Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Ultrastructural localization of thyrotropin (TSH) in the porcine anterior pituitary

Summary Two different immunocytochemical techniques based on specific antibodies against -subunits of porcine, rat and bovine TSH were applied at the ultrastructural level to identify the TSH cells in the porcine anterior pituitary and to compare the subcellular localization of the hormone.The post-embedding method on serial ultrathin sections revealed the localization of TSH in the granules of a specific cell type, negative for the other hormones. TSH was found in polyhedral cells characterized (i) by their content of granules that were the smallest of all the cell types examined, and (ii) by their flattened or slightly dilated RER cisternae. The pre-embedding method applied to isolated cells permitted a good penetration of antisera and the maintenance of antigenicity in sites inaccessible to the post-embedding method. Thus, immunoreactivity of TSH was detected in the secretory granules, the cytoplasmic matrix and in portions of the rough endoplasmic reticulum, in association with some membranes and inside some saccular structures.     

Influence of the size of inoculum on various growth phases inAspergillus oryzae

1) The duration of the lag phase of filamentous fungi should be expressed either as the time elapsing until exponential growth starts or until any growth takes place, but not until growth enters the linear phase or until mycelium formation is measurable.2) Exponential growth ofAspergillus oryzae can be established over a wide measurable range at a high rate of multiplication in vigorously aerated and agitated cultures where dispersed growth is obtained. Under such circumstances it is apparent that the rate of multiplication observed is equal to that in the non-measurable range which allows a determination of the lag phase with small inocula.3) The lag phase ofAspergillus oryzae is hardly affected by the size of the inoculum (conidia or mycelium) under various conditions of cultivation.4) Between inocula of 8 × 10⁷ and 4 × 10³ conidia per 100 ml and between 12.5 and 0.0008 mg mycelium per 100 ml the specific rate of growth in the exponential phase, and/or the rate of growth in the linear phase, and/or the maximum yield decreased with decreasing size of inoculum.5) The mentioned effects are dependent upon various factors. a) Trace elements markedly counteract the effects; however, the carry-over of these with large inocula is not sufficient to account for the observed phenomena. b) The higher the sugar concentration (at relatively low concentration of ammonium sulphate) the more pronounced are the effects, especially on maximum yield of mycelium. c) The effect on maximum yield is more pronounced if the cultures are strongly agitated and aerated. d) There is a tendency for early autolysis in the case of small inocula if the cultures are agitated (vibro mix and shaken cultures). e) Cultures originating from small inocula appear to be more easily influenced by the environment than cultures from large inocula in view of the larger variation of the results and the effects of products of heat sterilization where small inocula are used.6) Extremely large inocula (112 × 10⁷ and 288 × 10⁷ conidia per 100 ml) stimulate growth rate and maximum yield in substrates with 40 g/liter of maltose with or without trace elements. But at 80 g/liter of maltose a reversal of this effect is observed.7) Extremely small inocula (up to 20 reproductive units per 100 ml), in substrates poor in trace elements, can cause a marked increase in growth rate and maximum yield over those cultures originating from inocula 100 to 10,000 times larger.The experiments reported in this paper were carried out at the Department of Agricultural Bacteriology and Fermentation, Swiss Federal Institute of Technology, Zürich, Switzerland.     

Effects of isoproterenol on the dense core and perigranular membrane of atrial specific granules

Summary Following subcutaneous injections of isoproterenol hydrochloride (ISO), atrial cells present a large number of partly degranulated or completely clear specific granules enclosed by an intact membrane. Such profiles were never encountered in normal controls and might suggest ISO-induced release of a secretory product. Permeability of perigranular membrane was tested using the extracellular macromolecular tracer horseradish peroxidase (HRP). Reaction product was entirely absent within granules of atrial cells in which the sarcolemma was made permeable to HRP molecules by the ISO injections. This seemed to be the case even in heavily labelled cells in which the peroxidase had penetrated the mitochondrial membranes. In atrial cells impermeable to the tracer, the specific granules closely apposed to the sarcolemma were always HRP-negative. The release mechanism of a possible secretory substance from the specific granules is discussed.     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bbeta subunit into a replacement for yeast eEF1Balpha

