Isoenzymes and histochemistry of non-specific esterases in normal and cancerous skin

Synopsis Non-specific esterases in normal and carcinomatous skin of the mouse have been investigated electrophoretically and histochemically. Three esterase bands were obtained on electrophoresis from homogenates of normal skin; homogenates of carcinomas showed an accumulation of esterase-Ia and esterase-Ib.^* However, using several ester substrates, substrate-specific patterns were demonstrated in the electrophoresis separations and histochemically in tissue sections. On the electrophoresis separations, -naphthyl acetate, -naphthyl acetate, 6-bromo-2-naphthyl acetate, naphthol AS acetate, naphthol AS-D acetate and naphthol AS-LC acetate gave rise to similar patterns, but with -naphthyl propionate as subsmate, more esterase-Ib was indicated and with 5-bromo-indoxyl acetate a distinctive preponderance. Peripheral or uniformly distributed staining was found histochemically in tumour epithelium using -naphthyl acetate, -naphthyl propionate and -naphthyl acetate, whereas with the substrates of naphthol AS acetate, naphthol AS-D acetate and indoxyl acetate an intermediate pattern of staining related to keratinization was obtained.     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

L' α-Glycérophosphate-déshydrogénase du moustique Culex pipiens: Génétique formelle,linkage et étude de populations

THE -CLYCEROPHOSPHATE-DEHYDROGENASE IN THE MOSOUITO CULEX PIPIENS: HEREDITY, LINKAGE AND POPULATION STUDIES. Culex pipiens -glycerophosphate-dehydrogenase is coded by an autosomal locus which is shown to have a demonstrable activity in nymphs just prior to imago emergence and in adults of any age and sex. The -Gpd locus has two codominant alleles, -Gpd ^1.00 and -Gpd ^1.30, and heterozygotes present three isozymes. The -Gpd locus is localized on the same chromosomes as the two linked esterase loci Est-2 and Est-1, at 37.05 and 44.88 cross-over units, respectively, of these loci. Population studies seem to indicate that the -Gpd locus has a low variability in Culex pipiens as in most other organisms.     

Esterases in histochemistry and ultrahistochemistry

Synopsis The histochemical identification of individual esterases is a problem that has not yet been overcome. Inhibitors and different substrates reveal different patterns of distribution. 8-hydroxyquinoline acetate is a useful substrate in ultrahistochemistry. There is evidence of a relationship between esterase distribution and function.ACTH adrenocorticotropic hormone - 5Bri–O-2 5-bromoindoxyl acetate - 5Br–4ClI–O-2 5-bromo-4-chloro indoxyl acetate - cAMP cyclic adenosine monophosphate - DFP di-isopropyl-fluorophosphate - hCG human chorion gonadotropin - HS-2/4 thiol acetate/butyrate - I-O-2/4 indoxyl acetate/butyrate - N-O-2/3/4 -naphthyl acetate/propionate/butyrate - N-O-2 -naphthyl acetate - N-S-2/9 -naphthyl thiolacetate/nonanoate - NAS-O-2 naphthol AS acetate - NASD-O-2 naphthol AS-D acetate - 4NP-O-2/3 p-nitrophenyl acetate/propionate - 4NP-S-2 p-nitrophenyl thiol acetate - P-O-2 phenyl acetate - Q-O-2/4 8-hydroxyquinoline acetate/butyrate - Q-S-2/4 8-mercaptoquinoline acetate/butyrate - TBA-S-2/9 -thiolbenzanilide acetate/nonanoate - TSH thyroid-stimulating hormone     

Esterases in the mosquito Culex pipiens pipiens L.: Formal genetics and polymorphism of adult esterases

The genetics of two esterase loci active in autogenous adults of the mosquito Culex pipiens pipiens L. has been studied by means of starch gel electrophoresis. Three alleles at the Est-1 locus and eight at the Est-2 locus are described. Both loci have a null allele. Active alleles are codominant and there is no hybrid enzyme in heterozygotes. The Est-1 locus codes esterases preferentially hydrolyzing -naphthylacetate and the Est-2 locus esterases preferentially hydrolyzing -naphthylacetate. Strains homozygous for both loci were selected. Linkage studies of the two loci have shown that they are not sex linked but are linked to each other, the crossover frequency being 8.6%. The polymorphism of two laboratory and two natural populations is described for both loci. Phenotypic distributions are in good agreement with Hardy-Weinberg expectations.This work was conducted at the Université des Sciences et Techniques du Languedoc (Laboratoire de Génétique Expérimentale des Populations), Montpellier, France, in partial fulfillment of the requirements for the degree of Docteur de spécialité.     

