Esterase Patterns Research Articles

Isoenzyme Patterns of Solanum Nigrum and the Cybrid Plant containing S. Nigrum Genome and S. Tuberosum Plastome

Transfer of chloroplasts from Solanum tuberosum into S. nigrum cell resulted in an atrazine sensitive cybrid plant. The shoots of this cybrid were bleached under atrazine stress. The cybrid displayed identical isoenzyme patterns that have been found in S. nigrum, and thus nuclei of the cybrid plant cells did not integrate any chromosomes or chromosome fragments from S. tuberosum nuclei. Under atrazine stress, differences in the isoenzyme expression were found in both the cybrid and original plants. In the peroxidase patterns, POX-4 was detected while POX-1 disappeared. Esterase patterns were less influenced, EST-4 was expressed in both plants under the influence of atrazine. Atrazine treatment of both cybrid and original plant shoots had specific effects on carbonic anhydrase, malate dehydrogenase, and glutamate oxaloacetate transaminase. The number and/or the staining intensity of bands corresponding to these isoenzymes decreased under atrazine stress more in cybrid plants (atrazine sensitive) than in S. nigrum (atrazine resistant).

Mechanisms of Resistance to Organophosphorus and Carbamate Insecticides in Oriental Fruit Moth Populations (Grapholita Molesta Busck)

Mechanisms of resistance to organophosphorus and carbamate insecticides were investigated in larvae and adults of the oriental fruit moth,Grapholita molesta(Busck). Comparative studies with an N-methyl carbamate (carbofuran) tested with or without synergists indicated that more than one factor contributed to insecticide resistance in the oriental fruit moth. Increases in esterase activity in both larvae and adults of resistant strains toward α-naphthyl acetate (5.5-fold for third instar larvae, 3.9-fold for adults), β-naphthyl acetate (4.6-fold for third instar larvae, 3.8-fold for adults), α-naphthyl butyrate (4.2-fold for third instar larvae, 5.5-fold for adults), and β-naphthyl butyrate (2.6-fold for third instar larvae, 2.8-fold for adults) model substrates and differences between strains in esterase patterns resolved using isoelectric focusing indicated that esterases were major factors in the resistance of the oriental fruit moth to insecticides. Differences between resistant and susceptible strains in inhibition of acetylcholinesterases by guthoxon (6-fold for third instar larvae, 10.5-fold for adults), carbofuran (33-fold for third instar larvae, 53.5-fold for adults), carbaryl (43.8-fold for third instar larvae, 1757.4-fold for adults), and methomyl (3.3-fold for third instar larvae, 8-fold for adults) indicated that target site insensitivity was another major resistance factor. Measurements of penetration of radiolabeled carbofuran in adults suggested that a decrease in insecticide uptake may supplement resistance provided by other mechanisms. Measurements of glutathione transferase, mixed-function oxidase, and aliesterase activities failed to demonstrate that these factors were involved in resistance. Because resistance is expressed in both larvae and adults, monitoring for resistance is appropriate with these insecticides in either life stage.

Response of Three Greenbug (Homoptera: Aphididae) Strains to Five Organophosphorous and Two Carbamate Insecticides

Insecticide resistance in insects often is associated with elevated acetylcholin esterases and carboxylesterases. Three greenbug, Schizaphis graminum (Rondani), strains with different general esterase patterns were tested for differences in insecticide response. The standard strain had no noticeable elevated esterase activity. Pattern 1 and pattern 2 strains had different elevated esterase patterns. Toxicity of 5 organophosphorous (chlorpyrifos, dimethoate, disulfoton, malathion, and parathion) and 2 carbamate (carbofuran and methomyl) insecticides were tested using surface residue bioassays. Esterase pattern 1 greenbugs had significantly higher LC50s for each of the organophosphorous insecticides, but the LC50 for the 2 carbamate insecticides were not higher than the standard strain. Esterase pattern 2 greenbugs had significantly higher LC50s for 4 of the 5 organophosphorus insecticides (chlorpyrifos, dimethoate, disulfoton, and parathion) and the 2 carbamate insecticides, but the LC50 for malathion was not higher than the standard strain. Pattern 2 greenbugs had significantly higher tolerance to disulfoton than did pattern 1 greenbugs. This indicates that certain strains of the greenbug are cross resistant to many of the organophosphorous and carbamate insecticides currently labeled for greenbug control in wheat and sorghum. There is an urgent need to find alternative control measures.

