Biomedical subjects

H R Morris

Publications and source records attributed to H R Morris.

At least 19 recordsLinked to original sources

PubMed1995

Structural analysis of the oligosaccharides derived from glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities.

Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Gal beta 1-4GlcNAc (lacNAc), GalNAc beta 1-4GlcNAc (lacdiNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacdiNAc), Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewisx), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (lacdiNAc analogue of Lewisx). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.

Amino Acid Sequence↗

PubMed1995

A unique multifucosylated -3GalNAc beta 1-->4GlcNAc beta 1-->3Gal alpha 1- motif constitutes the repeating unit of the complex O-glycans derived from the cercarial glycocalyx of Schistosoma mansoni.

The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R-->Gal beta-1-->3GalNAc, and type 2, R-->Gal beta 1-->3(R-->GlcNAc beta-1-->6)GalNAc, core structures via repeat units of (3GalNAc beta 1-->4(Fuc alpha 1-->2Fuc alpha 1-->2Fuc alpha 1-->3)GlcNAc beta-1-->3Gal alpha-->)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1-->4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.

Animals↗

PubMed1995

Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification.

The developmental expression of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid-containing glycosphingolipids (KDN-gangliosides) in rainbow trout testis during spermatogenesis was studied using a monoclonal antibody, mAb.kdn3G, which recognizes the KDN alpha 2-->3Gal beta 1-->epitope. A major KDN-ganglioside found in mature sperm, (KDN)GM3, KDN alpha 2-->3Gal beta 1-->4Glc beta 1-->Cer (where Cer is ceramide), was expressed in testis throughout all stages of its maturation. On the contrary, four new KDN-gangliosides which were reactive with mAb.kdn3G were not detected in mature sperm, although they were identified in immature testis and expressed during spermatogenesis. The structures of these KDN-gangliosides were established by chemical, enzymatic and immunochemical methods as: (i) (KDN)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer; (ii) (KDN, Neu5Ac)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(Neu5Ac alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer and Neu5Ac alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer; (iii) (KDN) GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(KDN alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer; and (iv) (KDN,Neu5Ac)GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer. (KDN)GD1a and (KDN,Neu5Ac)GD1a first appeared at an early stage of spermatogenesis, but (KDN)GD1 alpha and (KDN,Neu5Ac)GD1 alpha were not expressed until 2 months prior to spermiation. While (KDN)GM3 was previously shown to contain only 4-sphingenine (d18:1) acylated with a C16:0 fatty acid, the new KDN-gangliosides discovered in this study were composed of 4-hydroxysphinganine (t18:0) or 4-sphingenine (d18:1), and were acylated with a C24:1 or C16:0 fatty acid. A possible function of these KDN-gangliosides is suggested.

Acylation↗

PubMed1995

The sulphated carbohydrate-protein linkage region isolated from chondroitin 4-sulphate chains of inter-alpha-trypsin inhibitor in human plasma.

Inter-alpha-trypsin inhibitor (ITI) in human plasma has a unique structural architecture composed of three polypeptide chains (H1, H2 and L chains), which are linked to each other through a chondroitin 4-sulphate chain. The structure of the carbohydrate-protein linkage region of the chondroitin 4-sulphate chain attached to the L chain was investigated. The peptide-chondroitin sulphate fraction was isolated by anion-exchange chromatography after exhaustive digestion with lysyl endopeptidase and then V8 protease. The chondroitin 4-sulphate chain was released from the peptides by beta-elimination using NaB3H4 and then digested with chondroitinase ABC. These treatments resulted in a single 3H-labelled hexasaccharide alditol fraction derived from the linkage region which had been associated with the L chain. Chemical and enzymatic analyses as well as fast-atom bombardment-mass spectrometry (FAB-MS) analysis revealed that the 3H-labelled hexasaccharide alditol had the following structure: delta HexA-alpha 1-3GalNAc(4-sulphate)beta 1-4GlcA beta 1-3Gal(4-sulphate)beta 1-3Gal beta 1-4Xyl-ol (where delta HexA is 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and Xyl-ol is xylitol). The structure contained the novel 4-sulphated Gal residue, which was previously demonstrated in a linkage hexasaccharide isolated from chondroitin 4-sulphate of rat chondrosarcoma (Sugahara et al., J. Biol. Chem., 263, 10168-10174, 1988) and of whale cartilage (Sugahara et al., Eur. J. Biochem., 202, 805-811, 1991). The above disulphated hexasaccharide alditol was the only component detected in the linkage region fraction of the chondroitin 4-sulphate chain of ITI, which implies some biological significance of this novel structure.

Alpha-Globulins↗

PubMed1994

O-glycosylation mimics N-glycosylation in the 16-kDa fragment of bovine pro-opiomelanocortin. The major O-glycan attached to Thr-45 carries SO4-4GalNAc beta 1-4GlcNAc beta 1-, which is the archetypal non-reducing epitope in the N-glycans of pituitary glycohormones.

The NH2-terminal domain of pro-opiomelanocortin, designated as the 16-kDa fragment, is highly conserved throughout the vertebrate family and is likely therefore to have an important functional role. Bovine 16-kDa fragment is a 77- residue glycopeptide, which has been found to be glycosylated at threonine 45 and asparagine 65. Available evidence suggests that glycoforms lacking glycans at the O-linked site are processed in the intermediate pituitary at -Arg49-Lys50- to give the residue 1-49 amino-terminal peptide and a carboxyl-terminal glycopeptide referred to as Lys1 gamma 3-melanotropin. Glycoforms carrying O-glycans remain unprocessed in the intermediate pituitary. Thus O-glycosylation is likely to play an important role in controlling the fate of the NH2-terminal portion of pro-opiomelanocortin, thereby affecting the biological events that are influenced by peptides and glycopeptides derived from this domain. In a recent study (Siciliano, R. A., Morris, H. R., McDowell, R. A., Azadi, P., Rogers, M. E., Bennett, H. P. J., and Dell, A. (1993) Glycobiology 3, 225-239), we sequenced the N-glycans attached to Asn-65 of bovine 16-kDa fragment and demonstrated that the acidic components contain, in addition to neutral antennae, a single SO4-4GalNAc beta 1-4GlcNAc beta 1- antenna, which is characteristic of the pituitary glycohormone N-glycans (Baenziger, J. U., and Green, E. D. (1988) Biochim. Biophys. Acta 947, 287-306). We now report the structural characterization of the O-linked oligosaccharides found in bovine 16-kDa fragment. The major component, which constitutes about 80% of the O-glycan population, is a novel sulfated tetrasaccharide, which carries the same sulfated epitope as the N-glycans. This is the first time that the SO4-4GalNAc beta 1-4GlcNAc beta 1- moiety has been observed in O-glycans, and it raises the interesting possibility that the beta-N-acetylgalactosaminyltransferase responsible for the addition of N-acetylgalactosamine to the pituitary glycohormones (Smith, P. L., and Baenziger, J. U. (1988) Science, 242, 930-933) might be capable of glycosylating both N- and O-linked acceptors.

Amino Acid Sequence↗

PubMed1994

Effect of hemicholinium-3 on the hypothalamic concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase-stimulating substance.

Hypothalamus and plasma of salt-loaded rats, spontaneously hypertensive rats (SHR), and hypertensive reduced renal mass rats (RRM), and the plasma of patients with essential hypertension and of Milan hypertensive rats contain an increased concentration of a cytochemically detectable glucose-6-phosphate dehydrogenase (G6PD)-stimulating substance that has properties similar to that of a possible choline derivative di-methyl methylene immonium ion. Intracerebroventricular (i.c.v.) administration of hemicholinium-3 (HC-3) selectively blocks high-affinity neuronal choline uptake, inhibits brain acetylcholine (ACh) synthesis, and decreases arterial pressure in SHR through an inhibiting effect on hypothalamic cholinergic function. The experiments were performed to study the effect of centrally administered HC-3 on the content of the cytochemically detectable cholinelike substance in hypothalamus and plasma of SHR. HC-3 or saline was infused into the lateral cerebral ventricle for 6 days with a minipump in 14 SHR. On day 7, the hypothalamic and plasma concentration of the cytochemically detectable substance was significantly reduced in rats that received HC-3. The hypothalamic concentration was 225 +/- 95.6 x 10(8) G6PD U per hypothalamus (range 38.2-775) in SHR that received saline and 1.037 +/- 0.45 x 10(8) G6PD U (range 0.112-3.61) (p < 0.05) in SHR that received HC-3. The respective plasma concentrations were 284.9 +/- 26 U/ml (range 192-374) and 72.7 +/- 14.7 U/ml (range 24-119) (p < 0.05). The findings are consistent with the physicochemical evidence, which suggests that the cytochemically detectable substance is a choline derivative.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi.

The highly antigenic surface glycolipids of serovars of the Mycobacterium avium complex are glycopeptidolipids of the general structure [formula: see text] However, it has recently been shown [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063; Rivière, M., & Puzo, G. (1992) Biochemistry 31, 3575-3580] that the characteristic glycopeptidolipid of a strain of Mycobacterium xenopi is of the structure [formula: see text] [monosaccharide, 3-O-CH3-6-deoxy-alpha-L-talopyranose; tetrasaccharide, 2,3,4-tri-O-CH3-alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L- rhamnopyranose(1-->3)2,4-di-O-acetyl/lauryl-6-deoxy-alpha-L-glucopyranos e]. The type of analyses previously applied to the glycopeptidolipids of M. avium combined with fast atom bombardment mass spectrometry allowed the recognition of other variants of this structure in other strains of M. xenopi: [formula: see text] in which the monosaccharide is 2-O-acetyl-3-O-CH3-6-deoxy-alpha-L-talopyranose or 3-O-CH3-6-deoxy-alpha-L-talopyranose, and the disaccharide is 4-O-octanoyl(or decanoyl)-alpha-L- rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose or alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose. Recognition of serine-containing glycopeptidolipids substituted in one case with a tetrasaccharide and in another case with a disaccharide unit implies that M. xenopi, an important opportunistic pathogen, exists as a serocomplex in nature.

Amino Acid Sequence↗

PubMed1993

The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin.

The N-terminal glycopeptide of pro-opiomelanocortin (POMC), designated as the 16K fragment, is highly conserved throughout vertebrates from amphibians to mammals and is likely therefore to have an important functional role. In this paper, we report the first structural characterization of N-glycans attached to asparagine-65 of a 16K glycopeptide. The 16K fragment was isolated from bovine pituitaries and the N-glycans were analysed using fast atom bombardment mass spectrometry together with sugar and linkage analysis. Sulphated-N-acetylgalactosamine-capped antennae, typical of the pituitary glycohormones, were present in the major acidic components. The POMC oligosaccharides are distinct from those of the pituitary glycohormones because the sulphate is exclusively located on the 3-arm of biantennary structures and, in addition, a significant proportion of the molecules carry the Lewis x epitope. It is probable that these differences reflect the absence of a tripeptide motif in POMC which fully conforms to the criteria previously defined for the recognition sequence for the N-acetylgalactosamine transferase that is specific for the pituitary glycohormones [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA, 89, 329-333]. It remains to be seen whether the Lewis x epitope is involved in selectin-mediated events, but previous studies suggest that the sulphated moieties are unlikely to play a major role in clearance. The Lewis x epitope is also present in the neutral N-linked oligosaccharides, together with a variety of other antennae including a rarely found fucosylated GalNAc-GlcNAc structure.

Acetylgalactosamine↗

PubMed1993

A possible connection between an increased concentration of a cytochemically detectable substance in the hypothalamus of the spontaneously hypertensive rat and certain cerebral cholinergic disturbances.

The hypothalamus of the spontaneously hypertensive rat (SHR) contains an increased concentration of a cytochemically detectable choline-like substance, the physicochemical properties of which are similar to the choline analogue dimethyl methylene immonium. In the hypothalamus of the adult SHR there is an increased turnover of acetylcholine in the cholinergic pressor posterior hypothalamic nucleus and the neurons of the dorsomedial cholinergic depressor nucleus are atrophic, possibly due to diminished cholinergic activity. Both of these changes are pressor. It is suggested that an increased hypothalamic content of a choline-like analogue might either be responsible for, or an indicator of, these abnormalities.

Acetylcholine↗

PubMed1992

Localization of O-GlcNAc modification on the serum response transcription factor.

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.

Acetylglucosamine↗

PubMed1992

Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family.

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin-Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Amino Acid Sequence↗

PubMed1992

Derivation of the amino acid sequence of rat C-reactive protein from cDNA cloning with additional studies on the nature of its dimeric component.

Rat C-reactive protein (CRP) is unique among mammalian CRPs in being a glycoprotein and in containing a covalently linked dimer in its pentameric structure. To investigate these features, cDNA clones encoding rat CRP were isolated from an expression library, and the primary structure of the protein was derived. Taken along with the results of Northern blotting, we conclude that a single mRNA of approximately 2,500 nucleotides codes for a precursor of rat CRP with a signal sequence of 19 amino acids and a polypeptide of 211 amino acids, the latter sharing extensive homology with human, rabbit, and mouse CRPs. The deduced sequence agreed with results obtained from partial microsequencing and mapping by fast atom bombardment-mass spectrometry. Two potential sites for N-glycosylation (Asn-128 and Asn-147) and a C-terminal heptapeptide (Leu-205 to Ser-211, containing two cysteines at positions 208 and 209) were unique to rat CRP. The protein was also shown to be composed of five apparently identical monomers, two of which form a dimer linked by two interchain disulfide bonds involving Cys-208 and Cys-209. These same cysteines form an intrachain disulfide bond in the other three monomers. The primary structure of rat CRP and the basis of dimer formation have, therefore, been elucidated.

Amino Acid Sequence↗

PubMed1992

The effect of a cAMP analogue on Ca2+ ionophore-, antigen- and agonist-induced inositol phosphate release in rat basophilic leukaemia (RBL-1) cells.

The effect of the stable cAMP analogue 8-Br-cAMP on leukotriene D4 (LTD4)-, 5'-N-ethyl-carboxamidoadenosine (NECA)-, antigen- and Ca2+ ionophore-induced inositol phosphate (IP) production was studied in RBL-1 cells. The cAMP analogue significantly inhibited LTD4- and antigen induced-IP production, thus supporting the hypothesis of a negative interaction between cAMP and phosphoinositide breakdown in blood cells. Ionophore-induced IP release, which was blocked by a 5-lipoxygenase inhibitor and by a LT-receptor antagonist, and therefore is probably mediated by LTs, was also inhibited by 8-Br-cAMP. NECA-induced IP release was not significantly inhibited by the cyclic nucleotide, thus showing that the effect described herein is not a general action on receptor-activated phospholipase C. 8-Br-cAMP did, however, inhibit GTP gamma S-induced IP release in permeabilised RBL-1 cells, thus suggesting that the inhibition does not occur at the receptor level but might be due, at least in part, to an effect on some receptor-coupled G proteins.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.

Acetylglucosamine↗

PubMed1992

Identification of albumin in breast tumor cytosol as a factor involved in the stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase (reductive) activity.

Estradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) is the enzyme responsible for the interconversion of estrone (E1), and the more biologically potent steroid, estradiol (E2), and has a crucial role in regulating breast tissue concentrations of E2. It has previously been shown that breast tumor cytosol is able to preferentially stimulate the reductive conversion of E1 to E2 in cultured MCF-7 breast cancer cells. In this study the stimulatory factor(s) from breast tumor cytosol have been partially purified by gel filtration and affinity chromatography. Human serum albumin (HSA) has been identified as a component of this bioactive fraction. Subsequent testing of commercially purified HSA preparations has revealed the ability of some preparations to be highly stimulatory. The albumin present in breast tumor cytosol may therefore be a contributing factor to the observed stimulation of reductive E2DH activity in cultured MCF-7 cells. Such a mechanism may account in part for the higher concentrations of E2 which are observed in breast tumors in vivo.

Breast Neoplasms↗

PubMed1992

Synergistic interaction of growth factors and albumin in regulating estradiol synthesis in breast cancer cells.

Estradiol 17 beta-hydroxysteroid dehydrogenase acts to convert estrone to the biologically active estrogen, estradiol, in breast tumors and MCF-7 breast cancer cells in vitro. In this study we have examined the ability of albumin to influence the effect of growth factors (insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha)) and cytokines (interleukin (IL)-1, IL-6) on estradiol 17 beta-hydroxysteroid dehydrogenase activity in MCF-7 cells. IGF-I (80 ng/ml) or albumin (30 micrograms/ml) stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity by 144% and 102% (p less than 0.01). The combination of IGF-I and albumin, however, produced a marked (704%) synergistic stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase activity. EGF or TGF alpha failed to stimulate estradiol 17 beta-hydroxysteroid dehydrogenase activity and no synergism with albumin was detected. IL-1 (10 ng/ml), but not IL-6, also stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity and acted synergistically with albumin to stimulate enzyme activity. MCF-7 cells were shown to specifically bind 125I-albumin and binding is increased by pretreatment of cells with IGF-I (80 ng/ml) for 48 h. It is concluded that the synergism that results from treating MCF-7 cells with albumin and IGF-I may result from increased albumin uptake and subsequent biological effect.

Albumins↗

PubMed1991

Characterisation of the N-linked oligosaccharides of the light chain of human glycoprotein IIb by f.a.b.-m.s.

The glycosylation of the light chain (GPIIbL) of glycoprotein IIb, one of the glycoproteins constituting the receptor for fibrinogen, fibronectin, and the von Willebrand factor on platelet cell surfaces, was investigated using fast-atom-bombardment mass spectrometry (f.a.b.-m.s.). Complex-type N-glycans were observed, attached to Asn-60. The most abundant oligosaccharide is a disialylated biantennary structure substituted with fucose on the chitobiose core. Mono-sialylated biantennary, and di- and tri-sialylated triantennary structures were found as minor constituents of the N-glycan population. The amino acid sequence of GPIIbL was fully mapped by f.a.b.-m.s., thereby providing the first direct evidence for the absence of O-glycosylation.

Amino Acid Sequence↗

PubMed1991

Fast-atom-bombardment mass spectrometry of sulphated oligosaccharides from ovine lutropin.

The positive- and negative-ion f.a.b.-mass spectra and the fragmentation of sulphated oligosaccharides derived from ovine lutropin are described. Negative-ion f.a.b.-m.s. of methylated derivatives offers a sensitive and rapid method for screening glycans for sulphation, for defining the location of sulphated residues, and for sequencing sulphated branches. Positive-ion f.a.b.-m.s. gives complementary data on non-sulphated branches in both complex and hybrid-type sulphated structures.

Animals↗