Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Determination of the Cellular Mechanisms Regulating Thermo-Induced Stem Growth in Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a species with a cold requirement for the initiation of reproductive development (thermoinduction). Work in this laboratory has been focused on elucidating the biochemical and molecular mechanisms underlying the bolting or rapid stem elongation response that is an intricate part of reproductive development in this species. In the present paper the cellular basis for thermo-induced stem growth was determined. Evidence is presented indicating that bolting results from the production of new cells that elongate to their original length before thermoinduction. This increase in cell division occurs in the pith and cortex approximately 0.5 to 5.0 millimeters below the stem apex. For at least the early stages of thermo-induced stem growth, enhanced cell elongation does not appear to be a factor because average lengths of pith cells from stems of thermo-induced plants were similar or less than noninduced controls. In addition, both the amount of increase in the production of new pith cells and stem growth were positively correlated with the length of the cold treatment. Two other lines of evidence are presented corroborating previous assertions (JD Metzger [1985] Plant Physiol 78: 8-13) that gibberellins mediate thermo-induced stem growth in field pennycress. First, treatment of noninduced plants with gibberellin A₃ completely mimicked the effects of a 4-week cold treatment on mitotic activity in the pith and cortex. Second, very little increase in the production of new cells was observed in the pith and cortex of thermo-induced plants of a gibberellin-deficient dwarf mutant of field pennycress. It is also shown that the influence of photoperiod on stem growth is mediated by an effect on the final length that cells ultimately attain.     

Utilization of pretreated coir pith for the optimized bioproduction of cellulase by Aspergillus nidulans

The present study is aimed at simultaneous cellulase synthesis and coir pith degradation by Aspergillus nidulans using coir pith as chief substrate. The lignocellulosic biomass, coir pith is known to be an excellent carbon source for microbial cellulase production under solid state fermentation. The alkali pretreatment with sodium hydroxide was seen to enhance enzymatic hydrolysis. The effect of coir pith weight, moisture content, initial pH and growth temperature on cellulase activity and yield were investigated by response surface methodology (RSM) employing a four-factor-five-level central composite design (CCD). The results of Fourier transform infrared spectroscopy (FTIR), X-Ray diffraction (XRD) and Scanning electron microscopy (SEM) of coir pith showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Maximum carboxy methyl cellulase activity (CMCase) of 28.64 U/g and cellulase yield of 66.32% were achieved with 8 g coir pith at 70% moisture content and 40 °C temperature with pH 5 as evident from run numbers 25 and 30. Filter paper (FPase) and cellobiase (CBase) activities of 10.23 U/g and 4.31 U/g respectively were observed on the 11th day after the inoculation.     

Histochemical Studies of Sclereid Induction in the Shoot of Rauwolfia Species: I. CYTOCHROME OXIDASE

Results of histochemical tests for cytochrome oxidase activityin four species of Rauwolfia have been reported. The cells beneaththe terminal shoot meristem constitute the pith. Histochemicaltests showed intensified enzymatic activity in those cells ofthe pith which would differentiate as sclereid initials. Similarcytochrome oxidase activity also occurred in the sclereid initialsand the developing sclereids. The cytochrome oxidase activitywas associated with two types of particulate formation, thegranular and rod-shaped. The ground parenchymatous cells ofthe pith and the leaf-base showed very little enzymatic activityof cytochrome oxidase. The characteristic indophenol blue colorationdue to cytochrome oxidase activity did not appear in controlsections. Physiological changes correlated with morphogeneticexpression of some pith cells demonstrate that the physiologicalchanges occur before the initiation of sclereids in the morphologicallyhomogeneous parenchymatous cells of the pith. Intensified cytochromeoxidase activity was also recorded in the meristematic tissuesof shoot apex, procambium, axillary buds and the laticiferouscells of Rauwolfia.     

Characterization of the ribonuclease activity on the skin surface

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin^® although not by the ribonuclease inhibitor SUPERase· In?. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.     

Regulation of alpha-amylase activity in bean stem tissues

α-Amylase activity was assayed in 1-centimeter sections taken from bean (Phaseolus vulgaris var. Kentucky Wonder) hypocotyls and epicotyls at measured distances from the cotyledons. The activity was low throughout the hypocotyl for the first 7 days. An increase was first observed with etiolated hypocotyls in the basal region, becoming higher in the more central regions by 14 to 17 days. By 21 days the activity was highest in the upper region, but had decreased in the lower regions. A comparable pattern was observed for the epicotyl from etiolated seedlings, the activity increasing first in the region closest to the cotyledons. These increases were associated with loss of cells from the pith in the hypocotyl and epicotyl of both dark- and light-grown plants. Since the changes were observed in tissues virtually devoid of starch, it is hypothesized that the control mechanism is related to the cellular disassembly associated with the mobilization of materials released during senescence rather than to a regulation by the enzyme's substrate or products.     

Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth

The dehydrodiconiferyl alcohol glucosides A and B are factors isolated from transformed Vinca rosea tumor cells that can replace the cytokinin requirement for growth of tobacco (Nicotiana tabacum) pith and callus cells in culture. These factors, present in tobacco pith cells, have their concentrations elevated approximately 2 orders of magnitude after cytokinin exposure. Biosynthesis experiments showed that these compounds are not cell wall fragments, as previously suggested, but are produced directly from coniferyl alcohol. Their synthesis is probably associated with the existing pathway for cell wall biosynthesis in both Vinca tumors and tobacco pith explants. The pathway requires only two steps, the dimerization of coniferyl alcohol by a soluble intracellular peroxidase and subsequent glycosylation. Biosynthetic experiments suggested that dehydrodiconiferyl alcohol glucoside breakdown was very slow and control of its concentration was exerted through restricted availability of coniferyl alcohol.     

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ⁰ cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.     

Evidence for multiple ribonucleases in crude extracts from cultured mosquito cells

Crude extracts from Aedes albopictus (mosquito) cells appear to contain several ribonuclease activities, which can be differentiated on the basis of heat-stability, pH optima and the effects of divalent cations. Ribonuclease activities which can be detected on polyacrylamide gels differ with respect to molecular weight, and various subcellular fractions appear to have distinct ribonuclease activities. Zinc chloride and heparin are effective inhibitors of ribonuclease activity in crude extracts. These results provide useful criteria for further characterization of the major ribonucleases present in cultured mosquito cells.     

Cytotoxic effect of structurally modified bull seminal ribonuclease (AS RNase)

Chemical modifications of bull seminal ribonuclease (AS RNase) cause considerable changes in its cytotoxic activity. However, binding of AS RNase on cells has been changed little or not at all. Every modification (oxidation, reduction, carbomethylation, succinylation and maleylation) inhibited ribonuclease and aspermatogenic and embryotoxic activity of AS RNase. The common cytotoxic effect was also changed to some degree. Inhibition of incorporation of ³H-thymidine to BP-8 tumor cells in culture was observed after application of native, oxidized, reduced, and carboxymethylated AS RNase.     

Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

Structures and functions of adventitious roots in species of the genus Philodendron Schott (Araceae)

We discuss here the anatomical variations of the arrangements and compositions of stele types observed in different roots types in four populations of the three species of Philodendron as probable adaptations to their habitats. Terrestrial individuals of P. corcovadense have cylindrical steles while rupicolous individuals have lobate steles with dispersed internal cortical parenchyma. The Philodendron species sampled showed polyarch structures. The crampon roots of P. oblongum and anchor roots of P. cordatum show medullated protosteles, with the former species having a reduced pith with sclerified parenchyma cells while the latter has a wide pith and parenchyma cells with only slightly thickened walls. The feeder roots of P. cordatum also show a medullated protostele—although a central vessel is present until approximately 60 cm from the apex that later disappears, forming a parenchymatous pith. We conclude that the different root types reflect adaptations of the subgenera Philodendron and Meconostigma to their different habits and habitats, such as in P. corcovadense, where the roots of rupicolous individuals have lobate steles while the roots of the terrestrial plants have cylindrical steles.     

DEVELOPMENT OF CHAMBERED PITH IN STEMS OF PHYTOLACCA AMERICANA L. (PHYTOLACCACEAE)

An integrated microscopic (light and electron microscopy) and macroscopic investigation of chambered pith development was made of Phytolacca americana L. Terminal internodes have a solid pith cylinder in contrast to the alternating diaphragms and chambers occurring in subjacent pith. Macroscopically, chambers and diaphragms of any one internode are of equal size. Microscopically, diaphragms vary in height within an internode (from 1–6 cells high). Nevertheless, all diaphragms become thicker circumferentially (5–12 cells high) and connect with long files of intact peripheral pith cells. Diaphragm cells have a large centrally positioned vacuole with a thin, parietal layer of cytoplasm; nuclei, mitochondria, endoplasmic reticulum, and unidentified organelles differentiate in the cytoplasm of diaphragm cells. Although schizogenous activity has most often been implicated as the mechanism by which chambered pith develops in vegetative organs of angiosperms, the results of this study show that cavities in pokeweed result from both schizogenous and lysigenous mechanisms. Schizogeny is suggested by the fact that central pith cells of terminal internodes are longer and thinner walled than peripheral pith cells arranged in vertical files, thus indicating elongation of cells as a possible result of internode elongation. The precise developmental pattern and arrangement of chambers and diaphragms also suggest schizogenous processes. Lysigenous or enzymatic activity is indicated by the fact that cavities are bounded by broken cells, and wall fragments and organelles are often found within enlarging cavities. Chamber formation occurs continuously acropetally and centrifugally in the central pith. A comparison of diaphragms is made with Liriodendron tulipifera and Juglans nigra in an attempt to resolve differences in structure and terminology regarding the differentiation of chambered and diaphragmed pith.     

Structural changes of dinoflagellate chromosomes by pronase and ribonuclease

When cells of the dinoflagellates Prorocentrum micans and Gyrodinium cohnii are exposed to the proteolytic enzyme pronase or alternatively to ribonuclease, the structure of chromosomes is markedly altered. These changes have been observed electron microscopically in thin sections and spreads. Treatment of cells with pronase removed the bulk of nonfibrillar chromosome material completely unmasking fine chromosomal DNA fibres. Pronase had similar effect also on the dense material which is in contact with chromosomes; fibrillar loops protruding from chromosomes were exposed. However, pronase had no effect on the structural integrity of chromosomes. On the contrary, treatment of cells with ribonuclease loosened the package of chromosomal fibres. Thin sections showed that the tight package of longitudinal periodic structures seen in untreated chromosome was relaxed; chromosome extended longitudinally and formed a linear array of balls. When ribonuclease-treated chromosomes were spread, they were substantially more stretched than untreated chromosomes because of uncoiling of two oppositehanded spiral chromatid bundles. The effect of ribonuclease treatment suggests that unknown RNA species have an important role in the maintenance of permanent condensation of dinoflagellate chromosomes. On the other hand, proteins removable by pronase are also present. Most probably they are not linked to the chromosome structure but represent the matrix of nuclear activity.     

Interaction of onconase with the human ribonuclease inhibitor protein

One of the tightest known protein-protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease inhibitor protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with K_i = 0.15 μM in a solution of low salt concentration.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Evidence for a senescence-associated gene induced by darkness

Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H₂O₂ via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.     

Characterization of the mycoplasma membrane proteins: V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50 % of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group.Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [¹²⁵I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core.Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

Ultrastructural Changes in Potato Tuber Pith Cells during Brown Center Development

Electron microscopy revealed that subjecting `Russet Burbank' potato (Solanum tuberosum L.) plants to 2 days of cool temperature growing conditions (18°C days/10°C nights) did not produce visible damage or changes in tuber pith tissue when compared to warm-grown tubers (23°C days/18°C nights). However, damage to some tuber pith cells was observed after 5 days of cool treatment. Eight days of cool treatment produced extensive alterations in cell structure. The cytoplasm of the cool-treated tuber pith cells had become highly vesiculated and there was evidence of complete destruction of amyloplast membranes and tonoplasts. In many cases the starch grains appeared to be undergoing hydrolysis suggesting total disruption of normal cell function. Sixteen days of cool treatment were sufficient to produce visible brown center development in all cool-grown tubers examined. Electron microscopy of these tissues revealed that, although some organelles were still present, the cytoplasm had become extremely vesiculated and lacked any resemblance to that of tissue from warm-grown tubers. Gross, irregular thickening of cell walls was also detected.     

Site-specific folate conjugation to a cytotoxic protein

Conjugation to folic acid is known to enhance the uptake of molecules by human cells that over-produce folate receptors. Variants of bovine pancreatic ribonuclease (RNase A) that have attenuated affinity for the endogenous ribonuclease inhibitor protein (RI) are toxic to mammalian cells. Here, the random acylation of amino groups in wild-type RNase A with folic acid is shown to decrease its catalytic activity dramatically, presumably because of the alteration to a key active-site residue, Lys41. To effect site-specific coupling, N^δ-bromoacetyl-N^α-pteroyl-l-ornithine, which is a folate analogue with an electrophilic bromoacetamido group, was synthesized and used to S-alkylate Cys88 of the G88C variant of RNase A. The pendant folate moiety does not decrease enzymatic activity, enables RI-evasion, and endows toxicity for cancer cells that over-produce the folate receptor. These data reveal a propitious means for targeting proteins and other molecules to cancer cells.