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.

Isozyme Variation among Isolates of Mucor piriformis

Isozyme polymorphisms among 59 isolates of Mucor piriformis from pears and nectarines were examined by vertical polyacrylamide gel electropho- resis. Among the enzyme systems tested, six enzyme activities, catalase, cx-esterase, glucose-6-phosphate de- hydrogenase, lactate dehydrogenase, malate dehy- drogenase and superoxide dismutase were used for detailed analyses. The variability of isozyme patterns revealed seven electrophoretic phenotypes for all the enzymes. No distinguishing morphological features were associated with the different electrophoretic phenotypes. In contrast, there was a correlation be- tween esterase, glucose-6-phosphate dehydrogenase and malate dehydrogenase banding patterns and the mating potency of the isolates from pears; those with mating abilities (plus and minus isolates) were clearly different from those without mating abilities (neutral isolates). In fruit inoculation pathogenicity studies, the plus and minus isolates were equally pathogenic to the neutral isolates. The 30 isolates from nectar- ines proved to be very homogeneous genetically, they did not reveal any polymorphisms for the 6 enzymes investigated. The potential application of the isozyme analysis to provide biochemical markers for Mucor species is discussed.

Cluster Analysis by Measurement of Peroxidase and Esterase from Citrus Flavedo.

The zymograms of peroxidase (POD) and esterase (EST) from seventy-two kinds of Citrus fruits and the fruit of both Poncirus and Fortunella were obtained by polyacrylamide gel electrophoresis. It was found that there was little variance in the band pattern of POD and EST of the fruits harvested after September. There were at least three bands of POD and approximately ten of EST in each cultivar. The density of each band for EST was measured with a densitometer. There was a characteristic band for POD identifying pummelos. The band patterns of EST were also characteristic among each species or cultivar. Two kinds of ootachibana (Citrus otachibana Hort. ex Tanaka) were shown to be different cultivars from each other according to their EST zymograms and peel oil analysis. Cluster analysis based on EST was done and discussed together with POD.

赤色系Ｔｅｔｒａｎｙｃｈｕｓ属ハダニ６種の簡易識別法の試み

Six species of spider mites common in Japan, show a close similarity in both color (red) and morphology. It is difficult to separate these species even with experience. To solve this problem, we analyzed their allozyme patterns of esterases, and tried to find morphological characters that may distinguish the species. Esterase patterns showed species-specific characteristics. Morphological characters of living adult females also provide a key to discriminate the six species.

Developmental pattern of esterases in Apis mellifera L honey bees. I. Stage-dependent changes of esterase isoenzymes in Africanized workers

