranes of rabbit small intestine bind ouabain-³H (high specific activity) with an affinity so great that a subsequent washing in ouabain-free medium has little effect on binding; lateral membrane binding is not apparent with low specific activity ouabain-³H, and inulin-³H and ouabain-³H (low specific activity) in the cores of the villi do not equilibrate with the intercellular spaces. Preliminary tracer measurements of ouabain-³H and inulin-¹⁴C spaces also agree with these findings As ouabain is a specific inhibitor of active sodium transport, these observations provide direct support for the view that lateral membrane pumping of sodium into the intercellular spaces causes, through osmotic forces on water, a flow of fluid out of these spaces into the interstitium.     

STUDIES OF CYTOCHEMICAL LOCALIZATION OF SPECIFIC DEHYDROGENASES IN FROZEN,DISEASED TISSUES OF PINUS

Intracellular activity of individual dehydrogenases in frozen tissues of Pinus monticola and Cronartium ribicola was demonstrated by supplying a specific substrate and the appropriate pyridine-nucleotide-linked coenzyme. Freezing broke cell permeability barriers releasing endogenous coenzymes and substrates which had produced nonspecific enzymatic reduction of nitro blue tetrazolium by miscellaneous dehydrogenases throughout fresh tissues. Freezing enhanced specificity by accentuating the differences between control and treatment sections. Succinic, ethanol, glutamic, α-glycerophosphate, isocitric, lactic, malic, glucose-6-phosphate, and 6-phos-phogluconate dehydrogenases and NAD and NADP diaphorases were localized within cells of the blister rust fungus and its western white pine host. NAD- and NADP-linked forms of glutamic, isocitric, and malic dehydrogenases were also detected. The distribution and activity of the enzymes are described for cell types of host and pathogen. β-Hydroxybutyric and pyruvic dehydrogenases were not detected. Calcium and magnesium (5 × 10⁻³ m final conen) and zinc (1.5 × 10⁻⁵ m final concn) had little or no effect on localization. Amytal increased reduction by 6-phosphogluconate, glutamic, and ethanol dehydrogenases while azide depressed the reaction for the last enzyme. Cyanide augmented diformazan formation with succinate. Transhydrogenase was eliminated as a likely contributor to spurious localization in these frozen tissues. Enzymatically produced diformazan appeared on the surface of lipid droplets in cells of both organisms in fresh and frozen sections. The use and interpretation of data from frozen and fresh tissues in tetrazolium cytochemistry are discussed.     

CYTOCHEMICAL LOCALIZATION OF PEROXIDATIC ACTIVITY OF CATALASE IN RAT HEPATIC MICROBODIES (PEROXISOMES)

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H₂O₂, and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H₂O₂ appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂ by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H₂O₂, completely abolished microbody staining in the absence of H₂O₂. Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.     

小麦细胞核仁中rRNA基因转录的超微定位

真核细胞核仁中rRNA基因转录位点是长期以来未能解决的问题。以小麦细胞为研究材料,应用常规电子显微镜技术,观察了小麦细胞核仁纤维中心(Fibrillar centers,FC)内染色质的超微结构;并通过DNA抗体阐明了核仁中DNA位于纤维中心、致密纤维组分(Dense fibrillar component,DFC)以及两者的过渡区域;应用RNA聚合酶I相关转录因子UBF(Upstream binding factor)抗体所做的分析显示,小麦细胞核仁中UBF位于FC与DFC的过渡区域以及DFC中,在FC中没有UBF的存在;进一步借助于RNA/DNA杂合体抗体选择性地直接标记核仁中rRNA基因的转录位点,结果表明了小麦细胞核仁rRNA基因的转录位点是在FC与DFC的过渡区域及DFC中。     

甘蔗叶不同部位ATP酶活性细胞化学定位

甘蔗叶片,叶鞘和肥厚带韧皮部 ATP 酶活性定位于筛管、伴胞的质膜、内质网和某些伴胞细胞基质、小囊泡和发育成熟的液泡上;叶片韧皮部薄壁细胞、厚壁细胞和厚壁通道细胞质膜及小囊泡中亦显示有 ATP 水解产物;维管束鞘细咆与厚壁细胞或厚壁通道细胞所构成的细胞间隙上也存在有 ATP 酶活性反应产物沉淀。甘蔗叶片大、中、小三种维管束,从小维管束到大维管束,面向细胞间隙的细胞表面上的 ATP 酶活性逐渐增强,而维管束鞘细胞质膜上的 ATP 酶活性则趋于减弱;同一维管束内则以韧皮部细胞的 ATP 酶活性最强。维管束鞘细胞与叶肉细胞之间存在很多的胞间连丝,并表现出高的 ATP 酶活性。讨论了 ATP 酶活性的分布状态与叶肉细胞的光合产物向韧皮部运输的关系。     

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN GRANULE FRACTIONS FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes     

The Extrafloral Nectaries of Cotton: II. CYTOCHEMICAL LOCALIZATION OF ATPASE ACTIVITY AND CA2+ -BINDING SITES, AND SELECTIVE OSMIUM IMPREGNATION

Localization of ATPase activity in the extrafloral nectariesof cotton (Gossypium hirsutum) shows most pronounced granularstaining at the plasmalemma of the sieve tube companion cellsbelow the nectaries and at the plasmalemma of cells in the secretorypapillae, particularly in the apical cell. Staining was muchmore intense in mature, secreting nectaries than in young, non-secretingones. When CaCl₂ is included in the fixation and washing solutionswith the aim of localizing calcium binding sites, prominentamorphous electron dense globules are seen at the plasmalemmaof the cells in the papillae, but also associated with othermembranes. Both granular and amorphous deposits are seen whenstaining for Ca²⁺-ATPase activity. Selective osmium impregnationshows pronounced staining of the ER and nuclear envelope insecreting nectaries but not in young non-secreting ones. Key words: Adenosine triphosphatase, Gossypium hirsutum, Nectary, Secretion     

CYTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE MITOTIC APPARATUS OF HELA AND SARCOMA 180 TISSUE CULTURE CELLS

ATPase was localized in distinct regions of the mitotic apparatus of HeLa and Sarcoma 180 tissue culture cells. ATPase was demonstrated in the metaphase spindle of HeLa and Sarcoma 180 cells fixed in cold buffered 2 per cent formalin (pH 6.5 to 6.8) containing 2 x 10^-3 M CaCl₂. A high concentration of ATPase was frequently observed at the poles of the spindle. ATPase was also demonstrated in the interzonal region of both cell types during anaphase. The narrowing of the band of ATPase activity localized in the interzonal region during telophase indicates that ATPase activity is associated with the central spindle. In polar views of Sarcoma 180 cells fixed in cold, unbuffered, 2 per cent formalin, ATPase was frequently localized in granules in the region of the inner circumference of the ring of chromosomes formed at metaphase. ATPase in the mitotic apparatus of HeLa and Sarcoma 180 cells was shown not to be due to non-specific alkaline phosphatase. Mitotic apparatus ATPase in Sarcoma 180 cells was suppressed by an —SH inhibitor.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.