Apical plasma membrane-bound enzymes of rabbit uterine epithelium. Pattern changes during the periimplantation phase

In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5-8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals. All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the "receptive state", 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.     

Enzym-Topochemie von Frühentwicklung und Implantation des Kaninchens

Zusammenfassung Untersucht werden Frühstadien der Entwicklung des Kaninchens von der Ovulation bis zur Implantation, einschließlich Tube und Uterus. Registriert wird die Verteilung von Glykosidasen (-Galaktosidase, Neuraminidase, Glukosaminidase, -Glukuronidase, -Glukosidase), vor allem im Hinblick auf den Stoffwechsel von Mukosubstanzen. Die verwendete Methode zum Neuraminidasenachweis ist ein erster Versuch zur histochemischen Lokalisation dieses Enzyms.Ergebnisse. In Furchungsstadien sind -Glukuronidase und Glukosaminidase auffällig aktiv. Eine Funktion bei den wichtigen morphogenetischen Prozessen dieser Phase wird vermutet. Das Epithel der Tube zeigt vor allem eine Aktivität von Glukosaminidase und -Galaktosidase, die möglicherweise eine Beziehung zur Bildung der Mukoproteidschicht haben. Im Uterusepithel sind in allen Stadien -Galaktosidase und Glukosaminidase aktiv; -Glukuronidase tritt vor allem vor dem Eintritt des Keims in den Uterus und bei der Implantation hervor. Für die Auflösung der Blastozystenhüllen und für die Implantation ist wahrscheinlich die -Galaktosidase-, -Glukuronidase-und Glukosaminidaseaktivität der Trophoblastsprosse von Bedeutung. Neuraminidase ist dagegen vor allem im Uterusepithel lokalisiert.

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Enzyme topochemistry of the early development and implantation in the rabbitII. Glycosidases

Summary Early stages of development and the surrounding tissues of the Fallopian tube and the uterus are studied in the rabbit from ovulation to implantation. The topochemistry of the following glycosidases is demonstrated: -galactosidase, neuraminidase, glucosaminidase, -glucuronidase, -glucosidase. The distribution given for neuraminidase is the result of preliminary attempts to develop histochemical procedures for this enzyme.Results. In cleaving eggs, the activities of -glucuronidase and glucosaminidase are dominant. The epithelium of the Fallopian tube shows an activity of glucosaminidase and -galactosidase possibly correlated to the formation of the mucoproteid layer. These enzymes are also demonstrated in the epithelium of the uterus in all stages, whereas -glucuronidase has its maximum activity before eggs enter the uterus and at implantation. In the trophoblastic knobs, these three enzymes could have a physiological role in the processes of dissolution of blastocyst coverings and implantation. Neuraminidase activity is found in the epithelium of the uterus.

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Abkürzungen EDTA Äthylendiamintetraazetat - FBB Fast Blue B Salt (Echtblausalz B) - MS Mukosubstanz, -en - NA Neuraminsäure, Salinsäuren - NAase Neuraminidase - Nitro-BT Nitro Blue Tetrazolium Chloride - nMS neutrale Mukosubstanz, -en - p.c. post coitum - h.p.c., d p.c. Studen p.c., Tage p.c. - PMS Phenazinmethosulfat - PVP Polyvinylpyrrolidon - sMS saure Mukosubstanz, -en - v/v Mischungsverhältnis zweier Volumina - w/v Gewicht pro Endvolumen     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Uteroglobin in the rabbit

Summary Rabbit uterine uteroglobin (UGL) was studied by electrophoretic and immunological methods following normal copulation, after ovariectomy and progesterone treatment, 17-oestradiol and combined progesterone treatment, 17-oestradiol treatment alone and after HCG-induced pseudopregnancy. Electrophoretic studies show the amount of ULG in uterine secretions, the immunological investigations indicate the intracellular localization of ULG and the distribution of ULG-positive cells in the endometrium.No obvious differences were found between the uteri 7 days after injection with chorion-gonadotropin and those 7 days following normal copulation. No differences could be demonstrated between the uteri of animals 35 days following ovariectomy and subsequent progesterone treatment on Days 31–33 and those of normal 7 d. post coitum (p.c.) animals. Uteri from animals treated with progesterone on Days 2–5 p.c. contained more ULG-positive cells than controls. 17-oestradiol treatment with and without subsequent progesterone treatment resulted, in both gravid and ovariectomized animals, in the formation of a tall columnar endometrial epithelium. Treatment with 17-oestradiol on Days 1 and 2 p.c. led to a decrease in the number of UGL-positive cells at 7 days p.c. Even after ovariectomy with 17-oestradiol substitution, UGL-positive cells were still present in the endometrium. However a secretion of any magnitude could not be detected. The importance of differentiation between synthesis and secretion (= release) as distinct phases of the glandular response is especially emphasised by the latter findings.Supported by Grant No. Ki 154/5-6, Deutsche Forschungsgemeinschaft     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Rhizogenesis in stem discs of Malus pumila rootstock M9 “Jork”: I. Hormonal and environmental effects on root induction and callus formation