Une etude detaillee a ete faite des variations des isozymes d'esterase au cours de l'ontogenese des ouvrieres d'abeilles Apis mellifera africanisees. Les profils electrophoretiques des esterases des embryons, des larves, des nymphes et des adultes ont ete determines. Le debut et la duree d'activite de chaque isozyme ont egalement ete releves et mis en relation avec les changements critiques survenant dans le processus de developpement. Afin d'obtenir des ouvrieres d'âge connu, trois reines ont ete encagees avec des rayons pendant des periodes de 6 heures. Les ouvrieres prelevees aux diverses phases du developpement ont ete broyees dans du Tris-HCI a 0,02 M, pH 7,5, et soumises a une electrophorese sur gel d'amidon a 10 % prepare avec le meme tampon. L'esterase a migre pendant 4-5 heures a 7 °C, a 5 V/cm. Le tampon utilise de migration etait du Tris-HCI a 0,3 M, pH 7,5. Les bandes d'esterase ont ete detectees en lumiere UV apres incubation du gel dans une solution d'acetate ou de butyrate de 4-methylumbelliferyle. Les variations de l'activite isozymique detectees par electrophorese nous permettent de distinguer sept phenotypes d'esterase (figs 1-3): i) le phenotype embryonnaire, qui a le plus petit nombre d'esterases (Est-1, Est-2, Est-4); ii) le phenotype des jeunes larves (stades L1, L2, L3, L4) avec cinq esterases (Est-1, Est-2, Est-3, Est-4, Est-6); iii) le phenotype des larves L5, caracterise par l'activite de 6 esterases (Est-1, Est-2, Est-2a, Est-3, Est-4, Est-6); iv) le phenotype des prenymphes (PP1, PP2 et PP3) et des nymphes aux yeux blancs, roses et rose fonce (Pw, Pp, Pdp) avec six esterases (Est-1, Est-2, Est-2a, Est-3, Est-4, Est-5); v) le phenotype des nymphes aux yeux bruns (cuticule blanche, cuticule legerement et moyennement pigmentee: Pb, Pbl, Pbm), caracterise par l'activite de sept esterases (Est-1, Est-2, Est-2a, Est-3, Est-4, Est-5, Est-6); vi) le phenotype des nymphes aux yeux bruns et a la cuticule fortement pigmentee (Pbd) et des adultes âges de 1 et 2 jours (A1 -A2) avec six esterases (Est-1, Est-2, Est-3, Est-4, Est-5, Est-6) et vii) le phenotype des adultes (A3-A35), caracteristique des ouvrieres âgees de 3 a 35 jours, avec sept esterases (Est-1, Est-2, Est-2a, Est-3, Est-4, Est-5, Est-6), bien que trois d'entre elles, Est-2a, Est-4 et Est-5, presentent une faible activite. Ces resultats montrent que les changements de l'activite isozymique sont lies aux divers stades de developpement. Comme le montre la figure 1, le debut de l'activite d'Est-3 et Est-6 suit l'eclosion. Le debut de l'activite d'Est-2a coincide avec le debut du 5 e stade larvaire. Les prenymphes se forment au moment ou Est-5 est detecte, moment qui coincide aussi avec l'absence d'activite d'Est-6. L'emergence a lieu durant une periode caracterisee par l'interruption de l'activite d'Est-2a, qui reprend juste apres le debut de la vie adulte. Les phenotypes d'esterases decrits ici sont donc des marqueurs des phases de developpement, puisque celles-ci se caracterisent par des groupements particuliers d'isozymes. Ceci laisse supposer pour ces derniers l'existence d'un role dans les evenements critiques de l'ontogenese.

Geographical Distribution of Meloidogyne Species (Nematoda: Tylenchida) in Tobacco Fields of Japan

The distribution of root-knot nematodes was surveyed in tobacco fields of Japan. In 1992 and 1994, 219 populations of root-knot nematodes in tobacco fields covering the northern most area of Honshu to the Ryukyu Is. were identified on isozyme patterns of esterase and malate dehydrogenase. In this work, four Maloidogyne species, M. incognita, M. arenaria, M. hapla and M. javanica, were found. In M. arenaria, two distinct esterase phenotypes, one (A-1) and two (A-2) band types, were found. Meloidogyne incognita was widely found from southern Tohoku to the Shinetsu districts and southward to the Ryukyu Is. This species was recorded for the first time in Gifu, Nagano, Fukushima, Yamagata and Miyagi Prefs. Meloidogyne arenaria (A-2) was widely found from the Ryukyu Is. to Akita and Iwate Prefs., however, its distribution in Kyushu and the Ryukyu Is. was patchy. This species was found for the first time in Akita and Iwate Prefs. Meloidogyne arenaria (A-1) was mainly concentrated in Kyushu and the Ryukyu Is., and was found in one locality in Shikoku. Meloidogyne hapla was distributed from Shinetsu to the Kanto distinct northward to Aomori Pref. Meloidogyne javanica was found only in the Ryukyu Is. In areas where the distribution of these species overlapped, two or three species were often found in a single field.

Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.

Electrophoretic patterns of esterases and lactate-and l-malate-dehydrogenases from Flavobacterium species

Esterases and lactate-(LDH) and l-malate-(MDH) dehydrogenases of 64 strains of 13 species of Flavobacterium (particularly 46 strains of F. meningosepticum belonging to 15 different serotypes) were analyzed by horizontal polyacrylamide-agarose gel electrophoresis. Twenty-five esterase types were detected; these enzymes were distinguished by their spectra of hydrolytic activity towards five synthetic substrates (hydrolytic type) and their electrolytic mobilities (electrophoretic type). No variant of LDH was detected in all the strains of Flavobacterium. No variant of MDH was detected in three species: F. aquatile, F. branchiophilum, and F. breve and in serotype I, J and L. of F. meningosepticum. These results within the genus Flavobacterium permit precise identification, which may be useful for characterization of strains kept in collections as well as for epidemiological studies.

