Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by β-carboline and indole derivatives

β-Carboline derivatives inhibited both indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase activities from various sources. Among them, norharman is most potent for both enzymes from mammalian sources. Kinetic studies revealed that norharman is uncompetitive (K_i = 0.12 mm) with l-tryptophan for rabbit intestinal indoleamine 2,3-dioxygenase, and linearly competitive (K_i = 0.29 mm) with l-tryptophan for mouse liver tryptophan 2,3-dioxygenase. In addition, some β-carbolines selectively inhibited one enzyme or the other. Pseudomonad tryptophan 2,3-dioxygenase was inhibited by a different spectrum of β-carbolines. Such a selective inhibition by the structure of substrate analogs is more evident by the use of indole derivatives. Indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibited a potent inhibition for mammalian tryptophan 2,3-dioxygenase, while they moderately inhibited the pseudomonad enzyme. However, they showed no inhibition for indoleamine 2,3-dioxygenase. These results suggest the difference of the structures of the active sites among these enzymes from various sources.     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Comparison of stimulus-response (V-log I) functions in five types of lepidopteran compound eyes (46 species)

Summary Stimulus intensity-response relations (V-log I curves) were electrophysiologically (ERG) determined for the compound eyes of 46 lepidopteran species belonging to five different groups: butterflies (22 species), hesperids (3 species), diurnal sphingids (2 species), diurnal moths (3 species) and nocturnal moths (16 species). The V-log I curves were fitted to the Naka and Rushton equation, in whichn represents the slope of the linear part of each curve. The slopes so determined range fromn=0.35 (the shallowest slope) in nocturnal moths with the greatest dynamic range ton=0.54 (the steepest slope) in diurnal moths andn=0.53 in butterflies both of which have narrow dynamic range. Hesperids (n=0.41) and diurnal sphingids (n=0.38) have intermediate values between butterflies and nocturnal moths.The ratio of rhabdom to retinula volume is significantly higher in nocturnal moths (70–75%), however, those of butterflies and of diurnal moths are very small (2–5%), and hesperids and diurnal sphingids show intermediate ratio (ca. 25%).The slopes of V-log I curves are inversely proportional to the ratio of rhabdom to retinula volume in the various eye types. In all groups except diurnal moths, the light intensities which produce maximal and saturated responses are nearly the same, therefore the nocturnal moths which have the lowest threshold to light increase their sensitivity to dim light mainly by decreasing the slopes of V-log I curves.     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium

The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as α-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial β-oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Giant oil cells in the cotyledons ofPharbitis nil and other convolvulacean plants

Suspension of protoplasts (ca. 13–25 μm in diameter) that were isolated from the mesophyll of the cotyledons ofPharbitis nil, strain Violet, contained many large spherical or spheroidal bodies (ca. 100 μm in diameter). Microscopic observation of these bodies and some anatomic studies of the cotyledons during embryogenesis and after germination showed that these bodies are giant cells containing many oil drops stainable with Sudan dyes. Such giant cells were found in four otherPharbitis nil strains, Nepal, Tendan, Africa and Tokyo-kokei, and in six other Convolvulacean plants,Ipomoea batatas, cv. Koukei-14,Calystegia japonica, Calystegia hederacea, Calonyction aculeatum, Quamoclit pennata andCuscuta japonica.     

Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )