Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis,Neocallimastix frontalis,and Piromyces rhizinflata

-d-Glucosidase, -d-fucosidase -d-xylosidase, and -cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. -d-Galactosidase and -l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA₃ inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA₃, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - [9R-5P]DHZ 9--d-ribofuranosyl-dihydrozeatin-monophosphate - [9R-5P]iP 6-isopentenyl-9--d-ribofuranosyladenine-monophosphate - [9R-5P]Z 9--d-ribofuranosyl-zeatin-monophosphate - [9G]BA 6-benzyl-9--d-glucopyranosyladenine - [9G]DHZ 9--d-glucopyranosyl-dihydrozeatin - [9G]iP 6-isopentenyl-9--d-glucopyranosyladenine - [9G]Z 9--d-glucopyranosyl-zeatin - [9R]BA 6-benzyl-9--d-ribofuranosyladenine - [9R]DHZ 9--d-ribofuranosyl-dihydrozeatin - [9R]iP 6-isopentenyl-9--d-ribofuranosyladenine - [9R]Z 9--d-ribofuranosyl-zeatin - BA 6-benzyladenine - DHZ dihydrozeatin - ES⁺ LC-MS/MS HPLC coupled Electrospray Tandem Mass Spectrometry - f.m. fresh mass - mT 6-(3-hydroxybenzyl)adenine - IMA imazalil - iP isopentenyladenine - NAA 1-naphthalene acetic acid - NFT Nutrient Film Technique - (OG)[9R]DHZ O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin - (OG)[9R]Z O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin - (OG)DHZ O--d-glucopyranosyl-dihydrozeatin - (OG)Z O--d-glucopyranosyl-zeatin - PAR Photosynthetic Active Radiation - PBZ paclobutrazol - PRO prochloraz - TDZ thidiazuron - TRI triflurnizole - Z zeatin     

The quaternary structure of four crustacean two-hexameric hemocyanins: immunocorrelation,stoichiometry, reassembly and topology of individual subunits

Summary Two-hexameric (2×6) hemocyanins from the brachyuran crabsCancer pagurus andCallinectes sapidus, the freshwater crayfishAstacus leptodactylus and the lobsterHomarus americanus were isolated and dissociated into native subunits.The subunits of each hemocyanin were analyzed by electrophoresis and immunology. Three immunologically distinct subunit types, which were termed, and, could be identified in each case. They were isolated preparatively, and interspecifically correlated. Subunit is subdivided into several electrophoretically distinct isoforms which are immunologically closely related (Astacus) or identical (other species). InAstacus andCancer one of these isoforms was shown to dimerize and to act as inter-hexamer bridge. It represents a fourth subunit type termed. A fifth, diffuse component, which in PAGE migrated at the position of a dimer, was identified in the crossed immunoelectrophoretic patterns as denatured hemocyanin.A common feature of the four hemocyanins is the presence of 4 copies of and 8 copies of/ within the 2×6 particles. The: ratio is 4:4 in the two Astacidea and 6:2 in the two Brachyura. exists in 2 copies inAstacus andCancer which means that a single dimer- is present in a two-hexamer. This leaves 2 monomeric copies inAstacus and 4 inCancer.Every subunit from the four species except ofAstacus - was capable to form hexamers in reassembly experiments. If subunit combinations were tested, hetero-hexamers were formed preferentially. Two-hexamers were reconstituted only in the presence of all subunit types and the native subunit stoichiometry was required to obtain twohexamers in considerable yields. Factors limiting 2×6 reassembly are discussed.Authentic 2×6 molecules ofAstacus, Homarus andCancer hemocyanin were immunolabeled with subunit-specific antibody fragments (F_ab) or IgG molecules, and the resulting immuno complexes were studied in the electron microscope. A topological model of the quaternary structure of decapod 2×6 hemocyanins is derived, showing the position of each copy of the four subunit types. In this model, the inter-hexamer bridge- is surrounded by two and two subunits forming the central core of the dodecamer. Two additional and two additional subunits form the periphery together with one subunit occupying the peripheral short edges of each hexameric half structure. The model is discussed with respect to the current literature.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This paper is decicated to Professor Dr. Bernt LinzenPreliminary accounts of this work have been presented in the proceedings of a symposium at Tutzing 1985. Linzen B (ed) (1986) Invertebrate oxygen carriers. Springer, Berlin Heidelberg New York. This also includes: Stöcker et al. 1986; Markl et al. 1986) and in a review article (Markl 1986)     

Biotransformation of 5βH-pregnan-3βol-20-one and cardenolides in cell suspension cultures of Nerium oleander L.

Summary In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5H-pregnan-3ol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5H-steroidaglycones at C-3. The respective glucosides of the 5H-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5H-steroidglucosides.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - EtOAc ethylacetate - MeOH methanol - MS Murashige & Skoog     

Steroid synthesizing cellular sites in the ovary of the domestic pigeon Columba livia (Gmelin): a histochemical study.