Studies on Strains of Trichogramma evanescens Westwood from Different Regions of Eurasia

Strains of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) collected from six different geographical regions in Eurasia were compared in terms of their biological and morphological characteristics and electrophoretic patterns. Significant differences were observed between the strains for biological characteristics such as the proportion of female progeny, developmental period, longevity, proportion of adults emerging and fecundity. Strains from the Altai region in Russia were more fecund at all hygrothermal conditions in which they were reared (27 C, 80% relative humidity (RH); 27 C, 40% RH; 23 C, 80% RH; and 23 C, 40% RH). Poor adult emergence took place at 27 C and 40% RH in strains from Moldavia and Slovakia. Morphological examination of male antennae showed that strains from Moldavia and Slovakia had the highest ratio of the length of antennal hairs to the maximum width of flagellum. The patterns of esterases after electrophoresis indicated that strains differed in the mobility and i...

Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing

Esterases from Listeria monocytogenes strains isolated from cheeses were analyzed by starch gel electrophoresis. Five esterases, numbered from EST 1 to EST 5 in order of decreasing anodal migration, were identified. The EST 1, EST 3, EST 4, and EST 5 set was most active toward alpha-naphthyl propionate, while EST 2 was most active toward alpha-naphthyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed in the population. Twelve esterase patterns were defined and used to subdivide serotypes.

Properties of sake yeast mutants resistant to isoamyl monochloroacetate

Isoamyl monochloroacetate ( i-AmOCIAc) inhibited the growth of sake yeast. AOF10, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate ( i-AmOAc), also showed resistance to i-AmOClAc. Monochloroacetic acid formed by an esterase through the hydrolysis of i-AmOClAc was more effective at glycolysis inhibition than i-AmOClAc. Forty five mutants resistant to i-AmOClAc were isolated from a parental haploid strain (HL-69) derived from sake yeast. Three had less than 20% of the original activity of the esterase. All mutants fermented sake mash as well as the parent strain, HL-69, and accumulated 1.3 to 1.7-fold the levels of i-AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by HL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i-AmOAc and 1.3-fold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (1.6-fold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band ( EST2) were closely related to the activity of the esterase toward i-AmOAc.

Combination of biotyping and electrophoretic patterns of esterases for differentiation of nosocomial Serratia marcescens strains

A total of 292 Serratia marcescens strains isolated in seven Belgian hospitals between 1986 and 1992 were submitted to both biotyping by carbon source utilization and esterase electrophoresis typing. The strains were assigned to 11 biotypes (290 isolates) or were auxotrophic (2 isolates), whereas the various electrophoretic patterns of their esterases produced 54 zymotypes. The two typing methods correlate well, since biotypes embraced more than one zymotype, while each zymotype was restricted to a single biotype. Esterase electrophoretic patterns represent a potent marker for delineating outbreaks of nosocomial infections, whereas biotyping appears to be an appropriate method for screening of strains.

Stereoselective hydrolysis of xenobiotic esters by different cell lines from rat liver and hepatoma and its application to chiral prodrugs for designated growth suppression of cancer cells.

Stereoselectivity in the hydrolysis of racemic ethyl 2-phenylacetate derivatives by cultured cells of noncancerous cell lines from rat liver (BRL, BRL 3A, Clone 9, and ARLJ301-3), spontaneously or oncogene transformed rat liver cell lines (ARLJ301-3TR1, Anr4, Anr9-1, and Anr13-1), and cancer cell lines from rat hepatoma (H4-II-E, McARH7777, and MH1C1) and sarcoma (XC) was studied. A strong (R)-enantiomer preference was found in the hydrolysis of ethyl 2-hydroxy-(2c) and 2-methoxy-2-phenylacetate (3c) by the noncancerous and oncogene-transformed cells and an (S)-enantiomer preference for ethyl N-acylphenylalaninates with all the present cell lines. These inclinations were, however, not recognized with ethyl 2-methoxy-2-phenylpropanoate and ethyl N-difluoroacetyl- or N-trifluoroacetylphenylalaninate. Moreover, the R preference for 3c was reversed in the reaction by hepatoma cells. Thus, the stereoselectivity is influenced by both structure of acyl group and species of cell lines. The hepatoma cells were considerably different from the noncancerous or oncogene-transformed cells in stereoselectivity. This fact was consistent with the order of colony formation in soft agar cultures (index of malignancy) and the resemblance in actively stained esterase patterns in gel electrophoresis. The stereoselective hydrolysis leads to cell-specific activation of anticancer prodrugs. This has been confirmed for the first time by the stereoselectivity of Anr4 and H4-II-E cells in the hydrolysis of a chiral mustard ester, bis(2-chloroethyl)aminophenyl 2-methoxy-2-phenylacetate (14) and by the difference of IC50 values of (R)- and (S)-14 against the two cell lines.

