IDENTIFICATION OF PROCAMBIUM IN THE PRIMARY ROOT OF TRIFOLIUM PRATENSE (FABACEAE)

Histochemical and morphometric analyses were used to identify and define an early stage of procambial differentiation in 1.5–2.0-cm-long (48 hr after germination) primary roots of Trifolium pratense L. Esterase activity was used as a histochemical marker for early differentiation of procambium. Morphometric analysis of cell length and width as a function of distance from the root cap junction was performed on the same tissue using brightfield and Nomarski DIC optics. This combination of techniques allowed the identification of esterase activity in both the cell wall and cytoplasm and permitted the determination of the exact location, size, and shape of the histochemically stained cells within the apex. Esterase activity identified the proendodermis and procambial cylinder (six to seven cells in diameter) two to three cells proximal to the root cap junction. In this system, esterase activity proved to be an earlier marker for procambial differentiation than morphometric or cytological changes. It is suggested that these techniques will be useful in characterizing procambial pattern development in more complex shoot systems.     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

INITIATION OF THE VASCULAR SYSTEM IN THE FERTILE FLORET OF ANTHOXANTHUM ODORATUM (GRAMINEAE)

Procambium was initially isolated near the insertions of lemma and stamen primordia in the grass Anthoxanthum. The palea was initiated before its procambium. The acropetal, continuous differentiation of procambium involved in the siting of leaves on shoots of many other megaphyllous plants, does not occur in the rachilla of this grass. A portion of the vascular system of the fertile floret of Anthoxanthum became connected with the vascular system of the rest of the spikelet by basipetal differentiation of the procambial trace of the fertile lemma. A core of residual meristem persisted in the fertile floret above the procambial trace to the fertile lemma. Vascular continuity between the procambial trace to the fertile lemma and the procambial traces of the stamens was achieved by the differentiation of procambium from this core of residual meristem.     

Phenolic extracts from various Algerian plants as strong inhibitors of porcine liver carboxylesterase

Carboxylesterases (CE), expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The goal of this study was to study the effect of phenolic compounds from several plants from the Algerian Atlas used traditionally in Arab folk medicine on the enzymatic activity of porcine liver carboxylesterase. The plants have shown a potent inhibition of carboxylesterase (CE) enzymatic activity in a concentration-dependent manner. Results indicate that the Phenolic extracts from these plants lead to the inactivation of the CE pI = 5.1 with K_i values in micromolar range (1.4–38 μM). These results encourage further biological investigation and identification the inhibitors responsible for this activity.     

ONTOGENY OF FOLIAR VENATION IN EUPHORBIA FORBESII

Six species of Euphorbia endemic to the Hawaiian Islands have disjunct veins as a normal component of their foliar anatomy. An ontogenic study of the foliar venation of one of these species, E. forbesii, showed a normal development of the foliar procambium as determined by previous studies of dicotyledonous leaves. The disjunct veinlets are isolated early in the histogenesis of the intersecondary veins when certain procambial cells fail to differentiate into vascular tissue. It appears that these cells develop into normal parenchymatous cells of the ground tissue. It is suggested that these cells are physiologically distinct from the rest of the procambial cells. In no instance was a tracheary element seen which appeared to have arisen independently of the normal procambial reticulum.     

Biochemical mechanisms of resistance in strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) resistant to malathion and chlorpyrifos-methyl.

The acetylcholinesterase, carboxylesterase, and cytochrome P450 monooxygenase activities of three strains of Oryzaephilus srinamensis (L.) were examined to better understand biochemical mechanisms of resistance. The three strains were VOS49 and VOSCM, selected for resistance to malathion and chlorpyrifos-methyl, respectively, and VOS48, a standard susceptible strain. Cross-resistance to malathion and chlorpyrifos-methyl was confirmed in VOS49 and VOSCM. Acetylcholinesterase activity was not correlated to resistance among these strains. VOS49 and VOSCM showed elevated levels of carboxylesterase activity based on p-nitrophenylacetate, alpha-naphthyl acetate, or beta-naphthyl acetate substrates. PAGE zymograms showed major differences in caboxylesterase isozyme banding among strains. VOSCM had one strongly staining isozyme band. A band having the same Rf-value was very faint in VOS48. The VOS49 carboxylesterase banding pattern was different from both VOSCM and VOS48. Cytochrome P450 monooxygenase activity was based on cytochrome P450 content, aldrin epoxidase activity, and oxidation of organophosphate insecticides, all elevated in resistant strains. The monooxygenase activity varied with insecticide substrate and resistant strain, suggesting specific cytochromes P450 may exist for different insecticides. The monooxygenase activity of the VOS49 strain was much higher with malathion than chlorpyrifos-methyl as substrates, whereas VOSCM monooxygenase activity was higher with malathion than chlorpyrifos-methyl as substrates. Results are discussed in the context of resistance mechanisms to organophosphate insecticides in O. surinamensis.     

