Enhancement of adventitious root formation in mung bean cuttings by 3,5-dihalo-4-hydroxybenzoic acids and 2,4-dinitrophenol

3,5-Dihalo-4-hydroxybenzoic acids enhanced adventitious root formation in mung bean (Vigna radiata L.) cuttings. 3,5-Diiodo-4-hydroxybenzoic acid was more active than 3,5-dichloro-4-hydroxybenzoic acid, increasing the number of roots formed by about 4-fold. 2,4-Dinitrophenol also enhanced significantly adventitious root formation in mung bean cuttings. The phenolic compounds were active with or without indole-3-acetic acid. The possible mechanism by which these phenolic compounds enhance rooting is discussed.Abbreviations CCCP carbonyl cyanide 3-chlorophenylhydrazone - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DNP 2,4-dinitrophenol     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

Localization of acid organelles in frog skeletal muscle fibers

By means of fluorescent and phase-contrast microscopy the distribution of acid membrane organelles in normal and vacuolated frog skeletal muscle fibers has been studied. The vacuolation of the T-system was produced by loading and subsequent removal of glycerol (80-110 mM), or it appeared as a result of Zenker's necrosis. Acridine orange (AO) was used as a marker for acid intracellular compartments. AO accumulated in granules localized near the nuclear poles (more seldom around the nucleus)' and in the intermyofibrillar spaces. Typically the AO granules make up short longitudinal chains or regular pairs, where the distances between neighboring granules are short-dated to sarcomere lengths. Almost all granules emit in red, but about one third of them simultaneously emit in green, which is characteristic of AO monomers. In the vicinity of necrotic boundary or under the influence of brefeldin A, a green component of fluorescence appears in most granules. Treatment with monensin leads to granule disappearance. Vacuoles accompanying the glycerol treatment or developing of necrosis do not accumulate AO and exert no effect on the localization of AO-granules. The nature of cellular organelles accumulating AO in skeletal muscle fibers is discussed.     

Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.     

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.     

Metabolism of 4-Pyridone-3-Carboxamide-1-ß-D- Ribonucleoside Triphosphate and Its Nucleoside Precursor in the Erythrocytes

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-β -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-β -D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 ± 0.6 μ M to 74.9 ± 9.17 and 4PYR monophosphate increased from 5 μ M to 254.7 ± 13.9 μ M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5′nucleotidase-mediated dephosphorylation.     

Metabolism of 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate and its nucleoside precursor in the erythrocytes

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-beta -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 +/- 0.6 micro M to 74.9 +/- 9.17 and 4PYR monophosphate increased from 5 micro M to 254.7 +/- 13.9 micro M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5'nucleotidase-mediated dephosphorylation.     

Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation

Stress granules are RNP-containing particles arising in the cytoplasm in response to environmental stress. They are dynamic structures assembling and disassembling in the cytoplasm very rapidly. We have studied whether the cytoskeleton is involved in the formation of stress granules. Stress granules were induced in CV-1 cells by sodium arsenate treatment and visualized by immunofluorescent staining with antibodies either to the p170 subunit of eIF3 or to poly(A)-binding protein. Treatment with sodium arsenate for 30-120 min led to assembling of stress granules in a majority of CV-1 cells. Disruption of MT array with nocodazole treatment abolished arsenate-induced formation of stress granules. A similar effect was induced by the microtubule-depolymerizing drug vinblastine, though the influence of the microtubule-stabilizing drug paclitaxel was opposite. Nocodazole treatment did not prevent arsenate-induced phosphorylation of the eIF-2alpha factor, essential for stress granule formation, suggesting that the presence of intact MT array is required for granule assembly. Unexpectedly, treatment of cells with the actin filament-disrupting drug latrunculin B slightly enhanced stress granule formation. We propose that stress granule formation is microtubule-dependent process and likely is facilitated by the motor protein-driven movement of individual stress granule components (e.g., mRNP) along microtubules.     

Aspergillus oryzae AoSO Is a Novel Component of Stress Granules upon Heat Stress in Filamentous Fungi

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi.The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.     