Est-7, a set of genes controlling green tissue esterases in wheat and related species

Summary Analysis using isoelectric focusing of Chinese Spring wheat genetic stocks revealed a set of coleoptile and leaf esterase loci, designated Est-7, on the long arms of the group 2 chromosomes. A survey of 38 other hexaploid genotypes revealed onyl a single variant, at Est-D7. Homoeoloci were found on chromosome (arm) 2HL of Hordeum vulgare, 2RL of Secale cereale, 2R ^m of S. montanum, 2U of Aegilops umbellulata, 2E of Agropyron elongation and 2V of Dasypyrum villosa.     

Carboxylic ester hydrolases in mitochondria from rat skeletal muscle

Summary A mitochondrial pellet, prepared from rat skeletal muscle, contained a number of carboxylic ester hydrolase isoenzymes. The esterases which split -naphthyl acetate were organophosphate sensitive, whereas two out of three indoxyl acetate hydrolysing enzymes were resistant to both organophosphate and organomercury. The activity of the indoxyl acetate esterases was enhanced by the non-ionic detergents Tween-40 and Lubrol. After freezing, thawing and high speed centrifugation most of the -naphthyl acetate splitting enzymes were found in the supernatant, indicating that the enzymes are loosely bound to mitochondrial membranes.     

Esterase Patterns and Phylogenetic Relationships of Drosophila Species in the Saltans Subgroup (Saltans Group)

The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using and naphthyl acetates as substrates, they were classified in 18 -esterases (they hydrolyse the -naphtyl substrate), 15 -esterases (they hydrolyse the -naphtyl substrate) and 1 /-esterase (it hydrolyses the and -naphtyl substrates). Among the -esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to and naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.     

Über die Eignung von Naphthyl-α-l-fucosiden zur Untersuchung der α-l-Fucosidasen

Zusammenfassung Verglichen mit 1- und 2-Naphthyl--d-glucosid,--d-galactosid,--d-glucuronid,--d-N-acetylglucosaminid,--d-glucosid,--d-galactosid und--d-mannosid werden 1- und 2-Naphthyl--l-fucosid schneller oder im gleichen Ausmaß von Homogenaten verschiedener Rattenorgane hydrolysiert. Trotzdem fällt der histochemische Nachweis der -l-Fucosidasen methodenunabhängig im Gegensatz zu dem der anderen Glykosidasen überwiegend negativ aus. Ursache dafür ist die massive Hemmung der -l-Fucosidase durch Aldehydfixation und Diazoniumsalze; die Inhibitionsrate liegt bei 90% bzw. zwischen 85 und 98%; die - und -d-Glucosidase, - und -d-Galactosidase, -d-Mannosidase, -d-Glucuronidase sowie -d-N-Acetylglucosaminidase werden durch Aldehydfixation oder Kuppler höchstens zu 70% gehemmt. Daher können 1- und 2-Naphthyl--l-fucosid für die histochemische Darstellung der -l-Fucosidase nicht einschränkungslos empfohlen werden. Kleine Mengen Dimethylformamid hemmen die meisten Glykosidasen nicht.Für biochemische Messungen der -l-Fucosidase eignet sich speziell 1-Naphthyl--l-fucosid und läßt sich an Stelle von p-Nitrophenyl--l-fucosid werwenden. Bei der fluorometrischen Untersuchung der -l-Fucosidase in Rattenorganen mit dem 2-Naphthylderivat ergeben sich bemerkenswerte Aktivitätsunterschiede.

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Suitability of naphthyl--l-fucosides for the investigation of -l-fucosidases

Summary In comparison with 1- and 2-naphthyl -d-glucoside, -d-galactoside, -d-glucuronide, -d-N-acetylglucosaminide, -d-glucoside, -d-galactoside and -d-mannoside 1- and 2-naphthyl -l-fucoside are hydrolyzed more quickly or to the same extent by homogenates prepared from freezedried cryostate sections of various rat organs. Nevertheless, when the fucosides are employed for the histochemical demonstration of -l-fucosidase mostly negative data were obtained independent on the method used, whereas all other naphthyl glycosides deliver positive results. The reasons for these discrepancies are the marked inhibition of -l-fucosidase by aldehyde fixation and diazonium salts. Then, -l-fucosidase activity is suppressed to 90% and between 85 and 98% respectively; the inhibition of - and -d-glucosidase, - and -d-galactosidase, -d-mannosidase, -d-glucuronidase and -d-N-acetylglucosaminidase by the fixative or coupling reagent does not exceed 70%. Therefore 1- and 2-naphthyl -l-fucoside cannot be recommended in general for histochemical purposes. Small amounts of dimethylformamide do not influence the activity of most of the glycosidases investigated.For biochemical measurements, however, especially 1-naphthyl -l-fucoside represents a suitable alternative in a fluorometric procedure instead of p-nitrophenyl -l-fucoside used for the photometric evaluation of -l-fucosidase. With the fluorometric method the enzyme was measured in rat organs, which posses remarkably different activities of -l-fucosidase.

