The Cytochemical Localization of Oxidative Enzymes : I. Diphosphopyridine Nucleotide Diaphorase and Triphosphopyridine Nucleotide Diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

A Histochemical Method for the Demonstration of Diphosphopyridine Nucleotide Diaphorase

The present investigation concerning the histochemical demonstration of DPN diaphorase follows the development of a new reagent, Nitro-BT, which has already been used successfully for the cytochemical localization of the succinic dehydrogenase system. The most consistently favorable results were obtained with the lactate-lactic dehydrogenase system buffered at pH 7.4. Using sections of rat kidney and stomach, it was found that the intensity of stain was optimal after 15 minutes incubation at 37°C., conducted aerobically. By appropriate variations in the substrate mixture it was possible to selectively demonstrate the histochemical distribution of certain DPN-linked dehydrogenases in addition to DPN diaphorase. This was made possible by the special distribution of some of these dehydrogenases which distinguished them from one another. Of the dehydrogenases studied the distribution pattern of β-hydroxybutyric dehydrogenase was the most singular. In the gastric mucosa β-hydroxybutyric dehydrogenase was restricted to the cells of the mucous lining epithelium and the gland necks; and in the kidney the enzyme was limited to the cells of the proximal convoluted tubule and thick limbs of Henle's loop. In contrast, lactic dehydrogenase like DPN diaphorase was demonstrable in almost all cytologic elements of both the stomach and the kidney.     

Histochemical Localization of Phosphatases in Mycoplasma gallisepticum

Histochemical studies of adenosine triphosphatase and acid phosphatase activity were performed on Mycoplasma gallisepticum. The adenosine triphosphatase activity appears to be localized in the bleb and infrableb regions exclusively and is associated with the cell membrane; acid phosphatase activity is localized in the infrableb region and does not appear to be membrane-associated. These findings are consistent with data from biochemical studies of Mycoplasma cell fractions but, unlike them, reveal that adenosine triphosphatase activity is restricted to a particular part of the cell membrane.     

The Histochemical Localization of Cholesterol in Formalin-Fixed and Fresh Frozen Sections

The premixed cholesterol reagent (acetic acid, sulphuric acid, ferric chloride and phosphoric acid) employed in a spectrophotometric method for serum cholesterol determinations was applied to frozen sections of various rat and rabbit tissues. The histochemical reaction was compared with the results obtained by the classical Schultz reaction. The same bluish-green color was observed after treatment of formalin-fixed and fresh cryostat sections incubated in 2.5% iron alum at 37 C for 3 days. The premixed cholesterol reagent is stable for years at room temperature if stored in a brown bottle and is thus readily available for routine use.     

石韦孢子叶片组织化学定位

利用冰冻切片法和徒手切片对石韦(Pyrrosia lingua)新鲜叶片中的葸琨类化合物、皂苷类化合物及多糖进行组织化学定位研究.结果表明:葸琨类化合物主要积累在叶片的栅栏组织中;表皮毛、海绵组织和韧皮部也有葸琨类化合物,木质部和表皮无明显显色反应.皂苷类化合物也主要积累在叶片的栅栏组织中;表皮毛、海绵组织、薄壁组织和韧皮部中也有少量皂苷类化合物,木质部无明显显色反应.厚角组织中蒽琨类物质含量比皂苷类物质含量高;多糖普遍存在于石韦叶肉中,维管束、薄壁组织和表皮有少量分布.     

Histochemical Localization and Nematoxicity of Terpenoid Aldehydes in Cotton

In healthy cotton, except for random occasional occurrence in cortical cells, terpenoid aldehydes (TA) are localized in the epidermis and, even there, are absent from the tip 2-4 cm of the root. Since constitutive TA do not occur in the endodermis and stele of the root, they cannot be effective agents against the development of the sedentary stage of the root-knot nematode, Meloidogyne incognita. Within 4 days after inoculation with the root-knot nematode, infection-induced TA accumulated in the endodermis and outer stele. These induced TA were thus localized where they could be effective against the sedentary stage of the nematode. Infection-induced TA accumulation was more rapid and occurred in more stele cells in a resistant cotton cultivar than in two susceptible cultivars.TA extracts from cotton were inhibitory to nematode movement. All second-stage larvae exposed to 1,000 ppm TA for 3 h became rigid, made no movement, and appeared dead. Washing these larvae to remove the TA and incubating them for an additional 24 h did not change their appearance. Shorter exposure times or lower TA concentrations allowed some larvae to recover. Exposing larvae to 10 ppm of TA for 24 h had little effect on them. TA extracted from G. arboreum, a cotton that does not methylate TA, were slightly less inhibitory to the root-knot nematode than TA extracted front G. hirsutum which partially methylates TA.     