Comparison of <Emphasis Type="Italic">Escherichia coli,Saccharomyces cerevisiae,Pichia pastoris,Spodoptera frugiperda</Emphasis>, and COS7 cells for recombinant gene expression

Expression of a rabbit liver carboxylesterase has been achieved in several different model systems including Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda, and COS7 cells. Although, recombinant protein was observed in E. coli sonicates, little or no enzymatic activity was detected. Similarly, no activity was observed following expression in S. cerevisiae. In contrast, active protein was produced in P. pastoris from S. frugiperda, following baculoviral infection and in COS7 cells following transient transfection of plasmid DNA. For the preparation of small amounts of protein for kinetic and biochemical studies, enzyme expressed in P. pastoris has proved sufficient. However, to produce large amounts of carboxylesterase for structural studies, baculoviral-mediated expression of a secreted form of the protein in S. frugiperda was the most efficient. Using this system, we have generated and purified milligram quantities of essentially pure protein. These results demonstrate that the choice of in vitro system for the generation of large amounts of active carboxylesterase, and probably most endoplasmic reticulum processed proteins, is crucial for high level expression and subsequent purification.     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Characterization of Stelar Initiation in Shoot Apices of Ferns

Procambium is commonly recognized as a vascular meristem inshoot apices of vascular plants. Prestelar tissue comprisingprovascular tissue (PVT) and pith mother cells (PMCs) immediatelysubjacent to the single cell layer of promeristem has been consideredto represent the initial stage of stelar differentiation precedingprocambium and rib meristem in ferns. In addition to characterizationof PVT and PMCs on the basis of cell morphology, cytologicalfeatures and developmental continuity with procambium and ribmeristem, four lines of evidence from studies of shoot apicesof Matteuccia struthiopteris and Osmunda cinnamomea supportthis interpretation of initial differentiation. (1) Differentialstaining by safranin-fast green and crystal violet-erythrosinshows that PVT and PMCs differ in colour reactions from promeristemand resemble procambium and pith meristem, respectively. (2)Comparative ultrastructural study reveals qualitative differencesin the cell membrane system, nuclei, cytoplasm, vacuoles andplastids between promeristem and PVT but similarity of PVT toprocambium. (3) Large droplets of tannins occur in promeristembut not in PVT, PMCs and procambium. (4) Cytochemical studyof the shoot apex of Osmunda shows that carboxylesterase activityis strongly demonstrated in PVT and procambial cells but notin promeristem cells and PMCs. These observations further substantiatethe interpretation that PVT represents initial vascular differentiationand PMCs reflect a commitment to pith development.Copyright1995, 1999 Academic Press Initial vascular differentiation, provascular tissue, differential staining, ultrastructure, tannins, carboxylesterase, shoot apex, Matteuccia struthiopteris, Osmunda cinnamomea     

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.     

ONTOGENY OF MAJOR VEINS IN THE LAMINA OF POPULUS DELTOIDES BARTR

The ontogeny of the major venation in the lamina of Populus deltoides Bartr. leaves was investigated in relation to the development of original procambial bundles, subsidiary bundles, and their derivatives. Serial sections and clearings were used to show that the midrib region is a composite structure consisting of several independent vascular bundles, each of which eventually diverges into the lamina to become a secondary vein. The sequence of events in the ontogeny of major secondary veins is: (1) an original procambial strand develops acropetally and becomes the precursor of the first vascular bundle of the midrib region of the lamina, (2) ground tissue at the forefront of acropetally developing subsidiary procambial bundles differentiates in a wavelike continuum; meristematic regions precede the acropetally developing procambial bundles, (3) discrete subsidiary bundles differentiate in the meristematic regions as they advance acropetally, (4) subsidiary bundles diverge obliquely in the lamina margin giving rise to the secondary veins in a basipetal fashion, and (5) subsequent differentiation and maturation of the secondary veins occurs within the lamina. The original procambial bundles and first-formed subsidiary bundles become the secondary veins of the uppermost portions of the lamina, the next-formed subsidiary bundles become the secondary veins of the middle portions of the lamina, and the last-formed subsidiary bundles become the secondary veins of the lowermost portion of the lamina.     

Recent changes in the distribution of carboxylesterase genes and associated chromosomal rearrangements in Greek populations of the tobacco aphid Myzus persicae nicotianae

We present data on the frequency of amplified E4 and FE4 carboxylesterase genes in Myzus persicae s.l. clones collected during the years 2002–2007 and 2012 in Greece. Most clones were of the tobacco aphid, Myzus persicae nicotianae. Samples from 2012 were genotyped with microsatellite DNA markers and a number of them were karyotyped. Aphid clones with amplified FE4 genes predominated in all years, whereas E4 was present in only 3.5% of all samples and always occurred in clones with FE4. Most of the clones examined showed high carboxylesterase activity levels (R2 resistant category). The results showed marked changes in the frequencies of the two carboxylesterase genes in the tobacco aphid populations compared to published data that were collected in Greece in the mid 1990s, when E4 was recorded on its own in 20% of all samples and in 32% of samples from tobacco. A parallel change in karyotype was also observed because the A1,3 translocation, which had a worldwide association with amplified E4 genes in the 1990s, was not detected in the clones analyzed in 2012. Possible causes for these changes are discussed, although selection as a result of pest management practices appears to be the major one. Novel chromosomal rearrangements were also found in M. persicae nicotianae clones. These rearrangements could be a result of clastogenic effects of nicotine, which could persist because of the holocentric nature of aphid chromosomes. The results are discussed in relation to rapid evolution events that have taken place in the tobacco aphid in Greece during the last two decades. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 455–470.     

The relationship between the carboxylesterase and monoacylglycerol lipase activities of chicken liver microsomes

The carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) and monoacylglycerol lipase (glycerol-monoester acylhydrolase, EC 3.1.1.23) activities, measured against ethyl butyrate and emulsified monooleoylglycerol respectively, were determined for chicken liver microsomes and highly purified chicken liver carboxylesterase. The activity ratio (ethyl butyrate activity/monooleoylglycerol activity) was approx. 5 for microsomes and approx. 400 for carboxylesterase. Homogenization of microsomes in 0.1 M Tris-HCl buffer (pH 7.92) released all of the ethyl butyrate activity and about half of the monooleoylglycerol activity into a soluble form. Both activities eluted from a Sephadex G-200 column with the same elution volume as that of pure carboxylesterase. This fraction (fraction B) had an activity ratio of approx. 15, an average pI of 5.01 (cf. 4.75 for carboxylesterase), and ran on polyacrylamide gel electrophoresis at pH 8.6 as a number of closely spaced esterase bands with mobilities considerably less than those of the esterase bands present in the carboxylesterase. Fraction B activities against both substrates were completely inhibited by diethyl p-nitrophenyl phosphate and completely precipitated by antibody to carboxylesterase. The remaining half of the monoacylglycerol lipase activity of microsomes was solubilized by treatment with 1.5% (w/v) Triton X-100. This solubilized monoacylglycerol lipase was completely inhibited by diethyl p-nitrophenyl phosphate, showing it to be a serine-dependent enzyme like the carboxylesterases. However, it had no detectable activity against ethyl butyrate, indicating that it is not closely related to the carboxylesterases.     

Esterases of the ox adrenal: their histochemical and biochemical distribution

Summary Esterases were demonstrated in the ox adrenals histochemically. The substrates used were -naphthyl acetate, and naphthyl acetate AS on fresh and fixed tissue. The highest activity was in the zona glomerulosa, the adrenaline-storing cells and the ganglia. The other parenchymal cells showed a moderate activity. The cytological localization of the enzyme was both diffuse and particulate and highly polarized in the adrenaline-storing cells. The particulate form persisted after formaldehyde fixation. The following inhibitors were used to differentiate between the various esterases histochemically and biochemically: DFP, E600, eserine, p-chloromercuribenzoate and 62C47.The cortex, adrenaline storing- and noradrenaline-storing cells were separated by dissection. Suitable marker were used to assess contamination. Total esterase, carboxylesterase, arylesterase, acetylesterase, acetylcholinesterase and cholinesterase were assayed biochemically in conjunction with the inhibitors. Acetylesterase showed the highest activity and was fairly evenly distributed as was carboxylesterase, although at a lesser level of activity. Aryl esterase was more abundant in the cortex than the medulla. The reverse was found for acetylcholinesterase and at a lower level of activity cholinesterase. The results were compared with the histochemical findings.     