     

Further genetic variation at the esterase loci of Drosophila virilis

Reexamination of the electrophoretic mobilities of esterases encoded by the Est- and the Est- alleles of Drosophila virilis was carried out in detail using both thin-layer agar gel and polyacrylamide slab gel electrophoresis. Many allelic products with fine differences in their electrophoretic mobilities were found and designated by a new system. Some esterases separable by the agar gel method were indistinguishable using the polyacrylamide gel method. But the polyacrylamide gel method uncovered two multiband homozygotes, ^(d).77 and ^(d)1.28. Some allelic frequencies on the basis of the new designation were estimated in two natural populations. As a result, it is proposed that the total scope of allelic variation at the two esterase loci of Drosophila virilis is composed of discrete distribution patterns of gene frequencies, each histogram of which shows a bell-shaped pattern.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Small enzymes with esterase activities from two thermophilic fungi, Emericella nidulans and Talaromyces emersonii

Extracellular esterase activities in Emericella nidulans and Talaromyces emersonii are attributed to small enzymes with molecular weights less than 10 kDa (microenzymes). A 1.6 kDa esterase accounted for most of the esterase activity observed in both organisms and one of them also contained a 4.1 kDa microenzyme with weaker esterase activity. These esterases were growth-associated and active towards fluorescein dibutyrate and -naphthyl acetate as well as tributyrin.     

Gynogenic lines of onion (Allium cepa L.): evidence of their homozygosity

Haploid induction via gynogenesis offers the possibility of using doubled haploid (DH) inbred lines in onion breeding. A first DH line that originated from the open-pollinated (OP) cultivar Dorata di Parma was obtained after overcoming difficulties associated with the haploidy of the regenerants. Spontaneous chromosome doubling occurs seldom in onion. The first DH line obtained was cloned and selfed to produce sufficient seeds for genetic studies. The homozygosity of the DH gynogenic line was revealed on the basis of the low standard deviations of the bulb traits polar diameter, shape index and weight with respect to those of the S₁ line or the OP cultivar. In the DH line, moreover, segregation of RAPD and alpha esterase markers was not noted. Out of four primers revealing polymorphism at 16 ge-netic loci in the OP cultivar Dorata di Parma, none produced polymorphism in the DH gynogenic line. The Est-1 locus, homozygous in 22 plants (Est-1 ^1/1 in 3 and Est-1 ^2/2 in 19) and heterozygous (Est-1 ^1/2) in 11 plants of the OP cultivar, always carried the same alleles in the DH line. We also tested genetic stability during micropropagation of a second halpoid line obtained via gynogenesis from var. Senshyu Yellow. Seventeen plants of this line were tested to detect changes occurring during the tissue culture process. Again no polymorphism was observed. The high genetic homogeneity observed in the two gynogenic lines of onion could be related to the absence of the callus phase during the gynogenic process.     

Genetics of esterases in Drosophila. IV. Slow-migrating S-esterase in Drosophila of the virilis group

A slow-migrating -esterase (S-esterase) is described which has been detected in Drosophila montana, Drosophila imeretensis, and some stocks of Drosophila virilis when mixtures of - and -naphthyl acetate are used as substrates in histochemical reactions after electrophoresis. Sexual dimorphism for S-esterase has been demonstrated. This esterase is contained in male genitalia only, predominantly in the ejaculatory bulb (waxy plug). It appears 3–4 days after emergence of flies. In hybrids between S⁺ and S⁰ species, the activity of the slow esterase is either decreased or inhibited. An autonomous synthesis of the S-esterase in the ejaculatory bulb was established by transplantation of imaginal genital discs into larvae of different Drosophila stocks. Based on analysis of physicochemical and immunochemical properties, S-esterase is suggested to be an independent fraction of esterase, possibly dimeric, which does not cross-react with -esterase antiserum.     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.