Accessibility of the carbohydrate moiety of membrane-bound rhodopsin to enzymatic and chemical modification

Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.     

Heterogeneity of horse transferrin: the role of carbohydrate moiety

Homozygous horse transferrin (Tf O) is highly heterogeneous. In starch gel electro-phoresis it gives at least 9 zones. Two main components (2a and 4b) were purified by rivanol and ammonium sulphate precipitation, DEAE-Sephadex chromatogra-phy and SP-Sephadex chromatography. Molecular weights of 75 200 and 80 500 for components 2a and 4b, respectively, were determined by sedimentation equilibrium ultracentrifugation. Amino acid compositions of the two components were similar, and there were no differences in the N -terminus (glutamic acid followed by glu-tamine) and the C -terminus (valine). Differences were found in carbohydrate composition between components 2a and 4b. Component 2a contained 10 moles of sugar components per mole of protein (4 hexoses, 4 hexosamines and 2 sialic acids), while component 4b contained twice the number of both total carbohydrates and individual sugar components. Carbohydrates were identified as mannose and galac-tose (ratio mannose:galactose = 1.5:l), N -acetylglucosamine and N -acetylneu-raminic acid. At present it is not clear whether the difference between the two components resides solely in the difference of carbohydrate contents. It is proposed that component 2a has one diantennary glycan, while component 4b has two.     

糖化血红蛋白、糖化血清蛋白与急性脑梗死预后的相关性

目的:分析急性脑梗死患者临床预后与糖化血红蛋白(HbA1c)水平及糖化血清蛋白(GSP)水平的相关性。方法:以80例健康体检者为对照组,以80例急性脑梗死患者为观察组,对比两组入组时HbA1c水平及GSP水平差异。并根据改良Ran Kin评分将观察组分为轻症组(50例)及重症组(30例),对比两组间入组时HbA1c水平及GSP水平差异。并对观察组入组时HbA1c水平及GSP水平与治疗前后改良Ran Kin评分差值的相关性。结果:观察组HbA1c水平及GSP水平均明显高于对照组(P0.05),而重症组HbA1c水平及GSP水平同样明显高于轻症组(P0.05),同时观察组入组时HbA1c水平及GSP水平与治疗前后改良Ran Kin评分差值存在显著的负向直线相关性(P0.05)。结论:急性脑梗死患者入组时HbA1c水平及GSP水平与患者预后存在显著的相关性,可用于与评价患者的临床预后。     

Invertase immobilization via its carbohydrate moiety

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.     

Influence of equine growth hormone,insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E₂), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO₂. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E₂, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO₃-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E₂, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E₂, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.     

生长激素和催乳素放射免疫测定法的建立与应用

目的：建立测定大鼠垂体和血浆中生长激素（GH）和催乳素（PRL）含量的高特异性、高灵敏度的双抗放射免疫测定（RIA）法;研究急性低氧对垂体激素GH和PRL的作用。方法：用氯胺－T法进行抗原放射性碘标记;采用平衡饱和加样程序的双抗RIA法测定。结果：用该方法测定急性低氧（0．5h）时血浆和垂体GH和PRL含量,7km低氧,垂体GH含量明显升高（P＜0．05）,血浆则相反;7km低氧,明显降低垂体和血浆PRL含量（P＜0．01）;而5km低氧对GH和PRL的作用与对照组比无统计学差异。结论：本双抗RIA法具有高特异性、高灵敏度及简便易行等特性;用该法测定提示急性低氧可抑制大鼠GH和PRL的分泌。     

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

a-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.     

The effect of periovulatory imbalance of prolactin on the expression of prolactin receptors in rat ovary cells

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in the prolactin level in the blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.     

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Glycosylated rat prolactin: isolation and structural characterization.

Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.     

Homology modeling of rabbit prolactin hormone complexed with its receptor

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex. Proteins 27: 459–468, 1997. © 1997 Wiley-Liss, Inc.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

Bovine corpus luteum is an extrapituitary site of prolactin production

Prolactin (PRL) is known to be synthesized not only in the anterior pituitary, but also in other organs including the ovary. Among its various functions, PRL is regarded as the most important constituent of the luteotropic complex in rodents and pigs. The purpose of the present study was to determine whether PRL is produced locally in bovine corpus luteum (CL) and to determine its possible roles in CL. In the present study, we examined changes during the luteal phase in (1) the expressions of PRL and PRL receptors (long form: l-PRLR, short form: s-PRLR) in CL and (2) the localization of PRL in CL. We also measured the levels of PRL mRNA in cultured luteal cells and luteal endothelial cells. Furthermore, the effect of PRL on progesterone (P4) and prostaglandin (PG) F2alpha production by cultured bovine luteal cells was examined. Semiquantitative RT-PCR analysis revealed that the mRNAs for PRL and its two receptors, l- and s-PRLR, were expressed in all luteal stages examined. PRL mRNA expression was less in the regressed stage (days 19-21 after ovulation) than in the other stages. Both l-PRLR and s-PRLR mRNA expressions were higher in the late luteal stage (days 15-17) than in the other stages, while the ratio of l-PRLR to s-PRLR was less in the regressed stage than in the other stages. PRL mRNA was also detected in cultured luteal cells and luteal endothelial cells. PRL protein was immunohistochemically detected only in CL of the mid- and regressed stages. It was detected in smooth muscle cells of the intraluteal arterioles and endothelial cells but not in luteal cells and other cell types of CL. Exposure of cultured luteal cells obtained from mid-stage CL (days 8-12) to bovine PRL (100, 200 ng/ml) for 24 hr did not affect P4 and PGF2alpha production by the cells. The present study demonstrates for the first time the expressions of PRL and PRLR mRNA in bovine CL throughout the luteal phase. The overall results strongly suggest that the bovine CL is an extrapituitary site of PRL production.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

Specific Binding of Glycosylated β-Galactosidase to Bovine Brain Synaptic Membrane

The binding of a series of glycosylated beta-galactosidases to a fraction rich in synaptic membrane of bovine brain was examined. beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal) was found the most effective in binding to synaptic membrane, followed by that modified with beta-D-glucopyranoside, whereas the enzyme modified with p-aminophenyl derivatives of alpha-D-galactopyranoside, alpha-D-glucopyranoside, and alpha- and beta-L-fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4 degrees C and the optimal pH was approximately 4.0. The binding of beta-D-Gal beta-gal was inhibited by free p-aminophenyl beta-D-galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 +/- 35 nM and 27.5 +/- 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.     

Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.