Enzyme polymorphism in the ant genus Tetramorium mayr and its social parasites (Hymenoptera: Formicidae)

Eleven enzyme-encoding loci were examined by electrophoresis in 19 species of tetramoriine ants. GPI and PGM were found to be highly variable in some species, confirming the suitability of these enzymes for population genetical studies. The mainly slave-making ant genus, Strongylognathus showed great similarities in most enzyme phenotypes to the non-parasitic genus Tetramorium. On the other hand, the two inquiline parasites, Anergates atratulus and Teleutornyrmex schneideri differed strongly from each other and from their Tetramorium hosts in allozyme characters. Based on enzyme similarities, the evolution of the inquilines from slave-making ancestors of the genus Strongylognathus appears unlikely. Both esterase patterns and the apparent fixation of T. caespitum and T. impurum for different electromorphs in MDHP could possibly be used as diagnostic characters to distinguish biochemically between workers of these two very similar species.

Polymorphism of Esterases in Free-living Amoebidae

Summary In free-living Amoebidae the pattern of esterases examined by polyacrylamide gel electrophoresis is represented by multiple bands. Only slight differences have been found in the number of fast electromorphs in patterns of 7 strains attributed to Amoeba proteus: three bands in the K strain and four — in all the other strains examined (C, B, L, F. Da and Bk). In this respect, A. lescherae does not differ from the K strain, and A. discoides is identical to the other A. proteus strains studied. Together with our earlier data, this allows one to consider A. lescherae and A. discoides as strains of A. proteus, rather than separate species. Three other Amoeba species, A. borokensis, A. amazonas and A. indica, differ from A. proteus in the number of fast and/or intermediate electromorphs. In A. leningradensis, in contrast to A. proteus and other Amoeba species, one of the subgroups of slow esterases is absent. Chaos carolinense, while lacking a different slow esterase subgroup than Amoeba leningradensis, is distinguished from A. proteus and Polychaos dubium by its higher number of fast electromorphs. In P. dubium the number of both intermediate and fast electromorphs is lower than in Amoeba proteus. Thus, the analysis of electrophoretic patterns of esterases, in addition to other taxonomic criteria, proved to be useful not only for intergeneric and interspecific differentiation of free-living Amoebidae, but also for the detection of erroneously classified species and strains.

Identification by isozymes of nine populations ofTulipafrom Greece

SummaryTwo enzymatic systems: esterase and malate dehydrogenase were studied on nine populations of tulip from different parts of Greece and naturalized in Crete. The results showed that the analysis of esterase banding patterns permitted a clear distinction of the populations because esterase patterns were readily reproducible. The banding of malate dehydrogenase is poor and this isozyme permits a complementary distinction of populations.

Electrophoretic Analysis of Protein and Selected Enzymes of Acacia Seeds from Saudi Arabia

The water-soluble, diluted salt-soluble and ethanol-soluble proteins of seed extracts from eight Saudi Arabia acacia species were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Qualitative differences were evident in the electrophoretic patterns of all protein fractions. Also, the anodic isoenzyme patterns of amylase, glutamate oxaloacetate aminotransferase (GOT), non-specific esterase (EST) and phosphorylase in seed extracts were separated by polyacrylamide gel electrophoresis (PAGE). The studied enzymes were found to be highly polymorphic. It is clear from the results that the isoenzyme patterns can be used together with protein banding patterns as markers to estimate both genetic variation at biochemical level and taxonomical relationship among the species.