Elongation factor subunit eEF1B (formerly EF-1 in plants and EF-1 in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK). CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes. eEF1B and the related protein eEF1B (formerly EF-1' in plants and EF-1 in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1 in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes. Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1B. A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bs, but not eEF1B. These interactions between CDK and eEF1B prompted investigations into the in vivo consequences of this relationship. Expression of cDNAs encoding rice or pea eEF1B subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1B gene, as was previously observed for the human eEF1B. However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1B, with alanine allowed the pea protein to substitute for eEF1B function in vivo. In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast. Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1B can serve as the guanine nucleotide exchange factor for eEF1A.Communicated by C. P. Hollenberg     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Ultrastructure of the pituitary gland (pars distalis) in sockeye salmon (Oncorhynchus nerka) during gonad maturation

Summary The fine structure of the various hormone-producing cell types (with the exclusion of the prolactin cells) in the pituitary gland (pars distalis) of migratory sockeye salmon is described. All fish were in an advanced stage of sexual maturation. In the proximal pars distalis five cell types were distinguished: growth hormone cells, ACTH cells, gonadotrops, vesicular cells, and chromophobe cells. Gonadotrops were also found throughout the rostral pars distalis. A conspicuous feature of the gonadotrops was the presence of two kinds of secretory inclusions: small electron-dense granules (200–375 m) and large, relatively electron-translucent globules (400–2 000 m). The large vesicular cells, so called because of their conspicuous vesicular endoplasmic reticulum, were numerous and often appeared to contain some small granules. It is argued that they may represent a second type of gonadotropic cell, which, in earlier stages of gonad development, contains many granules but becomes largely degranulated near the time of reproduction when the other gonadotrops (globular gonadotrops) abound. The chromophobes, which were smaller and far less abundant than the vesicular cells, also appeared to contain small granules (120–280 m). They are probably thyrotrops.The assistance of Mr. S. Killick, of the International Pacific Salmon Fisheries Commission, who helped in the collection of salmon, is gratefully acknowledged.     

Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells

Proteolytic cleavage of recombinant human interferon- (IFN-) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN- was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN- polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN- polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln¹³³-Met¹³⁴. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln¹³³ occurred during the later stages of the culture resulting in a heterogeneous IFN- polypeptide population with 'ragged' C-termini.     

Plasmalemma structure in relation to microfibril biosynthesis in Oocystis

Summary The plasmalemma of Oocystis apiculata, W. West when freezeetched has been shown to bear granules of several sizes. At the earliest stage of development the outer face of the plasmalemma of the naked autospore has small (8.5 nm diameter) granules aligned in rows, in pairs. These rows are stacked together forming extensive granule-bands over the plasmalemma surface. The orientation of these granule-bands corresponds exactly to one of the major microfibril directions. Occasionally, the bands are reduced to patches, some of which are at right angles to each other. Banding of granules on the inner plasmalemma face of naked autospores is also seen. During development the plasmalemma is seen to change so that in the final stages it bears reticulate invaginations, the granule bands occurring within them. The significance of the granulebands in terms of cellulose microfibril biosynthesis is discussed.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Protein kinase C isoenzymes in mouse Harderian gland. Differential expression of the α- and ε-isoforms during pregnancy

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-,-,- and-. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the - and -isoforms. The 80-kDa native form of PKC- almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C- appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the - and -isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.     