Developmental anatomy of foliage leaves,bracts, calyx and corolla inPharbitis nil

The structure and ontogeny of the foliage leaves, bracts, bracteoles, calyx and corolla ofPharbitis nil were investigated, with special reference to the development of the lamina and the procambium. Reproductive organs used are those of a terminal inflorescence and axillary flowers induced by a single 16 hr dark period given to the seedling. The foliage leaf consists of the petiole and the broad lamina. Bracts show various forms and structures, which fluctuate from a lower leafy bract to an upper scaly one in a terminal inflorescence. The sepal is scaly. The corolla is funnel-shaped, and composed of five wedge-shaped petals. In the lamina of the foliage leaf primordium, marginal growth is followed by active growth by the plate meristem, and procambial strands of lateral veins differentiate from the residual meristem. The primordium of the lowest bract of the terminal inflorescence has already been initiated before the dark period, and develops into the bract, the residual meristem disappearing after the treatment. The leafy bract shows marginal growth and growth by the plate meristem similar to that of the foliage leaf, but of short duration. The activity of marginal growth of the scaly bract and the sepal decreases rapidly and procambial strands of lateral veins differentiate acropetally from highly vacuolated cells. The activity of marginal growth of the petal decreases gradually, and derivatives of the marginal meristem divide as a plate meristem. The corolla tube is initiated by co-operation of interprimordial growth and marginal growth of petal primordia.     

马桑内酯对粘虫体内蛋白质和消化酶的影响

用马桑内酯分别对粘虫采用注射和饲喂处理,48 h后测定粘虫不同部位消化酶活性及蛋白质含量,以探讨马桑有效成分的作用及其杀虫机理.结果显示:(1)注射羟基马桑毒素处理后粘虫组织中蛋白质含量(与丙酮处理比较)均降低,皮组织降低幅度最大为29.47%,饲喂处理后各组织中蛋白质含量均升高,其中皮组织的升高幅度最大为56.87%;但该毒素对粘虫体内蛋白酶和羧酸酯酶的影响不明显.(2)注射与饲喂马桑亭、马桑宁,粘虫体内蛋白质含量均明显比对照处理升高,蛋白酶活性增强,其中饲喂马桑亭处理的粘虫血淋巴中蛋白质含量升高最大为511.49%,蛋白酶活性增强幅度最大为4 640.26%.马桑亭和马桑宁均可使粘虫组织中羧酸酯酶活性降低,其中饲喂马桑宁处理降低幅度最大为82.94%.结果表明:羟基马桑毒素对粘虫体内蛋白质代谢的影响与用药方式有关,马桑亭和马桑宁处理后的粘虫体内蛋白质含量增高、蛋白酶活性增强及酯酶活性降低均达到显著水平.     

ONTOGENY OF THE CLOSED DICHOTOMOUS VENATION OF REGNELLIDIUM

Pray , Thomas R. (U. South. California, Los Angeles.) Ontogeny of the closed dichotomous venation of Regnellidium . Amer. Jour. Bot. 49(5): 464–472. Illus. 1962.—The venation of the pinna of Regnellidium consists of a flabellate series of dichotomizing vascular strands, branches of a single pinna trace. At the laminal margin, the entire venation is closed by a marginal vein. The development of the pinna was investigated primarily by means of paradermal sections. During early development, the organization of the marginal meristem and its derivatives is very similar to that previously described for Nephrolepis. The arrangement of cells in the embryonic pinna is predisposed for the differentiation of dichotomizing procambial strands. During the final phases of pinna development, the marginal meristem is altered in such a manner as to result in a submarginal band of elongated cells which differentiates a marginal procambial strand connecting the tips of the dichotomizing veins. Relatively late in pinna ontogeny and after the entire procambial venation pattern has been delimited, the marginal meristem becomes inactive. The possible correlation between extended marginal growth and dichotomizing veins is discussed.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.