To establish a protocol suited for the molecular characterization of root induction the influences of explant position, etiolation state and orientation as well as temperature and light intensity on root and callus formation were investigated. Depending on these factors stem discs of Malus incubated on indole-3-butyric acid containing medium and subsequently on hormone-free medium regenerated roots and callus of filamentous and smooth texture to a varying extent. Concentration and incubation duration of indole-3butyric acid strongly affected rooting and the production of smooth callus. Moreover smooth callus was profuse at the low light levels applied during rooting. Rooting efficiency decreased and filamentous callus increased between 19°C and 25°C. Explants close to the shoot apex displayed reduced rooting efficiency and profuse filamentous callus. There was a strong effect of explant orientation on root and filamentous callus formation.Abbrevations IBA indole-3-butyric acid - IAA indole-3-acetic acid - BA 6-benzylaminopurine - MS Murashigeand Skoog - rts roots - rt rooted - rtd rooted discs     

The histochemical distribution of hydroxysteroid dehydrogenases in the human foetal membranes at term

Summary The histochemical distribution of various hydroxysteroid dehydrogenases in human, term, foetal membranes has been investigated using the tetrazolium dye, Nitro-B.T.The trophoblastic layer was the most active, showing 3-, 3-, 11-, 16- and 17-hydroxysteroid dehydrogenase activities, a pattern of activity similar to that of the placental villous trophoblast.The amniotic epithelium showed weak 3-, 3-, 16- and 17-hydroxysteroid dehydrogenase activity; weak 3- and 3-hydroxysteroid dehydrogenase activity was noted in the connective tissue layers.All activity demonstrated was N.A.D.-linked.     

Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Differentiation of muscle and the determination of ultimate tissue size

Summary The factors that determine ultimate muscle size have been studied using a model that involves two strains of mice, which had been especially bred for largeness (QL) and for smallness (QS). The difference in muscle size was not found to be due to a difference in fibre size, but due to a difference in fibre number. The muscles from the QL mice contained about 30% more fibres.The reason for this increased fibre number was investigated. During early development, the fusion of mononucleated presumptive myoblasts to form multinucleated myotubes took place at the same time in both QL and OS mice, as indicated by the appearance and increase in activity of ATP: creatine phosphotransferase. At this stage fibre (and cell) number and size could be determined by measuring nuclear number and protein/DNA ratio respectively. No difference in fibre (and cell) size could be found in mice at 5 days before birth, newly born, or at 20 days of age. At these ages it was found that the muscles from the QL mice contained a greater number of nuclei (muscle cells). The amount of RNA/nucleus was used as an index of protein synthetic rate, and no difference could be found between the large mice and the small mice.It was concluded that, in the case of the QL mice, the increased fibre number was not brought about by: (i) a delay in time of fusion of presumptive myoblasts; (ii) a smaller number of myoblasts fusing to form myotubes; or (iii) extensive fibre formation after fusion. Differences in fibre number, and hence muscle size, must therefore, presumably be caused by initial differences in the rate of proliferation of myoblasts before fusion.     

Multifrequency behavioral patterns and the phase attractive circle map

With relative phase as a collective variable or order parameter, phase attractive dynamics can capture the temporally coherent behavior of a large number of different experimental systems. We present results from multifrequency coordination experiments in humans showing: a) that phase attraction persists especially for low order frequency ratios; b) that short-term jumps from one phase relation to another occur within a frequency ratio; c) that the most stable frequency-ratios are low order; and d) that transitions frequently occur from higher order (e.g. 52, 43) to lower order (21, 11) frequency ratios. We study a modified sine circle map with built-in phase attractive dynamics that qualitatively accounts for these results. In this phase-attractive map, patterns arise from competition between external driving and intrinsic phase attractive dynamics. The relative strength of extrinsic and intrinsic parameters determines the width of Arnol'd tongues, thereby influencing the delay or acceleration of irregular behavior. Behavioral complexity is inversely proportional to tongue width, thus accounting for the relative difficulty of performing different multifrequency behaviors and why errors in such behavior are often seen to occur.     