Electron microscopic and biochemical research on the vacuoles formed in the erythrocytes of frogs as affected by neutral red and novocaine

No lysosomes were found in the frog intact erythrocytes with electron microscope. Under the influence of neutral red (NR-8.7.10(-5) M) and novocaine (N-4.6.10(-3) M) segregation zones (vacuoles) including these substances are formed. Using electron microscopy and morphometry the action of NR and N for 5 minutes up to 48 hours was found to provoke the formation of four types of vacuoles differing in their morphology: with electron-transparent content, with amorphous inclusions and membrane whorls. The dynamics of vacuole formation, of their changes and amount were followed depending on the time of exposition of these substances. Biochemical investigation of both NR and N isolated vacuoles showed in these some activities of lysosomal marker enzymes--acid phosphatase and N-acetyl-beta,D-glucosaminidase. Ultrastructural investigation of acid phosphatase localization in the isolated vacuoles revealed the histochemical reaction product mainly in electron-translucent vacuoles (primary lysosomes) and partly in electron dense ones (secondary lysosomes). On the ground of the above studies a conclusion is made that in frog erythrocytes treated with NR and N lysosome formation is induced to be followed by the induced autophagocytosis and heterophagocytosis. Some possible ways of the vacuolar system formation in frog erythrocytes and the origin of lysosomal hydrolases are discussed.     

Plant regeneration on cultured root segments of Cephaelis ipecacuanha A. Richard

Summary Simple one step micropropagation system for Cephaelis ipecacuanha A. Richard was developed using root cultures grown in vitro. Adventitious shoots were directly formed on the cut end of root segments without callus formation, on phytohormone-free B5 solid medium in the dark. When the shoots attached with root segments were further cultured under 16 h light / 8 h dark, they developed into plantlets, which could be transplanted to soil. The regenerated plants grew well in a greenhouse with showing normal appearance and accumulated alkaloids. The influence of auxin on adventitious shoot formation was also investigated.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - B5 Gamborg B5(Gamborg et al. 1968) - WP woody plant (Lloyd and McCowm 1980) - RC root culture (Thomas and Davey 1982) - HF phytohormone free - IAA indole-3-acetic acid - NAA 1-naphtaleneacetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - SEM scanning electron microscopy - C.V. coefficient of variation     

The Stimulatory Effect of Indole-3-Acetic Acid on the Uptake of Amino-Acids by Tissue 1Helianthus annuus

Indole-3-acetic acid was observed to bring about a prompt andmarked increase in the amount of ¹⁴C accumulated by segmentsof sunflower hypocotyl from solutions of labelled glutamic acid,glycine, and lysine. The curve relating magnitude of effectto indole-3-acetic acid concentration followed the comparablecurves for water uptake and extension growth. The accumulation of ¹⁴C was related to the external concentrationof glutamic acid by a curve which departed only slightly fromlinearity. The percentage increase in ¹⁴C accumulation broughtabout by auxin did not decline to any appreciable extent withincreasing external concentration of glutamic acid. Under nitrogen the amount of ¹⁴C taken up from solutions oflabelled glutamic acid in 1·75 hour was cut down by approximatelyone-third, and the auxin effect was abolished. The Q₁₀ for ¹⁴Caccumulation between 16° C. and 26° C. was 1·2in the absence of indole-3-acetic acid, and was 1·3 inits presence. When net water uptake was eliminated by the addition of mannitolto the external solution, ¹⁴C accumulation in auxin-free mediawas not depressed. The percentage increase in ¹⁴C accumulationbrought about by auxin, however, was markedly reduced. The fate of the ¹⁴C accumulated was investigated by means ofchromatography on resin columns and on filter paper. About 30–40percent, of the ¹⁴C was in the form of glutamic acid after approximatelya hours' treatment. No marked difference in the level of glutamicacid was observed between auxin-treated and control segments.The effect of auxin was more evident on the amounts of otherradioactive derivatives, as yet unidentified. It was observed that, not only was the amount of CO₂ evolvedin respiration higher in the presence of indole-3-acetic acid,but that this CO₂ was richer in ¹⁴C, i.e. in auxin-treated tissueglutamic acid formed a larger proportion of the substrate respired. The possible implications of these observations are discussed.It is pointed out that indole-3-acetic acid may have achievedits effect by stimulating a transfer process, by lessening adiffusion resistance, or by promoting a process or processeswhich, by removing free amino-acids within the cell, maintainan inward diffusion gradient.     

Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A

Stress granules are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA-binding proteins that include several translation initiation factors. Stress granule assembly occurs in the cytoplasm of higher eukaryotic cells under a wide variety of stress conditions, including heat shock, UV irradiation, hypoxia, and exposure to arsenite. Thus far, a unifying principle of eukaryotic initiation factor 2alpha phosphorylation prior to stress granule formation has been observed from the majority of experimental evidence. Pateamine A, a natural product isolated from marine sponge, was recently reported to inhibit eukaryotic translation initiation and induce the formation of stress granules. In this report, the protein composition and fundamental progression of stress granule formation and disassembly induced by pateamine A was found to be similar to that for arsenite. However, pateamine A-induced stress granules were more stable and less prone to disassembly than those formed in the presence of arsenite. Most significantly, pateamine A induced stress granules independent of eukaryotic initiation factor 2alpha phosphorylation, suggesting an alternative mechanism of formation from that previously described for other cellular stresses. Taking into account the known inhibitory effect of pateamine A on eukaryotic translation initiation, a model is proposed to account for the induction of stress granules by pateamine A as well as other stress conditions through perturbation of any steps prior to the rejoining of the 60S ribosomal subunit during the entire translation initiation process.     

The inherent property of polyhydroxyalkanoate synthase to form spherical PHA granules at the cell poles: the core region is required for polar localization

Only the PHA synthase is required for formation of spherical intracellular PHA granules emerging at cell poles. This study aims to assign the polar targeting signal in the PHA synthase and to provide insight into molecular mechanisms of granule formation. Random in-frame insertion mutagenesis indicated dispensable and essential regions suggesting that only the N terminus (<100 aa) is dispensable and forms a random coil structure. The inactive PHA synthase (C319A) is still localized to cell poles, indicating that the nascent PHA chain does not serve as an anchor or signal for subcellular localization and granule formation. Deletion of the N terminus did neither affect subcellular localization nor PHA granule formation. The deletion of the hydrophobic C terminus (68 aa) did not impact on subcellular localization of the PHA synthase, but abolished PHA synthase activity. The structural protein PhaP1 was found to be not required for subcellular localization and initiation of granule formation. PhaP1 only localizes to the cell poles, when PHA granules are formed. These data suggested that the PHA synthase itself localizes to the cell poles via its core region (93-521 aa), which is structurally constraint and comprises the polar positional information for self-assembly of PHA granules at the cell poles.     

The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation

Quorum sensing (QS) through signal chemical molecules is known to be essential to bacterial adhesion and biofilm formation. In this study, the QS ability of aerobic granules—a special form of biofilms used for biological wastewater treatment—was investigated and compared with that of conventional activated sludge flocs. A novel sectional membrane bioreactor was used together with a flow-cell to evaluate the possible influence of signal chemicals produced by the source sludge on the growth mode of bacterial cells. The results demonstrate the apparent production of QS chemicals from granules and its impact on initial cell attachment and granule formation. When granules were used as the signal-producing biomass, the attached-growth mode was dominant for the free cells, and the biofilm formation rate in the flow-cell was about ten times faster than in cases which used activated sludge as the signal source biomass. In addition, the intracellular extract from mature granules significantly accelerated the sludge granulation process. It is argued that the production and expression of QS signal chemicals from granules and granule precursors might have induced the gene expression of bacteria in suspension for attached growth rather than suspended growth, leading to granule formation and its stable structure.     

Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system

The evolution of a microbial community was investigated during sludge granulation using a wide range of micro-scale and molecular biology techniques. Experimental results demonstrate that polyphosphate-accumulating granules were successfully cultured during the anaerobic/aerobic cycle. Improvement in sludge sedimentation performance occurred prior to the formation of granular sludge and was not affected by change in granule size. Rod-shaped and filamentous bacteria appeared to initiate granule formation and generate the structures that supported further granule growth. It was observed that mature granules supported microbial populations that differed from nascent granules and were predominantly packed with coccoid bacteria. It was further observed that the diversity of the granular microbial community increased as the granules grew. Accumulibacter, Nitrosospira and Thauera were mainly responsible for nutrient removal while microorganisms such as Rhodocyclus and Hyphomicrobiaceae appeared to be primarily responsible for forming and maintaining the granule structure.     

Amino acid and lipid composition of refringent granules from the ameboid sperm of Ascaris suum (Nematoda)

Transformation of the spermatozoon of Ascaris suum from a spheroidal to an ameboid cell is associated with the formation of a motile pseudopodium and coalescence of the intracellular refringent granules. The pseudopodia of the ameboid spermatozoa contain filaments organized into dense patches, bundles, web-like or lace-like networks, as observed by electron microscopy. The morphology and chemistry of the refringent granules were investigated in subcellular fractions enriched for these structures. Isolated refringent granules were heterogeneous in size measuring from 0.5 X 0.6 to 2.3 X 3.5 microns. Each granule is surrounded by a 110 A thick layer. During fusion, the surfaces of the refringent granules form small extensions resembling micropodia. The process of fusion occurs at many sites on a given granule and simultaneous fusion of several granules was commonly observed. Amino acid analyses of the refringent granule proteins (RGP's) indicated: they are rich in aspartic acid or asparagine (48%), leucine (10%), serine (19%) and aromatic amino acids (11%). Gas-liquid chromatographic analyses of alditol acetate derivatives of monosaccharides released by mild acid hydrolysis showed the predominant sugars to be glucose (7.3 micrograms/mg protein), galactose (9.2 micrograms/mg) and N-acetylglucosamine (5.5 micrograms/mg). Lipid analyses indicated a complex mixture of glycerides, ascarosides and waxes, together with a major component that resembled free fatty acid in mobility on TLC.     

Studies on the NADPH oxidizing activity in polymorphonuclear leucocytes: The mode of association with the granule membrane the relationship to myeloperoxidase and the interference of hemoglobin with NADPH oxidase determination

Phospholipase C-treated polymorphonuclear leucocytes were used to study the properties of NADPH oxidase activity of stimulated polymorphonuclear leucocytes.A comparison of the effects of phospholipase C treatment of whole leucocytes on the NADPH oxidase activity with other granule enzymes showed that the activities of β-glucuronidase and acid phosphatase were un-affected, whereas the NADPH oxidase activity was stimulated 4-fold and myeloperoxidase was inhibited about 30%.The distribution of NADPH oxidase activity among subcellular fractions of polymorphonuclear leucocyte homogenates was unaffected by phospholipase C whereas the other enzymes were released into the medium in soluble form; β-glucuronidase > acid phosphatase and myeloperoxidase.A number of solubilizing agents and procedures were tested for their ability to release NADPH oxidase activity from granules of phospholipase C-stimulated polymorphonuclear leucocytes. All procedures used caused appreciable release of granule protein but no release of NADPH oxidase activity. Most of the procedures used strongly inhibited the oxidase activity. These results indicate that the enzyme is tightly bound to granule structures and that the integrity of these structures is required for activity.Some of the solubilizing agents used (KCI, guanidium chloride) were very effective in solubilizing myeloperoxidase.The differential response of myeloperoxidase and NADPH oxidase to treatment with phospholipase C or solubilizing procedures suggests that the two activities are not due to the same enzyme. However, definite conclusion cannot be drawn because of the complex nature of myeloperoxidase.It was found necessary to lyse any erythrocytes present as contaminants of polymorphonuclear leucocytes preparations, since hemoglobin was converted to methemoglobin during the NADPH oxidase assay and methemoglobin exhibits appreciable NADPH oxidase activity.     

Synthesis and secretion of the enamel matrix precursor by the kitten secretory ameloblast

Secretory ameloblasts in kitten molar tooth germs were examined with an electron microscope to analyze the synthesis and secretion processes of the enamel matrix precursor. The contents of the secretion granule were identified as fine granular material, which observed in both the rough endoplasmic reticulum and the Golgi cisterns, accumulated in the dilated margins of the innermost Golgi cistern and formed condensing vacuoles. The same kind of condensing vacuoles was also produced from the GERL cisterns. During the secretion granule maturation processes in the Golgi region, the contents accumulated densely and the granules grew smaller. In addition, granule-limiting membranes acquired fine, bristle coats. The mature secretion granules then migrated, along microtubules, into the surfaces of the Tomes processes and finally released their contents by a process of exocytosis at the type 1 face which faces the enamel growth region.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.