Histochemical Localization of Superoxide Dismutase Activity in Rat Brain

Histochemical localization of superoxide anion (O₂^·−) scavenging activity in rat brain was visualized by the tissue-blotting technique. The activity was thought to mainly depend on Cu/Zn-SOD, because the localization of the activity was identical with the immunohistochemistry of Cu/Zn-SOD and the localization of its mRNA in the brain. Moreover, the activity was dramatically decreased after treatment of Cu (I) chelater. The activity was detected in pyramidal cells of the cortex, granular, and mitral cells of the olfactory bulbs, pyramidal cell layer CA1 to CA3, and dentate gyrus of hippocampus formation and granular cells of the cerebellum. Moreover, the activity was detected in the pontine nuclei of brain stem. Olfactory bulbs, hippocampus, and cerebellum were believed to be bestowed high brain functions, i.e., long-term potentiation and long-term depression. A part of the function was regulated by a retrograde neurotransmitter, nitric oxide (^·NO). Our findings suggest that the SOD is colocalized with NO synthase in olfactory bulbs, hippocampus, and cerebellum, where ^·NO plays the important roles. In contrast, low SOD activity was observed in the axonal neurofiber bundles, although the regions contain a lot of membrane lipids, which was thought to be peroxidized by O₂^·− and related radicals such as ^·OH in the regions. From these findings, it was suggested that the SOD did not only play a role in protecting the neurons from endogenously formed O₂^·−, but also play a role in preservation of beneficial natures of ^·NO in the brain.     

Histochemical Localization OF Acid Phosphatase IN Germinating Maize Kernels

Sections of germinating maize kernels obtained by freezing technic were examined for acid phosphatase according to the histochemical method of Gomori. The embryonic axis, scutellum and aleurone layer were found positive for the enzyme. A microtechnical method for preformed phosphate, based on Sumner's colorimetric method for phosphorus, is described.     

A Calcium-Citro-Phosphate Technique for the Histochemical Localization of Myosin ATPase

A simple and sensitive calcium precipitation method is described for the histochemical demonstration of myosin ATPase in striated muscle. In this technique inorganic pyrophosphate is incorporated in the fixative to protect the sites of myosin ATPase activity, thus minimizing enzymatic inactivation during fixation. The incubation medium used contains Ca⁺⁺ (70 mM) as the capturing agent, ATP (4 mM) as substrate, citric acid (7.5 mM) and gelatin. During incubation, the enzyme catalyzes the hydrolysis of ATP to ADP and inorganic phosphate. The liberated phosphate is precipitated as “calcium-citro-phosphate”. The latter is then converted to black cobalt sulphide. The calcium-citro-phosphate is rapidly formed during incubation (within 10 min) and can not be washed off the tissue sections.     

山茱萸核果的解剖结构和组织化学定位

用解剖学和组织化学的方法研究了山茱萸 (MacrocarpiumofficinacleSieb .etZucc .)核果的解剖结构和皂甙、多糖的组织化学定位。结果表明 :山茱萸核果的外果皮革质 ,由一层被覆较厚角质膜的表皮细胞构成 ;中果皮肉质 ,由多列薄壁细胞构成 ,含色素细胞不均匀分布 ,靠近外果皮的薄壁细胞大多为含色素细胞 ,使果实呈现红色 ,向内的薄壁细胞体积渐渐增大 ,在较大的薄壁细胞以及维管束周围的薄壁细胞内常含有色素块。组织化学定位显示 :外中果皮的含色素细胞的色素块中含有丰富的皂甙和多糖 ,果实的中果皮的薄壁细胞在未成熟时就已经形成皂甙 ,并随着果实的成熟逐渐增加积累     

Histochemical Localization of Phenolic Deposits in Leaf Blades of Eragrostis racemosa

The distribution of phenolic deposits in the leaves of Eragrostisracemosa from different habitats was investigated during cultivationunder uniform environmental conditions. The plants retainedtheir leaf phenolic distribution pattern over a period of 8months. Plants from dry habitats had phenolic inclusions inboth epidermal layers, whereas phenolic deposits were absentor only occurred in the abaxial epidermal layers of plants occupyingmoist habitats. Eragrostis racemosa, narrow-heart love grass, Poaceae, phenolic deposits, epidermis, histochemistry, anatomy