Synopsis The ovary of the domestic pigeon,Columba livia, has been assayed histochemically for the localization of ⁵-3-hydroxysteroid dehydrogenase (⁵-3-HSDH), 17-hydroxysteroid dehydrogenase (17-HSDH), 11-hydroxysteroid dehydrogenase (11-HSDH), glucose-6-phosphate dehydrogenase (G6P-DH) and NADH-diaphorase activities during different periods of the reproductive cycle. ⁵-3-HSDH, 17-HSDH, 11-HSDH, G6P-DH and NADH-diaphorase activity was found in the theca interna of growing, atretic and postovulatory follicles, the granulosa of ovulatory, atretic and postovulatory follicles, and interstitial gland cells during the pre-incubation and the laying periods. During the incubation and squab feeding periods only ⁵-3-HSDH, G6P-DH and NADH-diaphorase activities were observed in the above mentioned cells. The steroidogenic potential of atretic follicles depends upon the type of atresia a follicle undergoes.     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass

A comparison of different habitats, biogas plant, rumen fluid and sewage sludge, for cellulolytic organisms indicated sewage studge was the best source. Enrichment cultura gave a mixed culture which exhibited CMCase activity as well as extracellular Avicelase, xylanase, -glucosidase, -xylosidase activities and cell-bound -glucosidase, and -xylosidase production in a synthetic medium with eleven different cellulosic and lignocellulosic substrates. The activity of extracellular -glucosidase and -xylosidase production was significantly higher than endogenous activities. Hemicellulases were induced better than cellulases. The anzyme system was stable under aerobic conditions. Of the different lignocellulosic substrates, kallar grass was the best inducer of extracellular enzymes.

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Résumé La comparaison de différents habitats: digesteur méthanique, fluide du rumen ou boue de station d'épuration, pour leur contenu en organismes cellulolytiques, indiquent que la boue de station d'épuration est la meilleure source. Une culture par enrichissement a produit une culture mixte qui a exhibé aussi bien une activité CMCase que des activitiés extracellulaires avicelasique, xylanasique, -glucosidasique et -xylosidasique et qu'une production de -glucosidase et de -xylosidase liées à la cellule, dans un milieu synthétique et pour onze substrats cellulosiques et lignocellulosiques différents. L'activité de la -glucosidase extracellulaire et la production de -xylosidase sont significativement plus élevées que les activitiés endogènes. Les hemicellulases sont mieux induites que les cellulases. Le système enzymatique est stable dans des conditions aérobies. Parmi les divers substrats lignocellulosiques, l'herbe Kallar est le meilleur inducteur d'enzymes extracellufaires.

     

Ultrastructural localization of gonadotrophic hormones in the porcine pituitary using the immunoperoxidase technique

Summary Using specific antibodies against subunits of porcine LH and FSH, the pituitary cells which produce these two gonadotrophins were identified in the porcine pituitary at the ultrastructural level using the immunoperoxidase technique. It was clearly shown that most of the gonadotrophic cells are responsible for the production of both FSH and LH. However, some cells, only positive for FSH or LH, indicate that the concentrations of FSH and LH vary from cell to cell. At the ultrastructural level, the FSH/LH cells contain one class of secretory granules strongly positive for both FSH and LH as well as large negatively stained dense bodies. These findings indicate that the one cell-one hormone concept may not apply to gonadotrophic hormones; a FSH/LH cell cannot be distinguished from a LH or a FSH cell without immunocytochemical identification of its hormonal content.Abbreviations p-LH porcine luteinizing hormone - p-LH porcine LH subunit - p-LH porcine LH subunit - p-FSH porcine follicule stimulating hormone - p-FSH porcine FSH subunit - b-TSH bovine thyrotropic hormone     

Structural and functional properties of proteasome activator PA28

The proteasome activator PA28 or 11S regulator is a protein complex composed of two different but homologous polypeptides, termed PA28 and PA28. The purified activator protein (_200 kDa) is a ring-shaped heteromultimer containing the two polypeptides, possibly with an _{3 3} stoichiometry. The activator, which by itself shows no hydrolytic activity elicits activation of the proteasome's multiple peptidase activities by binding to the terminal rings of the proteinase. In vitro, active PA28 can be reconstituted from isolated and subunits, yielding two different oligomers: with the single subunit, PA28 homomultimers with moderate stimulatory activity toward 20S proteasomes are obtained whereas isolated -subunits are unable to form oligomers and are devoid of stimulatory activity. However, in the presence of both subunits, heteromultimers form, concomitant with restoration of full stimulatory activity. The recent finding that PA28 modulates the proteasome-catalyzed production of antigenic peptides presented to the immune system on MHC class I molecules indicates a cellular function of the activator in antigen processing. Abbreviations: IFN – interferon; LMP – low molecular weight peptide; MHC – major histocompatibility complex.     

Flavonglykoside in Blüten vonPaeonia arborea undPaeonia suffruticosa

Summary Three glycosides have been isolated fromPaeonia arborea: kaempferol-3--glucoside-7--glucoside (Paeonoside), apigenin-7--glucoside, and apigenin-7-rhamnoglucoside (Rhoifolin).Paeonia suffruticosa also contains these three compounds but its main glycoside is kaempferol-3--glucoside (astragalin), which is present inPaeonia arborea only in traces.     

Immunohistological analyses of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases

The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, G_M3(Neu5Ac), G_M3(Neu5Gc) and G_M1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was G_M3(Neu5Ac) followed by moderately expressed G_M3(Neu5Gc) and G_M1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-G_M3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle. Abbreviations: BSA, bovine serum albumin; DAPI, 4, 6-diamidine-2-phenylindole-dihydrochloride; DTAF, dichlorotriazinylamino-fluorescein; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycolylneuraminic acid [53]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [54] and the nomenclature of Svennerholm [55]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gal1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse₃Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₅Cer, GalNAc1-3GalNAc1-3GAl1-4Gal1-4Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone

Summary Morphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3- and 3-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable.The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation.No significant differences (0.7<P<0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P<0.001). This increase was probably related to the large amounts of androgens present in the treated animals.Theca interna cells possessed small gap junctions; no significant differences (P>0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.The following trivial names have been used: Dihydrotestosterone: 5-androstan 17 ol-13 one; 3-androstanediol: 5-androstan 3,17 diol; 3-androstanediol: 5-androstan 3,17 diol     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase