Glycosylation of recombinant proteins: problems and prospects.
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Animals↗
Biomedical subjects
N Jenkins
Publications and source records attributed to N Jenkins.
A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12.
Melanocytes preferentially express an mRNA species, Pmel 17, whose protein product cross-reacts with anti-tyrosinase antibodies and whose expression correlates with the melanin content. We have now analyzed the deduced protein structure and mapped its chromosomal location in mouse and human. The amino acid sequence deduced from the nucleotide sequence of the Pmel 17 cDNA showed that the protein is composed of 645 amino acids with a molecular weight of 68,600. The Pmel 17 protein contains a putative leader sequence and a potential membrane anchor segment, which indicates that this may be a membrane-associated protein in melanocytes. The deduced protein contains five potential N-glycosylation sites and relatively high levels of serine and threonine. Three repeats of a 26-amino acid motif appear in the middle of the molecule. The human Pmel 17 gene, designated D12S53E, maps to chromosome 12, region 12pter-q21; and the mouse homologue, designated D12S53Eh, maps to the distal region of mouse chromosome 10, a region also known to carry the coat color locus si (silver).
Amino Acid Sequence↗
Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.
Recombinant human interferon-gamma production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.
Ammonia↗
Recombinant human interferon-gamma. Differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture.
Recombinant human interferon-gamma (Hu-IFN-gamma) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-gamma was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-gamma, both fully glycosylated IFN-gamma (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-gamma, non-glycosylated IFN-gamma was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-gamma increased from 3 to 5% after 3 h, to 30% of the total IFN-gamma present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-gamma was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-gamma glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-gamma production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-gamma.
Animals↗
Source of heterogeneity in secreted interferon-gamma. A study on products of translation in vitro.
A cDNA clone coding for human interferon-gamma (IFN-gamma) was subcloned into a transcription-translation vector. When the mRNA transcribed in vitro was added to a rabbit reticulocyte-lysate system, two polypeptides were synthesized: one corresponding in Mr to pre-IFN-gamma (18,000) and one with a lower Mr (12,000) which corresponds to a polypeptide arising from incorrect initiation of translation. When microsomal vesicles isolated from dog pancreas or Chinese-hamster ovary (CHO) cells were added to the translation system, translocation of the pre-IFN-gamma occurred, as judged by protection from exogenous proteinases. The resultant changes in the Mr of the translation products were indicative of signal-peptide cleavage and heterogeneous core glycosylation. When translation products were treated with N-glycanase, the higher-Mr products were no longer observed, consistent with removal of all oligosaccharide side chains, leaving a single core polypeptide. Glycosylation of the synthesized protein yielded both singly and doubly glycosylated products compatible with the glycosylation variants seen in secreted IFN-gamma. Quantitative differences were seen in the relative amounts of singly and doubly glycosylated products synthesized by dog pancreatic compared with CHO-derived microsomes. These data indicate that the relative amounts of IFN-gamma glycosylation variants are determined at an early stage in protein synthesis and that product variants may occur when IFN-gamma is expressed in cells derived from different tissues.
Animals↗
Absence of specific cell-surface binding of tissue plasminogen activator in uterine cells.
Tissue plasminogen activator (tPA), an arginine-specific serine protease, is an oestrogen-regulated protein in uterine and breast cancer tissue. It contains a domain which shares homology with epidermal growth factor (EGF). The aim of the present study was to determine whether specific tPA receptors or EGF receptors mediate the binding of tPA to cells and whether tPA possesses intrinsic mitogenic activity. The binding of 125I-labelled tPA to rat uterine and liver membranes was shown to be non-specific and could not be displaced by unlabelled tPA or EGF. Furthermore, acid washing of cell membranes did not unmask specific tPA-binding sites. In contrast, 125I-labelled EGF binding to both rat uterine and liver membranes was displaced in a dose-dependent manner by unlabelled EGF, and Scatchard analysis of the binding data revealed dissociation constant (Kd) values of 2.4 and 0.71 nM respectively. Unlabelled tPA (up to 20,000-fold excess) did not displace 125I-labelled EGF binding to these membranes. A study of the binding of 125I-labelled tPA and 125I-labelled EGF to endometrial carcinoma cells (Ishikawa), cervical carcinoma cells (HOG-1) and vulval carcinoma cells (A431) showed that up to a 100-fold excess of EGF or a 1000-fold excess of tPA did not displace 125I-labelled tPA binding to these cells. In contrast, 125I-labelled EGF binding was displaced by unlabelled EGF (Kd values for Ishikawa and HOG-1 cells were 2.72 and 1.92 nM respectively) but not by unlabelled tPA (1000-fold excess).(ABSTRACT TRUNCATED AT 250 WORDS)
Acids↗
Only tissue-type plasminogen activator is secreted by immature bovine Sertoli cell-enriched cultures.
Two molecular variants of plasminogen activator (PA): urokinase (uPA) and tissue-type plasminogen activator (tPA), have been reported to be synthesized in the rat testis. Data obtained in this study using monospecific antibodies raised against uPA and tPA in immunoblotting and bioimmunoassay protocols consistently demonstrate that only tPA (and not uPA) is synthesized by bovine Sertoli cell-enriched cultures, and is induced by bovine FSH. Zymographic analysis of conditioned medium on gels containing plasminogen and casein showed a dominant PA proteolytic band (72 kDa) which co-migrated with human tPA. A proteolytic band (43 kDa), which was also secreted by FSH-stimulated cells, was not present when protection was afforded from auto-proteolysis by aprotinin, and was therefore concluded to be a proteolytic fragment of tPA, and not uPA.
Animals↗
Characterization of a plasminogen activator inhibitor induced by glucocorticoids in immature bovine Sertoli cell-enriched cultures.
Plasminogen activation has been shown to be inhibited by the cell-specific production of a number of protease inhibitors belonging to the serine protease inhibitor family. In the bovine testis this inhibitor production is induced by glucocorticoids. Monospecific antibodies raised against the three known classes of plasminogen activator inhibitor were used to identify which type of inhibitor was secreted by bovine Sertoli cell-enriched cultures. Immunoblot analysis and [35S]methionine labelling of newly synthesized proteins revealed that a novel protein with an apparent molecular weight of 49 kDa, which shares antigenic determinants with placental and macrophage PAI and fibroblast protease nexin, is secreted in response to dexamethasone stimulation. This protein was shown by immunoadsorption to be a functionally active inhibitor of both tissue-type and urokinase-type plasminogen activators.
Animals↗
Modulation of plasminogen activation by glucocorticoid hormones in the rat granulosa cell.
Granulosa cells from immature rats primed with diethylstilbestrol (DES) showed a fivefold rise in the activity of secreted plasminogen activator (PA) in response to human FSH. The dose giving 50% of the maximum response (ED50) was 10 ng/ml. Glucocorticoid hormones significantly suppressed PA activity in both control cells and cells stimulated with FSH (ED50 = 50 nmol dexamethasone/l; 1 mumol corticosterone/l). Cortexolone (2.5 mumol/l) significantly ameliorated this suppression, indicating that the response to glucocorticoids is receptor-mediated. These data, together with the time delay required for glucocorticoids to take effect (5 h), suggest that glucocorticoids induce the production of a specific PA inhibitor in granulosa cells.
Animals↗
Effects of passive immunization against oestradiol-17 beta on some endocrine values of the male lamb.
Passive immunization of male lambs against oestradiol-17 beta from 2 to 16 weeks of age significantly elevated androgen concentrations in plasma and depressed the median eminence content of dopamine. Removal of endogenous oestrogens had no significant effects on plasma FSH, LH or prolactin concentrations or on testicular growth and hypothalamic content of GnRH. These results suggest that endogenous oestrogens may indirectly suppress testicular androgen secretion by exerting a stimulatory influence on hypothalamic dopaminergic neurones, which in turn may inhibit GnRH secretion by the median eminence.
Androgens↗
Corticosteroids suppress plasminogen activation in the bovine Sertoli cell.
Bovine FSH stimulated a six fold increase in secretion of plasminogen activator by immature bovine Sertoli cells with half-maximum response (ED50) at 145 ng/ml. Treatment with FSH and either dexamethasone, cortisol or corticosterone produced a dose-dependent suppression of PA activity, with ED50 values of 35, 320 and greater than 650 nmol/l respectively. Effects of dexamethasone required over 6 h incubation to become significant (P less than 0.001), and were blocked by inhibitors of RNA synthesis and translation. These data demonstrate direct effects of corticosteroids on Sertoli cells, resulting in the synthesis of antiprotease factors which antagonize the actions of FSH.
Adrenal Cortex Hormones↗
Management of obstructing carcinoma of the left colon by extended right hemicolectomy.
In obstructing carcinomas of the left colon, primary resection with immediate colocolonic anastomosis is associated with a high risk of anastomotic dehiscence, and therefore, it has become traditional to manage such patients with staged resection. In obstructing carcinomas of the right colon, immediate resection of the lesion with ileocolonic anastomosis is acceptable. The scope of right hemicolectomy can be extended to encompass obstructing carcinomas of the left colon by performing subtotal or total colectomy with ileosigmoid or ileorectal anastomosis. Herein, we have reported the outcome in 16 patients treated in this manner. The operative mortality was 12.5 percent, which compared favorably the mortality of other methods of surgical management. Extended right hemicolectomy achieves in one operation relief of intestinal obstruction, tumor resection, restoration of gut continuity, elimination of the risks of synchronous or metachronous colonic tumors, and a possible increase in the 5 year survival rate. In our experience, the frequent passage of liquid stools is not a problem. This approach justifies further study.
Aged↗
Barium in welding fume.
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Air Pollutants, Occupational↗
The functional maturation of the Sertoli cell and Leydig cell in the mammalian testis.
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Age Factors↗
Customers talk about health care.
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Contract Services↗
Treatment of the perineal wound after proctectomy by intermittent irrigation.
Between 1977 and 1983, 43 patients underwent proctectomy (40 patients for carcinoma, three for ulcerative colitis). The subsequent perineal wound received primary closure and the pelvic space was treated with intermittent irrigation and suction drainage using a double-lumen catheter. Primary healing of the perineal wound occurred in 56.4% of cases and in 89.7% of cases the perineal wound had healed completely by the sixth postoperative week. The relationships between perineal wound healing and the age and sex of the patient, the stage of the tumor, and the tumor distance from the anal verge were assessed. The period of hospitalization was significantly reduced when the perineal wound underwent primary healing. The results obtained in terms of wound healing are comparable with the best published results using continuous irrigation. Our method is advantageous in that it does not necessitate immobilization of the patient.
Aged↗
Effects of hemicastration at various ages and of oestradiol-17 beta on plasma concentrations of gonadotrophins and androgens, testicular growth and interstitial cell responses in prepubertal lambs.
The effect of the removal of one testis from cross-bred lambs at 1, 4, 8 or 12 weeks of age on plasma FSH, LH and testosterone was studied until 16 weeks of age. Hemicastration at all ages elicited a significant increase in plasma FSH compared to controls without a corresponding change in plasma LH or testosterone. The raised FSH after hemicastration at 1 or 4 weeks of age was suppressed to control levels between weeks 7 and 8; such a suppression was not observed in the 4 weeks following hemicastration at 8 or 12 weeks of age. The weight of the remaining testis had increased compared with the control by 12 weeks of age after hemicastration at 1 week (+ 69%), 4 weeks (+ 13%) and 8 weeks (+ 40%); hemicastration at 12 weeks of age also resulted in growth of the remaining testis at 16 weeks (+ 82%). The total androgen production of interstitial cells in response to ovine LH stimulation in vitro did not differ significantly between lambs of 1 and 12 weeks of age, or in animals of 4, 8 and 12 weeks of age after hemicastration at 1 week of age. Subdermal implantation of oestradiol-17 beta into 1-week hemicastrated lambs at the time of operation or at 6 weeks of age increased plasma oestradiol concentrations by approximately 2-4-fold, prevented the FSH and testicular growth responses to hemicastration and suppressed plasma LH and testosterone to levels lower than those in control lambs. The total androgen response of interstitial cells from the remaining testis of oestradiol-implanted lambs at 12 weeks of age was significantly reduced. We suggest that the pituitary-testis axis varies in sensitivity during the prepubertal period although the interstitial cellular response of the testis to LH stimulation remains constant.
Animals↗
Sex steroids and their relationship to binding proteins in the serum of the marmoset monkey (Callithrix jacchus).
A sex hormone binding globulin (SHBG) similar to human SHBG was identified in marmoset serum based on its gel electrophoretic mobility, isoelectric point and steroid binding properties. Levels of serum SHBG were measured in immature and mature males, immature females and females during the luteal phase and pregnancy; serum progesterone, 5 alpha-dihydrotestosterone (5 alpha-DHT), testosterone, oestradiol-17 beta and oestrone were also measured. Mean (+/- S.E.M.) concentrations of SHBG in immature males (336 +/- 19 nmol/l) were higher (P less than 0.01) than those in mature males (251 +/- 13 nmol/l), whereas values in the groups of females were similar (359 +/- 12, 395 +/- 17, 397 +/- 39 nmol/l in immature, non-pregnant and pregnant females respectively). There was an inverse relationship between SHBG and the levels of testosterone (r = -0.67) and 5 alpha-DHT (r = -0.86) in males, but the correlation was significant (P less than 0.05) only for 5 alpha-DHT. There was no correlation between levels of SHBG and oestrogens in males or between levels of SHBG and any of the steroids measured in females. Equilibrium dialysis was used to assess the percentage of steroid in serum in the unbound form. Mean percentage values for unbound testosterone and 5 alpha-DHT were lower in immature males than in mature males (P less than 0.01) and negatively correlated with levels of SHBG (r = -0.78, testosterone; r = -0.56, 5 alpha-DHT).
Androgens↗