Biomedical subjects

F A Simmen

Publications and source records attributed to F A Simmen.

At least 73 records · Page 4Linked to original sources

PubMed1990

Perinatal expression of type I IGF receptors in porcine small intestine.

The gastrointestinal tract of the pig undergoes enhanced growth as well as morphological and functional differentiation during the perinatal period. Concurrently, porcine neonates ingest physiologically significant amounts of insulin-like growth factor-I (IGF-I) via colostrum and milk. The objectives of this study were to examine newborn pig small intestine for the presence of high affinity, IGF-I receptors and to evaluate the possible contributions of maternally derived and locally produced IGF-I to receptor-mediated postnatal growth of intestine. The specific binding of 125I-IGF-I to membranes prepared from scraped intestinal mucosa was time, temperature, and pH dependent; optimal conditions were 48 h, 4 C, and a pH of 7.8, respectively. Several pure peptides were evaluated for competition with 125I-IGF-I in binding to intestinal membrane sites. The relative order of competition was IGF-I greater than insulin-like growth factor-II (IGF-II) greater than insulin, whereas bombesin and epidermal growth factor were noncompetitive. Chemical cross-linking of 125I-IGF-I to binding sites, followed by denaturing SDS-PAGE and autoradiography, demonstrated labeled protein complexes of Mrs 135,000 and 260,000. Both autoradiographic bands were diminished when excess unlabeled IGF-I or IGF-II was included in the binding step. Insulin at higher concentrations also slightly inhibited labeling of both membrane proteins. Membranes prepared from intestinal mucosa of piglets at days 0 (less than 2-h old, colostrum-deprived), 3, 5, and 21 postnatal were evaluated for developmental variations in specific binding of 125I-IGF-I. Binding was highest at birth, declined (-43%) by day 3, remained low at day 5, and increased by day 21. Receptor affinity was relatively invariant whereas receptor number (per mg membrane protein) was variable. Intestine wt increased disproportionately to body wt between days 0 to 3, postnatal. Radioimmunoassay of extracts of the corresponding intestinal mucosa revealed a significant increase in content of IGF-I by day 3 (P = 0.05), whereas RNA dot-blot hybridization demonstrated low and unchanging IGF-I mRNA abundance in intestine. The quantitative variations in IGF-I protein content and IGF receptor numbers temporally coincide with intestinal villous growth, cessation of intestinal transport of macromolecules (closure), and onset of maturation of intestinal function.

Animals↗

PubMed1990

Insulin-like growth factor-I and -II messenger RNA expression in muscle, heart, and liver of streptozotocin-diabetic swine.

We have investigated the effects of streptozotocin-diabetes and fasting in juvenile swine by monitoring IGF-I and -II gene expression in muscle, heart, and liver tissues. In diabetic pigs, IGF-I messenger RNAs (mRNA) were decreased by 50% in muscle and liver tissues, and by 70% in heart. The imposition of fasting on diabetic animals tended to further decrease IGF-I mRNA levels, and fasting alone also decreased IGF-I mRNA abundance in the three tissues (P less than 0.05). Insulin therapy restored IGF-I mRNA levels to normal in muscle and livers but was less effective in hearts of diabetic pigs. Relative IGF-I mRNA expression in heart and muscle tissues was 2-fold and 4-fold higher, respectively, than in liver tissues under normal conditions in these animals. Serum IGF-I concentrations and tissue extractable immunoreactive IGF-I levels were also measured. Serum IGF-I was markedly decreased in the diabetic state, dropping to 70% below control levels (P less than 0.01). Extractable IGF-I in liver declined by 50% with diabetes (P less than 0.01), and by 30% in muscle with diabetes and fasting (P less than 0.05), but no significant changes in heart levels of IGF-I protein were detected. Expression levels of IGF-II mRNAs in the three tissues were unaffected by diabetes or fasting. These results are consistent with earlier observations in rat liver and further demonstrate that IGF-I expression in muscle and heart is altered by diabetes and fasting, whereas IGF-II mRNAs do not change.

Animals↗

PubMed1990

Hormonal regulation of insulin-like growth factor gene expression in pig uterus.

The present studies were undertaken to define potential regulation of endometrial insulin-like growth factor-I (IGF-I) synthesis and secretion by steroid hormones and to correlate conceptus- and serum-derived estrogens with these events. Four experimental groups of gilts were studied: 1) prepubertal gilts administered estradiol (E) and/or progesterone (P4) daily for up to 15 days; 2) mature gilts ovariectomized (ovx) on day 2 of the estrous cycle, and then administered E, P4, or E plus P4 from days 4-11 after estrus; 3) unilaterally pregnant gilts on day 12 or 16 after the onset of estrus; and 4) cyclic gilts administered E on day 11 and hysterectomized 1, 6, 12, or 24 h after E. IGF-I mRNA was measured by blot hybridization of total cellular RNAs, while a specific RIA was used to quantitate levels of tissue and uterine luminal fluid (ULF) IGF-I. In prepubertal gilts, E or P4 increased uterine IGF-I mRNA levels and IGF-I protein. Mature, ovx gilts treated with E, P4, or E plus P4 had increased levels of endometrial IGF-I mRNAs and protein and luminal IGF-I. The magnitude of E or P4 induction was comparable and was not additive. Gravid horns on day 12 had higher amounts of IGF-I in ULF compared to day 12 nongravid horns or day 16 gravid and nongravid horns. However, no significant differences in the amounts of endometrial IGF-I mRNA and tissue IGF-I content between horns on each day were detected. Acute treatment of cyclic gilts (day 11) with E increased levels of endometrial IGF-I mRNAs and luminal IGF-I 1 h after treatment. The effect of E was transient and was reversed within 24 h of E injection. IGF-II and IGF-binding protein-2 mRNAs were regulated by E and P4 distinct from IGF-I mRNAs. These results indicate that E and P4 are involved in the normal regulation of uterine IGF-I synthesis and/or secretion. However, the relative contributions of serum and conceptus-derived estrogens in these processes during the preimplantation period in the pig remain to be defined.

Aging↗

PubMed1990

Stimulation by colostrum or mature milk of gastrointestinal tissue development in newborn pigs.

Porcine colostrum and milk were orally administered to newborn pigs to evaluate their gastrointestinal growth-promoting activity. Five piglets per treatment group were gavage-fed 5% lactose (L), defatted colostrum (C) or defatted mature milk (d 16 of lactation) (M) at 3-h intervals over a 24-h period. Colostrum and milk were administered on equivalent dry matter basis and all piglets received 1 mCi of [3H]thymidine per kg BW at the onset of feeding. Small intestines of C- and M-fed pigs were 1.6-and 1.3-fold, respectively, the weight of small intestines of those fed L (P less than .01). Total DNA content of intact small intestines were not different among groups; however, cpm [3H]thymidine/mg intestinal DNA of C and M piglets exceeded (P less than .05) that for L piglets. DNA content and cpm [3H]thymidine of intestinal mucosa did not differ among groups. Total protein in the intestines and intestinal mucosa of C pigs exceeded (P less than .01) that for L and M pigs. Total RNA in the small intestine and intestinal mucosa were similar for C and M groups but less (P less than .01) for L piglets. Stomach and pancreas weights among all pigs were similar, although C and M pigs exceeded L pigs in stomach (P less than .01) and pancreas (P less than .01) RNA content. In contrast, no differences in stomach DNA, protein and cpm [3H]thymidine or in pancreatic DNA, protein and cpm [3H]thymidine were detected. SDS-polyacrylamide gel electrophoresis was used to identify qualitative and quantitative differences in the protein compositions of porcine colostrum and mature milk.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Regulation of uterine and conceptus secretory activity in the pig.

Evidence is presented for the involvement of a number of specific uterine- and conceptus-derived proteins in endometrial differentiation and conceptus or fetal development. These secretory proteins include mitogens (insulin-like growth factor-I and -II, epidermal growth factor, uterine luminal fluid mitogen), binding and transport proteins (uteroferrin, insulin-like growth factor and retinol binding proteins, respectively), protease inhibitors (antileukoproteinase, plasmin/trypsin inhibitor), and trophoblastic specific proteins. Using immunological reagents and specific complementary DNA (cDNA) probes, the tissue origins of several of these proteins have now been identified. In addition, the temporal regulation of messenger RNA (mRNA) production for a number of these proteins has been elucidated. The results suggest that although circulating and locally produced steroid hormones may be involved in regulating the synthetic abilities of these tissues during pregnancy, other, as yet undefined, factors may also mediate these activities. In this paper we present a review of the current knowledge pertaining to the identity, physiological regulation and potential functions of pig maternal and conceptus secretory proteins during pregnancy.

Animals↗

PubMed1989

Characterization of ixodid tick salivary-gland gene products, using recombinant DNA technology.

Ticks secrete an array of lesion-maintenance factors into the host via the salivary glands while feeding, some of which elicit an immune response by the host that adversely affects the ability of the tick to feed and reproduce. Our approach to characterizing these factors has been to make expression libraries from mRNA of salivary glands (from unfed and 3-day-feeding Amblyomma americanum females) which will serve as sources of the genes (clones) that code for them. Thus far, we have detected 10 positive clones in primary screens using polyspecific antiserum from rabbits hyperimmunized to 3-day-feeding tick salivary glands. We also report making a cDNA library from whole unfed females, and a genomic library from whole unfed ticks, which will serve as additional sources of genetic information for characterizing salivary-gland secretory products. Immunoblots of salivary glands from A. americanum females feeding for various intervals (unfed, and 12, 48, 72, and 96 h) revealed the presence of several prominent polypeptides (90 & 45 kDa) when probed with the same rabbit antiserum that was used to screen the expression library. Ixodes dammini had several immunogens in common with A. americanum at 96 h (90, 45, 43 and 23 kDa). We plan to use monospecific antiserum raised to antigens detected in our immunoblots (e.g. 90 kDa) to further screen the expression libraries, in addition to using the polyspecific antiserum already in hand. We discuss the future use of the salivary-gland genes for characterizing secretory products which facilitate attachment to the host (cement) and maintain the lesion during the lengthy feeding interval.

Animals↗

PubMed1989

Insulin-like growth factor-I expression during early conceptus development in the pig.

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

The relationship of insulin-like growth factor-I with postweaning performance in Angus beef cattle.

This study was designed to evaluate the ontogeny of serum IGF-I (SI) concentrations and its relationship to animal performance in a 140-d postweaning feeding trial. Ninety-eight progeny representing six sires (three high and three low feed conversion) and two sexes (43 bulls and 55 heifers) with ad libitum access to feed were allocated by sire and sex to monitor individual weights and pen feed consumption. Blood serum samples were obtained at the beginning of test (average age of 230 d) and every 28 d thereafter until each animal reached a fat thickness (estimated by sonoray) of 8.9 mm. Individual serum samples were acid-ethanol extracted and measured for IGF-I peptide by heterologous RIA. Serum IGF-I concentrations differed (P less than .10) between high (H) and low (L) feed conversion progeny groups at the end of the first 28-d period (125.12 vs 89.52 ng/ml) and tended to differ at the conclusion of the second 28-d period (P less than .15). Weight gains of H and L groups tended to differ in the second and third 28-d periods (P = .11 and .10, respectively). Serum IGF-I concentrations differed (P less than .05) between bulls and heifers for the first through fourth 28-d periods (P less than .01, P less than .05, P less than .10 and P less than .01, respectively). Phenotypic correlations indicated that pens with higher mean SI concentrations at the beginning of the test consumed less feed and had lower cumulative feed:gain ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Differential growth factor content of uterine luminal fluids from large white and prolific Meishan pigs during the estrous cycle and early pregnancy.

Uterine luminal fluids (ULF) from Large White (LW) and prolific Chinese Meishan (MS) gilts were compared with respect to their peptide growth factor content during an estrous cycle and early pregnancy. Insulin-like growth factor-I (IGF-I) was quantitated by RIA; in vitro growth promoting properties of uterine luminal fluid mitogen (ULFM) were measured by [3H]thymidine incorporation into DNA of quiescent AKR-2B fibroblastic cells in culture. Peak concentrations (pg/microgram ULF protein) of IGF-I in ULF of Large White and Meishan gilts, respectively, were: estrous cycle, 9.8 +/- 1.4 (on d 10) and 39.7 +/- 7.8 (on d 12); gestation, 13.1 +/- 3.2 (on d 8 and 10) and 11.9 +/- 2.1 (on d 12), with differences among days (except d 10, P greater than .5) being affected by breed (P less than .10). For both breeds, there was a rapid decline in IGF-I concentrations by d 14 of the cycle and of pregnancy. Uterine luminal fluid mitogen activity was greater (P less than .01) for LW than for MS gilts on d 10 to 14 of an estrous cycle and gestation and diminished in a time-dependent manner in both breeds. No correlation was observed between IGF-I concentrations and uterine weights for either breed. In contrast, a negative correlation between uterine weight and ULFM activity was detected for cyclic (MS: r = -.855, P less than .10; LW: r = -.834, P less than .05) and pregnant (MS: r = -.806, P less than .10; LW: r = -.928, P less than .05) gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Maternal and neonatal somatomedin C/insulin-like growth factor-I (IGF-I) and IGF binding proteins during early lactation in the pig.

RIA for insulin-like growth factor-I (IGF-I) was performed on Tris-neutralized, acid-ethanol extracts of porcine, bovine, ovine and human mammary secretions, and porcine maternal and neonatal sera. High levels (50-500 ng/ml) of immunoreactive IGF-I were present in the colostrum of all three animal species. IGF-I was also identified in porcine milk, though at levels 10- to 100-fold reduced relative to that in colostrum. Maternal (pig) sera was characterized by IGF-I concentrations intermediate between that in colostrum and that in milk. IGF-I levels were relatively low in serum of newborn pigs and exhibited an approximately 1.4-fold increase between Days 3 and 7 of postnatal life. Fractionation of pig colostrum in nondenaturing, gel-filtration columns demonstrated association of endogenous IGF-I with two prominent binding proteins (Mr's of 150,000 and 50,000 for the complexes). A third immunoreactive component was also observed to elute in the column void volume fractions (Mr greater than 158,000). The 150,000 and 50,000 Mr complexes were also present in serum obtained from sows at term. In contrast, IGF-I immunoreactivity in porcine milk was localized exclusively in the 150,000 Mr complex. Incubation of porcine colostrum and milk with 125I-IGF-I revealed a prominent, unoccupied IGF binding protein corresponding to that of the 150,000 Mr complex, whereas serum obtained from sows at term displayed both the 150,000 and 50,000 Mr unoccupied forms. Fractionation of (pooled) serum obtained from porcine neonates immediately at birth revealed a heterogeneous pattern of IGF-I immunoreactivity which included both the 150,000 and 50,000 Mr forms. Qualitative differences in this chromatographic pattern were apparent in serum at 6 hr postnatal and after ingestion of colostrum had occurred. The unoccupied IGF binding proteins in newborn pig serum were solely of the small size class. These results demonstrate that mammary secretion of IGF-I and its binding proteins are temporally regulated during the period immediately surrounding parturition. Physiologic alterations in the serum IGF-I profile during early postnatal life may reflect in part the uptake and/or response of the neonate to maternal IGF-I.

Animals↗

PubMed1988

A uterine cell mitogen distinct from epidermal growth factor in porcine uterine luminal fluids: characterization and partial purification.

Uterine luminal fluids (ULF) from early (Days 10 and 12)-pregnant sows contain factors that stimulate DNA synthesis in a variety of cell lines. The major growth factor component in these fluids has been partially purified 200-fold by heat treatment, anion-exchange chromatography, and gel filtration using mouse embryo-derived AKR-2B fibroblasts as an indicator cell line. The ULF mitogen (ULFM) is a polypeptide with an apparent molecular weight of 4800; it is extremely heat stable and resistant to treatment with urea. This mitogen is also present in ULF from cycling sows but is not detectable in uterine cytosolic extracts or in serum isolated from pigs at Day 12 of pregnancy. The addition of this factor to medium containing 0.5% calf serum results in a 50% increase in final cell density of AKR-2B cells. ULFM appears biologically distinct from mouse and human epidermal growth factor (EGF), since its activity is not inhibited by antibody to mouse EGF and it does not compete for binding to human (A431) EGF receptors. In addition, the ULF factor stimulates DNA synthesis in human A431 epidermoid carcinoma cells, whereas EGF is inhibitory. Partially purified ULFM also stimulates DNA synthesis in primary cultures of pig uterine stromal cells. This mitogen activity is dose-dependent and is not inhibited by antibody to mouse EGF. Thus ULFM may act in concert with other peptide growth factors in regulating uterine growth and/or differentiation.

Animals↗

PubMed1988

Porcine insulin-like growth factor-I (pIGF-I): complementary deoxyribonucleic acid cloning and uterine expression of messenger ribonucleic acid encoding evolutionarily conserved IGF-I peptides.

In order to facilitate studies of insulin-like growth factor-I (IGF-I) expression during the pregnancy-associated development of uterus and mammary gland in the pig model, we have isolated several cDNA clones corresponding to porcine IGF-I (pIGF-I) mRNA. Sequence analysis of two cDNA fragments (sigf. 2 and sigf. 3) revealed an open reading frame encoding in order a putative 25 amino acid (aa) hydrophobic leader peptide, the mature (processed) 70 aa pIGF-I peptide and a 35 aa carboxy-terminal extension (E) peptide. The deduced aa sequence of the pIGF-I peptide is identical to human and bovine IGF-I but differs from that of rat and mouse at three and four residues, respectively. The sequences of the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf. 3 as a probe revealed multiple IGF-I mRNAs (including species of 8000, 2300, and 1200 nucleotides in length) in uteri of pregnant pigs. Highest levels of the uterine IGF-I mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were also observed. Mammary levels of IGF-I mRNAs and protein were considerably lower than that observed for uterus at the same time period. Thus, uterine production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine growth, elevated IGF-I in uterine fluids, and rapid embryonic development are observed.

Amino Acid Sequence↗

PubMed1987

Expression of the avian c-erb B (EGF receptor) protooncogene during estrogen-promoted oviduct growth.

Expression of cellular erb B protooncogene messenger RNAs has been analyzed in the oviducts of immature chicks during estrogen-promoted growth. Hybridization of oviduct total cellular RNA with viral-derived erb B oncogene probes demonstrated significant expression of c-erb B mRNA in oviduct cells of untreated chicks. Daily administration of estrogen (diethylstilbestrol) to chicks results in marked oviduct growth but did not appreciably affect expression levels of c-erb B messenger RNA in oviducts after 2, 4 or 6 days of treatment. Withdrawal of chicks from estrogen treatment resulted in termination of oviduct growth. However, c-erb B messenger RNAs were detectable in the nonproliferative tissue at 5 days after hormone withdrawal. Readministration of diethylstilbestrol, progesterone or diethylstilbestrol plus progesterone to hormone-withdrawn birds (secondary stimulation) also did not affect c-erb B messenger RNA levels in the oviduct. These results demonstrate significant expression of the cellular erb B (epidermal growth factor receptor) gene in the avian oviduct. However, EGF receptor messenger RNA synthesis is not modulated in the oviduct by steroid hormones.

Animals↗

PubMed1987

In vitro growth-promoting activity of porcine mammary secretions: initial characterization and relationship to known peptide growth factors.

We have examined the in vitro growth-promoting properties and growth factor content of porcine mammary secretions. Defatted, porcine colostrum stimulated the proliferation of fibroblast and epithelial-like cell lines of diverse species origins in serum-free medium and cellular DNA synthesis (4- to 119-fold) as monitored by uptake of 3H-thymidine into DNA of quiescent cells in culture. Porcine milk, although mitogenic, had reduced activity when compared with colostrum on an equivalent-volume basis. Furthermore, the relative mitogenic activity of milk, although still detectable at 3 wk, continued to decline with length of the lactation period. Fractionation of pig colostrum on gel-filtration columns revealed multiple peaks of (AKR-2B) fibroblast mitogenic activity (208, 66 and 4.6 kdaltons) and a heterogenous profile of epithelial cell mitogenicity. Polyclonal antibodies (IgG) specific for murine epidermal growth factor (EGF; the major mitogen in human and murine milk) or human platelet-derived growth factor (PDGF) did not inhibit the mitogenic activity of pig colostrum or milk, demonstrating lack of antigenic relatedness between the contributing porcine factors and mEGF or hPDGF. Also, we were unable to demonstrate similarity of the small Mr colostral factor with EGF by use of EGF radioreceptor assay. These results identify porcine colostrum and milk as sources of potentially important in vitro growth-promoting factors. The enhanced expression of these factors in early mammary secretions suggests their possible in vivo involvement in mammary and neonatal tissue growth processes.

Animals↗

PubMed1986

Length and sequence polymorphisms in the ribosomal gene spacer of the Hawaiian sea urchin, T. gratilla.

Blot-hybridization of sea urchin (Tripneustes gratilla) genomic DNA with a cloned rDNA probe revealed individual variation in the length of the rDNA repeat unit and also in the non-transcribed spacer sequences. The number of distinct rDNA repeat subclasses distinguishable within any one sea urchin was limited and usually 2 to 3. However, examination of a number of sea urchins indicated a large number of distinct rDNA repeat types in the population as a whole; all of the rDNA repeat types in nine individuals were different. The presence of limited heterogeneity in the rDNA repeats of single individuals, with may different repeat types in the population as a whole, suggests that rDNA variants can be rapidly and selectively propagated within a chromosomal lineage.

Animals↗

PubMed1985

Structural analysis of ribosomal RNA genes from the Hawaiian sea urchin species, Tripneustes gratilla.

Ribosomal gene sequences (rDNA) were isolated from the genomic DNA of the Hawaiian sea urchin Tripneustes gratilla by cloning in plasmid and phage vectors. The rRNA coding regions in four clones were localized by hybridizing Southern transfers of endonuclease-digested DNA with 32P-labeled 26S rRNA and 32P-labeled 18S rRNA. Three of the rDNA clones were isolated from a library of DNA from a single sea urchin and represent the two major types of rDNA repeats present in that individual's genome. Both repeat types appear identical within their rRNA coding regions but are dissimilar in an area of the nontranscribed spacer (NTS) adjacent to the 3' end of the 26S coding region. The cloned rDNA repeats were shorter than their genomic counterparts due to deletions occurring within internally repetitious NTS domains, probably as a result of unequal recombination during phage propagation.

Animals↗

PubMed1984

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells.

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.

Amino Acid Sequence↗

PubMed1984

Translation in Xenopus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins.

Translation of messenger RNAs (mRNAs) for human epidermal growth factor receptor (EGF-R) proteins was accomplished in a Xenopus oocyte translation system. Translation of these mRNAs in rabbit reticulocyte lysates and wheat germ extracts were unsuccessful, in accordance with the general difficulty in translating mRNAs for membrane-bound glycoproteins in conventional heterologous cell-free systems. Total poly(A)+RNA from a human epidermoid carcinoma cell line A431 was injected into oocytes from Xenopus laevis. After incubation in the presence of [35S]methionine, the oocytes were homogenized and the supernatant incubated with several different polyclonal antibodies (IgGs) against the human EGF-R. Immunocomplexes were bound to protein A-Sepharose, washed extensively, and electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. A protein of Mr = 160,000, corresponding to the EGF-R glycoprotein, was specifically precipitated from RNA-injected oocytes by each of the anti-EGF-R IgGs. This protein was not detectable in the immunoprecipitates from uninjected oocytes nor was it precipitated from RNA-injected oocytes by IgG from normal rabbit serum or IgG to an unrelated protein. A second protein of Mr = 100,000 was also immunoprecipitated from the homogenates of RNA-injected oocytes. This product corresponds to the EGF-R-related protein, present in A431 cells, which is structurally similar to the EGF-R (Weber et al., 1984). Thus, frog oocytes provide an efficient and generally applicable heterologous system for translation of mRNAs for large glycoproteins such as growth factor receptors. In addition, successful immunoprecipitation of translated products in this system provides a method for assay of mRNAs where biological activity of the proteins cannot be monitored.

Animals↗