Discrimination of LH,FSH, TSH and ACTH in dissociated porcine anterior pituitary cells by light and electron microscope immunocytochemistry

Summary Using the PAP unlabelled antibody method, LH, FSH, TSH and ACTH were localized at light microscope level in cultured cells dissociated from the porcine adenohypophysis. Antisera were shown to be specific for the subunits of the porcine glycoprotein hormones by radioimmunoassay and absorption studies. Using these antisera, it was found that LH and FSH were contained within the same cell, with TSH in a separate cell. When absorbed with LH, anti-porcine ACTH stained a separate distinct population of ACTH cells.Adjacent ultra-thin sections stained with anti-pLH and anti-pFSH, and examined at electron microscope level, showed that the ovoid, 150–400 nm secretory granules of the LH/FSH gonadotrophs contained both LH and FSH.The authors gratefully acknowledge the technical assistance of Carole Smith and Adrian Walsh     

Histochemical investigations on the human fetal subcommissural organ

Summary A histochemical and ultrastructural study was carried out on subcommissura organs from 42 human embryos and fetuses in order to characterize some large granulesTypical granules make their appearance in the rostral hypendymal region of the subcommissural organ (SCO) in fetuses of about 50 mm CRL. Although they appear in other SCO-regions later, the highest number of granules is always located towards the pineal gland.Typical granules are of spherical shape with a diameter of about 2 microns. The various histochemical reactions reveal a reactivity which differentiates the shell of the granules from the granule interior. Nucleoproteins are present in the shell together with phospholipids and/or lipoproteins. The interior of the granules can contain different materials such as glycogen or lipid or neurosecretory substance. Ultrastructural observations show that a granule consists of whorls of endoplasmic reticulum sparsely studded with ribosomes surrounding an interior containing either lipid or lipoprotein inclusions, large amounts of glycogen or simply cytoplasm.It is suggested that the concentric lamellar organelle (CLO) is a morphological entity that might be involved in secretory processes rather than being the secretory granules themselves.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.     

Enhanced acetyl esterase production by Fusarium oxysporum

Production of acetyl esterase (EC 3.1.1.6) by Fusarium oxysporum strain F3 was enhanced by optimization of growth conditions. Under optimal conditions, activities as high as 0.89U/ml of culture medium were obtained. The culture filtrate was equally active on p-nitrophenyl acetate and acetylxylan. The enzyme produced 71% deacetylation of acetylxylan in 2h at 40C. Activity was optimized at pH6.5 and at 55^C. The respective K_m values for p-nitrophenyl acetate and acetylxylan were 0.25mM and 1.05% (w/v) and the V_m values were 0.65 and 0.43mol acetate/min/mg protein.     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

Lectin-histochemical analysis of glycans in ovine and bovine near-term placental binucleate cells

Chorionic binucleate cells (BNC) occur in several ruminants including cow, deer, goat and sheep. They migrate through the chorionic tight junction to fuse with uterine epithelial cells and discharge their granules into maternal connective tissue. We have compared the BNC of near-term, resin-embedded, ovine and bovine placentae using 15 biotinylated lectins and an avidinperoxidase revealing system. There was pronounced conservation of saccharides between the two species. Several sub-types of N-glycan were present, with highly branched structures being abundant, as shown by Galanthus nivalis, Pisum sativum and Phaseolus vulgaris (leuko) agglutinins. Among the non-reducing terminal saccharides conserved were GalNAc1,3(Fuc1,2)-Gal1,4GlcNAc1-, GalNAc1,6Gal1-, Gal1,4GlcNAc-and Gal1,3GalNAc1- shown by Dolichos biflorus, Wisteria floribunda, Erythrina cristagalli, and Maclura pomifera agglutinins, respectively. Arachis hypogaea and Glycine max agglutinins tended to bind to bovine BNC at different stages of maturity, while fucosyl residues detectable by Tetragonolobus purpureus and Ulex europaeus-1 agglutinins were not observed in either species. The only major difference related to sialyl residues, with 2,3-linked sialic acid being present in bovine (Maackia amurensis, Limax flavus) and 2,6 sialic acid being present in ovine (Sambucus nigra agglutinin) cells. This conservation of glycan may be related to glycosylation of peptide hormones in the granules, and may thus be important in the targeting of these hormones to their receptors.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)