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey

Summary Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and -glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for -glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low -glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermysial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.     

Microbial degradation of dehydrodiconiferyl alcohol,a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran--aldehydic (II) and -carboxylic compounds, 2) phenylcoumaran--aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran--carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the -aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the -ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.Non-Standard Abbreviations DHP dehydrogenation polymer - DHCA dehydrodiconiferyl alcohol - DDQ dichlorodicyano-p-benzoquinone - DDHQ dichlorodicyano-p-hydroquinone - Ar aromatic - TLC thin layer chromatography - GC-MS gas chromatography-mass spectrometry     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

On the role of the invariant glutamine at position 54 in the human choriogonadotropin β subunit

The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone subunits from a variety of mammalian species. -Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single AG mutation in the LH gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH, with Arg replacing Gin-54, associated with the subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine , and the media were analyzed for holoproteins, which were characterizedin vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG(Glu-54) bound to almost as well as hCG wild-type, and the resulting heterodimer competed with [¹²⁵l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED₅₀s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG(Lys-54) associated very poorly with . Our results suggest that while Gin-54 in hCG participates in receptor binding, its major function appears to involve binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.     

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

Summary A fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15-hydroxylation of lithocholic acid to a new bile acid (3,15-dihydroxy-5-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3, 15-dihydroxy-5-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.Abbreviations LCA lithocholic acid (3-hydroxy-5-cholanic acid) - 3, 15-DHC (3, 15-dihydroxy-5-cholanic acid) - DMSO dimethyl sulfoxide - CHES 2-[N-cyclohexylamino]ethanesulfonic acid     

Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes

Values ofK _m were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal1-4GlcNAc 2-6sialyltransferase and Gal1-3(4)GlcNAc 2-3sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal1-3GalNAc 2-3sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values ofK _m were determined using rat and human asialo₁ acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal1-3(4)GlcNAc 2-3sialyltransferase. Antifreeze glycorprotein was used as the macromolecular acceptor for the porcine enzyme. Values forK _m were also determined using CMP-NeuAc as the variable substrate.Abbreviations NeuAc N-acetylneuraminic acid - Gal galactose - GlcNAc N-acetylglucosamine Enzymes: Gal1-4GlcNAc 2-6sialyltransferase, EC 2.4.99.1; Gal1-3(4)GlcNAc 2-3sialyltransferase, EC 2.4.99.5; Gal1-3GalNAc 2-3sialyltransferase, EC 2.4.99.4.     

Proteases in the human full-term placenta

Summary Aminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, -glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorinic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. -Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoclectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions. Immunocytochemical staining of dipeptidyl peptidase IV in the villous tree resembled the pattern obtained by catalytic cytochemistry except for the blood vessel endothelium and the x-cells of the basal plate. Fluorometrically, all proteases were more active in the villous tree than in the paraplacenta. The kinetic measurements revealed the highest hydrolysis rates for dipeptidyl peptidase IV followed by the aminopeptidases. In contrast to eatalytic cytochemistry all proteases were detectable when using fluorometry.Supported by the German Research Foundation (Sfb 174)A preliminary account of this work was presented at a Symposion on Progress in General, Applied and Diagnostic Histochemistry (Smolenice, Czechoslovakia on March 24–28, 1986).     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Mitochondrial genetic damage induced in yeast by a photoactivated furocoumarin in combination with ethidium bromide or ultraviolet light

Summary Ethidium bromide (EB) and ultraviolet light (UV) in combination are known to produce a synergistic induction of petite mutants in yeast. Two other agents were combined with EB, 3-Carbethoxypsoralene (3 CPs) activated by 365 nm light or rays. EB in combination with 3 CPs also resulted in an enhanced production of petite mutants. After the photoaddition of 3 CPs in exponential phase cells, recovery of the petite mutation during dark liquid holding was inhibited by the presence of EB producing an enhanced number of petite mutants. The behavior of mitochondrial antibiotic resistance markers after individual and combined treatments with EB and 3 CPs indicates a random loss of markers after EB and a preferential loss of a certain region for the 3 CPs photoaddition. The combination of the two agents leads to an additivity of total drug marker losses rather than a synergistic loss. The combination of EB with rays produced no enhancement in petite induction. A combination of UV and 3 CPs showed a synergistic interaction for petite induction. These results indicate that the three agents, EB, UV and 3 CPs photoaddition may share a common repair step for mitochondrial lesions.