Biomedical subjects

J Ilan

Publications and source records attributed to J Ilan.

At least 37 records · Page 2Linked to original sources

PubMed1993

Transgenic strategies in reproductive endocrinology.

The present discussion surveys some of the recently published studies utilizing transgenic strategies to address questions in reproductive endocrinology. Beginning with a brief introduction of the transgenic method itself, the following areas are covered: 1. Sexual development and Müllerian-inhibiting substance; 2. Hypogonadal mice and hypothalamic GnRH; 3. The GnRH neuron: generation of immortalized rare cell types; 4. Glycoprotein hormones: immortalized cells, development and evolution; 5. Growth hormone and reproduction; and, 6. Gestation and the insulin-like growth factors. In each section, the discussion attempts to be integrative with respect to the significance of the results to physiological, cellular and molecular biology. We believe this approach is appropriate, as transgenic science itself is necessarily an integration of all of these levels of investigation and participation from those working at all levels is needed.

Animals↗

PubMed1993

Differential expression of insulin-like growth factor-II in specific regions of the late (post day 9.5) murine placenta.

Insulin-like growth factor-II (IGF-II) expression has been implicated as a major determinant of fetal size during murine pregnancy. It remains unclear whether expression in the fetus, the placenta, or both is the overriding factor controlling growth. To gain further understanding of the placental contribution, we mapped IGF-II expression in the fetal vascular and trophoblastic portions of the late murine placenta (day 9.5-18.5). We found that, as in the fetus itself, vasculogenic mesenchyme, in this case derived from the allantois, was the strongest expressor of IGF-II. Trophoblast, on the other hand, while expressing somewhat less IGF-II, showed a dynamic pattern of IGF-II expression, which reflected its continuing differentiation during late pregnancy. Initially (days 9.5 and 12.5), the spongiotrophoblast, which is homologous to the cytotrophoblast columns and shell in early human pregnancy, strongly expressed IGF-II. Later, expression in the spongiotrophoblast was down-regulated as a new population, the so-called glycogen cells, emerged within the spongiotrophoblast (day 12.5-15.5) and went on to invade the mesometrial decidua. Glycogen cells, which are homologous to human intermediate trophoblast, strongly expressed IGF-II. Trophoblast lining the area of maternal-fetal exchange, the labyrinth, on the other hand, maintained a constitutive lower level of IGF-II expression throughout late pregnancy.

Allantois↗

PubMed1993

Muscle differentiation: insulin-like growth factors as positive modulators of myogenic regulatory genes?

The contribution of autocrine production of insulin-like growth factor II (IGFII) to myogenic differentiation was studied in the mouse myogenic C2 cells. Permissive C2 cells were stably transfected with a vector generating antisense RNA complementary to IGF RNAs. Our results show that: (1) accumulation of IGFII mRNA is dramatically decreased (this mRNA is undetectable by RNA blotting); (2) accumulation of MyoD mRNA also becomes undetectable by RNA blot analysis; (3) the ability of the cells to differentiate, including the activation of myogenin and myosin genes, is severely compromised; (4) expression of the MyoD genes and the ability to differentiate are restored following the addition of insulin or IGF to the cells. Our observations indicate that the autonomous differentiation of myogenic cells requires autocrine production of IGF and raise the possibility that IGFII positively regulates expression of the MyoD gene, one of the members of the myogenic regulatory factor family.

Animals↗

PubMed1992

Loss of tumorigenicity of rat glioblastoma directed by episome-based antisense cDNA transcription of insulin-like growth factor I.

Malignant glioma is the most common brain tumor. The molecular basis of glioma tumorigenicity has not been defined. Cultured glioma cells accumulate high levels of insulin-like growth factor I (IGF-I) transcripts. We asked whether IGF-I expression is coupled to tumorigenicity, using a combined in vivo/in vitro system employing antisense RNA for IGF-I. An antisense IGF-I expression construct in an expression vector that incorporates Epstein-Barr virus replicative signals and the ZnSO4-inducible metallothionein I transcriptional promoter was assembled. Stable glioma transfectants were derived from C6 glioma cells, which constitutively express IGF-I. B-104 neuroblastoma cells, derived originally from the same tumor but not expressing IGF-I, were also transfected as controls. In the absence of ZnSO4, the C6 transfectants expressed high levels of IGF-I mRNA and protein as detected by in situ hybridization and immunocytochemistry, respectively. Addition of ZnSO4 in the culture medium resulted in high levels of antisense transcript accumulation and dramatically decreased levels of endogenous IGF-I mRNA and IGF-I protein. Subcutaneous injection of either nontransfected C6 parental cells or C6 cells transfected with vector without IGF-I sequences into rats resulted in large tumors after 2 weeks, as did transfected and nontransfected B-104 cells. However, the rats injected with transfected C6 cells yielded no tumors after 40 weeks of observation. Two weeks after injection of the transfected C6 cells a small cyst was apparent in six rats. Histologic sections revealed a few glioma cells infiltrated by a large number of mononuclear cells. No infiltration of mononuclear cells was apparent in the glioma tumors resulting from injection of parental (nontransfected) cells, suggesting that the parental cells, but not the antisense IGF-I transfectants, escape the host immune response.

Animals↗

PubMed1992

The third IGF-II promoter specifies transcription of three transcripts out of five in human placenta.

Insulin-like growth factor-II (IGF-II) mRNA exists as multiple transcript size classes, such as 6.0, 5.3, 4.9, 3.2, and 2.2 kb mRNAs in various human tissues. Three different promoters, 2 different polyadenylation sites, and alternative splicing are involved in producing these multiple transcripts. Initiation of transcription at the 3 different promoters results in multiple mRNAs which contain identical coding regions but different 5'-untranslated regions (5'-UTRs). The first promoter is thought to direct expression of 5.3 kb mRNA in adult human liver. The second promoter region directs expression of 6.0, 3.2, and 2.2 kb mRNAs in human fetal tissues and several adult nonliver tissues. The third promoter specifies transcription of a 4.9 kb mRNA in various tissues. We isolated and sequenced a cDNA clone (pIGF-II-1-70) from a human placental cDNA library, which contains the IGF-II coding region and the 5'-UTR associated with the third promoter. By using a 5'-UTR-specific probe from the clone, we found that this third 5'-UTR is contained in the IGF-II mRNA of 2.2 kb and is absent in the 3.2 kb IGF-II mRNA. We also found an 0.9 kb transcript expressed in placenta, which hybridized strongly to the third 5'-UTR specific probe but not to IGF-II coding region probes. This finding might indicate the existence of an mRNA encoding an IGF-II-associated peptide.

Base Sequence↗

PubMed1992

Regulation of transcription by translational components in coupled translation-transcription cell-free system.

A coupled translation-transcription cell-free system was established from eukaryotic cells. The biosynthetic activity of this coupled system closely resembles the synthetic behavior of cells in vivo, and exhibits regulatory phenomena similar to that of intact cells. The translational system consists of rabbit reticulocyte lysate, or its components fractionated by centrifugation. The transcriptional portion consists of cockerel liver nuclei. Incorporation of amino acids into protein by the coupled system is linear for hours. Similarly, transcription in the coupled system is continuous for hours and is proportional with time. More than 90% of the transcriptional products are secreted into the incubation medium. The components of the translational system influence and regulate transcriptional activities. In the presence of ribosomes the nuclei transcribe mostly poly(A)+ RNA with alpha-amanitin sensitivity consistent with activation of RNA polymerase II. Hybrid selection experiments demonstrate authentic preproalbumin mRNA among the transcriptional products. The putative mRNA secreted into the medium in the coupled system is found on polysomes, indicating translation of de novo synthesized message. Addition of excess reticulocyte mRNP to the medium of the coupled system results in transcription of primarily ribosomal RNA, 5S RNA, and tRNA, the products of RNA polymerases I and III. These activities closely imitate the behavior of liver in vivo under conditions of nutritional shifts or hormonal influences. The coupled system transcribes, processes, and transports substantial quantities of RNA, about 1.6 micrograms/10(6) nuclei/h. Thus, a coupled system has been established that lends itself to the exploration of regulatory interactions of cell components as it appears to closely resemble the in vivo situation.

Animals↗

PubMed1991

Newly synthesized RNA: simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes.

The levels of several RNA transcripts in cultured hepatocytes are regulated by transcriptional and post-transcriptional mechanisms and are affected by growth hormone and insulin. We assessed the effects of these hormones on transcription rates and the stability of insulin-like growth factor I, actin, and albumin transcripts in intact cells of primary cultures of rat hepatocytes by analyzing thiol-labeled, newly synthesized RNA isolated by mercurated agarose affinity chromatography. The application of this concept to the measurement of transcript stability is presented in detail. The data indicate that growth hormone stimulates the transcription rates of insulin-like growth factor I, actin, and albumin genes. The stability of all three transcripts, particularly albumin, appears to be lower in growth hormone-containing medium than it is in insulin-containing medium. The experiments indicate that the rates of transcription and/or degradation of albumin mRNA are influenced by hormonal treatment. However, the cells maintain roughly constant albumin transcript levels independent of hormone treatment by compensatory changes in the rates of transcription and degradation.

Actins↗

PubMed1991

Unique 3'-untranslated sequence of insulin-like growth factor-I isolated from human placenta.

A cDNA clone of 525 bp corresponding to the 3'-untranslated region of insulin-like growth factor-I was isolated from a human placenta library. The sequence of this clone extended 200 nucleotides downstream from the previously reported 3'-end of IGF-IA cDNA, indicating the existence of IGF-IA transcripts having an even larger 3'-untranslated region. By using this clone for RNA transfer blot hybridization, it was shown that this longer 3'-untranslated region is included in the 7.5- and 5.0-kb transcripts, but not in the 1.1- and 0.9-kb transcripts. It is also apparent that transcripts bearing the extended 3'-untranslated sequence are highly expressed in human placenta.

Base Sequence↗

PubMed1991

Effects of actinomycin D and cycloheximide on transcript levels of IGF-I, actin, and albumin in hepatocyte primary cultures treated with growth hormone and insulin.

The stability of several RNA transcripts in cultured hepatocytes is known to increase when serum is omitted from the culture medium. In order to investigate possible mechanisms for this phenomenon, we examined the effects of actinomycin D and cycloheximide on the levels of actin, albumin, and insulin-like growth factor I transcripts in primary cultures incubated in serum-free medium. The levels of IGF-I and albumin transcripts per culture increased for the first 4 hours following addition of actinomycin D and then declined. The levels of actin transcripts and total RNA per culture declined immediately following actinomycin D addition in a manner consistent with exponential decay. IGF-I and albumin transcript levels were relatively unaffected by cycloheximide, while actin transcript levels increased 7-fold over 7 hours. The half-lives of actin transcripts and total RNA were calculated to be 4.6 to 7.7 hours and 11 to 19 hours, respectively, with no statistically significant correlation with hormone treatment. The data suggest that the stability of albumin and IGF-I transcripts, but not actin transcripts, is controlled in part by an actinomycin D-sensitive process.

Actins↗

PubMed1990

Expression of the insulin-like and platelet-derived growth factor genes in human uterine tissues.

The human uterus repeatedly exhibits cyclic biochemical and cytological changes during the reproductive period of life. These changes are the result of a well-characterized endocrine network involving the hypothalamus, pituitary, and ovary. The exact nature of the mechanism(s) by which the sex steroids act on the uterus remains to be elucidated. Possible local mediators of hormonal action on the uterus include polypeptide growth factors. Using the method of RNA transfer blot hybridization, we have analyzed tissue samples from the cycling human endometrium and tissue samples of human myometrium and myometrial benign tumor (leiomyoma) for the presence of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) RNA. All the uterine tissues examined possessed RNA for PDGF-B chain and IGF-I and -II. Two transcripts were observed for PDGF-B chain, four were observed for IGF-I, and eight were observed for IGF-II. Overall, the relative abundance of PDGF-B chain RNA was consistent in all of the uterine tissues examined. In contrast, IGF RNA relative abundance varied. IGF-I RNA was highest in late proliferative stage endometrium, and IGF-II RNA was highest in early proliferative stage endometrium. Both IGF-I and IGF-II RNAs were greater in amount of leiomyoma than in myometrium. The increased IGF-I RNA in late proliferative-stage human endometrium correlates with the known elevation of estradiol secretion by the ovary and the increased concentration of uterine estradiol receptors during this stage of the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Endometrium↗

PubMed1990

Hepatocytes express blood coagulation factor XII (Hageman factor).

The liver synthesizes blood coagulation factor XII (Hageman factor). The specific cell that expresses factor XII, however, has not been previously identified. We used primary rat hepatocytes cultured in serum-free medium to study the transcription, de novo synthesis, and secretion of factor XII. A 32P-labeled human factor XII complementary DNA probe was used for RNA blot hybridization. A single band of hybridization at 2.4 kilobases appeared in blots of polyadenylated RNA derived from 24-hour hepatocyte cultures. This corresponds to the known size of factor XII-processed primary transcript (messenger RNA). Cultured hepatocytes secreted labeled factor XII when tritiated leucine was added to the medium, indicating that the hepatocytes used 3H-leucine to synthesize factor XII de novo. In these hepatocyte cultures immunoreactive factor XII levels progressively increased in 24 hours and factor XII clotting activity increased in parallel. Cycloheximide inhibited the accumulation of both immunoreactive and coagulant factor XII. Secreted factor XII from the rat hepatocytes comigrated with authentic rat plasma factor XII at 80,000 molecular weight in a Western immunoblot. These data indicate that cultured hepatocytes transcribe, synthesize, and secrete authentic factor XII.

Animals↗

PubMed1989

Insulin-like growth factors and neonatal cardiomyocyte development: ventricular gene expression and membrane receptor variations in normotensive and hypertensive rats.

Defined factors regulating or influencing mammalian ventricular myocyte (cardiomyocyte) development are not known at this time. During early neonatal ventricular growth, cardiomyocytes begin a 'transition phase' of development toward cellular maturation (hypertrophy) that entails terminal proliferation and cellular binucleation. Insulin-like growth factor-I and -II (IGFs) are believed to play a major role in mammalian postnatal and fetal growth, possibly functioning in local environments which facilitate autocrine or paracrine tissue growth characteristics. Therefore, we examined the expression of the IGF genes and their corresponding membrane receptors in ventricles of normotensive and spontaneously hypertensive (SHR) rat pups during the first 7-14 days of age. We have determined: (1) by receptor crosslinking that neonatal ventricular membranes possess type 1 and type 2 IGF receptors; (2) by receptor binding analysis that type 1 IGF receptor concentration is elevated between days 1-7 in the SHR and shows an age-related decline in concentration and an increase in affinity in both strains; (3) by Northern blot analysis that neonatal rat ventricular tissue expresses primarily IGF-II RNA transcripts of 3.6, 2.3 and 1.7 kilobases (kb) in size, with low levels of IGF-I transcripts detected; (4) by slot-blot hybridization that SHR ventricles contain higher levels of IGF-II transcripts at 3 days of age; and (5) localized the IGF transcripts to ventricular myocytes by tissue in situ hybridization. These observations support a role for cardiomyocyte-produced IGFs that may be locally produced and act in an autocrine or paracrine fashion to modulate cardiomyocyte growth and maturation in the developing rat heart. Because both IGF receptor and IGF RNA transcript parameters differed in SHR hearts, genetically predisposed to hypertrophy, a potentially important biochemical alteration may be associated with the fetal/neonatal growth abnormalities of the developing heart in this rat strain.

Animals↗

PubMed1989

The interleukin 2 gene is expressed in the syncytiotrophoblast of the human placenta.

The lymphokine interleukin 2 is an important immune system regulatory glycopolypeptide. It is produced by antigen- or mitogen-stimulated T lymphocytes and is required for the proliferation or clonal expansion of activated T lymphocytes. In this report, it is demonstrated by RNA transfer blot hybridization that the poly(A)+ RNA population of the human placenta contains a 0.85-kilobase RNA transcript that specifically hybridizes to a human interleukin 2 cDNA probe. By using hybridization histochemistry in situ, it is further shown that interleukin 2 RNA transcripts are localized, primarily, to the syncytial (syncytiotrophoblast) layer of the human placenta. Possible roles for syncytiotrophoblast-produced interleukin 2 are suggested and discussed.

Autoradiography↗

PubMed1989

Expression of insulin-like growth factor I in cultured rat hepatocytes: effects of insulin and growth hormone.

The effects of GH and insulin the accumulation of insulin-like growth factor I (IGF-I) RNA transcripts and the secretion of immunoreactive IGF-I protein were studied in rat liver hepatocytes cultured in serum-free medium. GH at concentrations of 10 ng/ml or greater stimulated the accumulation of IGF-I RNA transcripts relative to actin transcripts in poly(A)+ RNA isolated from cultured hepatocytes. The time course of IGF-I transcript accumulation in response to GH appeared to be biphasic. The transcript levels rose dramatically during the first 2 h after exposure of the cultures to GH, declined between 3 and 6 h, but reaccumulated by 24 h after exposure to GH. The presence of insulin did not influence the effect of GH on the accumulation of IGF-I RNA transcripts, although insulin did elevate IGF-I transcript levels in the absence of GH. Analysis of RNA pulse-labeled with thiouridine followed by purification of the thiol-labeled RNA using mercurated agarose indicated that GH probably acts by increasing IGF-I transcription. Insulin also affected the release of immunoreactive IGF-I into the culture medium. In the presence of insulin, immunoreactive IGF-I accumulated in the culture medium to approximately the same extent over a 24-h period regardless of whether GH was also present. In the absence of insulin, immunoreactive IGF-I accumulated in the medium only if GH was present. The results suggest that insulin may play an important role in both IGF-I transcript accumulation and the secretion of IGF-I from cultured hepatocytes.

Animals↗

PubMed1988

Induction of vitellogenin in primary monolayer cultures of cockerel hepatocytes.

A primary monolayer culture system from cockerel hepatocytes was established. The cultures synthesize and secrete proteins that comigrate with authentic serum proteins on polyacrylamide gels and are found in the same relative abundance. Addition of estradiol increased the synthesis of apoprotein B, found in very low density lipoprotein, under all culture conditions. Vitellogenin synthesis could not be induced directly by estradiol. However, when serum was obtained from cockerels injected with estradiol 4 days before blood collection and included in the culture medium, the cultures secreted a protein identified immunologically as vitellogenin by affinity chromatography. Furthermore, addition of growth hormone or prolactin to cultured cockerel hepatocyte monolayers resulted in the synthesis and secretion of a polypeptide that comigrates with authentic vitellogenin on polyacrylamide gels.

Animals↗

PubMed1988

Isolation of an insulin-like growth factor II cDNA with a unique 5' untranslated region from human placenta.

Human insulin-like growth factor II (IGF-II) cDNA from a placental library was isolated and sequenced. The 5' untranslated region (5'-UTR) sequence of this cDNA differs completely from that of adult human liver and has considerable base sequence identity to the same region of an IGF-II cDNA of a rat liver cell line, BRL-3A. Human placental poly(A)+ RNA was probed with either the 5'-UTR of the isolated human placental IGF-II cDNA or the 5'-UTR of the IGF-II cDNA obtained from adult human liver. No transcripts were detected by using the 5'-UTR of the adult liver IGF-II as the probe. In contrast, three transcripts of 6.0, 3.2, and 2.2 kilobases were detected by using the 5'-UTR of the placental IGF-II cDNA as the probe or the probe from the coding sequence. A fourth IGF-II transcript of 4.9 kilobases presumably containing a 5'-UTR consisting of a base sequence dissimilar to that of either IGF-II 5'-UTR was apparent. Therefore, IGF-II transcripts detected may be products of alternative splicing as their 5'-UTR sequence is contained within the human IGF-II gene or they may be a consequence of alternative promoter utilization in placenta.

Amino Acid Sequence↗

PubMed1988

Insulin-like growth factor-I messenger ribonucleic acid in the developing human placenta and in term placenta of diabetics.

Fetal growth and development are dependent upon the growth and development of the placenta. Control of placental growth and development is little understood. Immunoreactive insulin-like growth factor-I and -II (IGF-I and IGF-II) have been shown to be released by human placental tissue and human placental membranes have been observed to contain specific receptors for these growth factors. Furthermore, we have demonstrated the presence of IGF-II mRNA transcripts in the developing human placenta and at gestational term in placentae of diabetics. Thus, the IGFs may have a regulatory role in the growth and development of the placenta via autocrine and/or paracrine mechanism(s) of action. In this report we demonstrate the presence of four differing size species of placental poly(A)+ RNA which specifically hybridize to an IGF-I probe originally isolated from an adult human liver cDNA library and localize IGF-I and IGF-II mRNA to syncytiotrophoblasts and fibroblasts, respectively, of the placenta by in situ hybridization. The major transcript is 7500 bases in size and the remaining three transcripts are 5000, 1100, and 900 bases in length with no apparent changes from these sizes throughout gestation and at term in diabetics. Quantification by densitometry of placental IGF-I mRNA detected by dot blot hybridization indicated that first and second trimester placentae each express more IGF-I mRNA relative to that expressed in placenta at term. These results suggest that there are developmental changes in the relative amount of IGF-I mRNA expressed in the human placenta. IGF-I is, therefore, most likely important early in gestation as a placental growth factor. This time period is critical for fetal development and growth, when embryonic induction, organogenesis, and rapid cell proliferation occur.

Female↗

PubMed1986

Synthesis of somatomedin C/insulin-like growth factor I by human placenta.

We have reported the presence of insulin-related poly A+ RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+ RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [35S]methionine as a label. From 2.0 X 10(6) cpm of specifically incorporated [35S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14,000 represented about 0.1% of total radioactivity in the translational products of poly A+ RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24,000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.

Female↗