Smooth side-match classified vector quantizer with variable block size.

Although the side-match vector quantizer (SMVQ) reduces the bit rate, the image coding quality by SMVQ generally degenerates as the gray level transition across the boundaries of the neighboring blocks is increasing or decreasing. This study presents a smooth side-match method to select a state codebook according to the smoothness of the gray levels between neighboring blocks. This method achieves a higher PSNR and better visual perception than SMVQ does for the same bit rate. Moreover, to design codebooks, a genetic clustering algorithm that automatically finds the appropriate number of clusters is proposed. The proposed smooth side-match classified vector quantizer (SSM-CVQ) is thus a combination of three techniques: the classified vector quantization, the variable block size segmentation and the smooth side-match method. Experimental results indicate that SSM-CVQ has a higher PSNR and a lower bit rate than other methods. Furthermore, the Lena image can be coded by SSM-CVQ with 0.172 bpp and 32.49 dB in PSNR.

Growth hormone stimulates transcription of the gene encoding the acid-labile subunit (ALS) of the circulating insulin-like growth factor-binding protein complex and ALS promoter activity in rat liver.

The growth-promoting activity of GH, the principal hormonal determinant of body size, is mediated by insulin-like growth factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa complex that contains IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of IGF-I and determines its bioavailability to the tissues. Formation of the 150-kDa complex depends upon the synthesis of ALS, which is synthesized primarily in liver and is regulated by GH. The present study demonstrates that GH stimulates ALS gene transcription in rat liver and ALS promoter activity in a rat hepatoma cell line. ALS messenger RNA (mRNA) and ALS nuclear transcripts were decreased to similar extents in the livers of GH-deficient hypophysectomized rats. GH increased hepatic ALS mRNA within 3-4 h to about 65% of the levels seen in sham-operated control rats. To confirm that GH stimulated ALS gene transcription, we transiently transfected an ALS promoter-luciferase reporter gene construct into H4-II-E rat hepatoma cells and primary rat hepatocytes. Recombinant human GH (hGH) stimulated promoter activity about 3-fold. In contrast, basal promoter activity was lower, and GH stimulation was absent when the ALS reporter construct was transfected into GH-responsive 3T3-F442A mouse preadipocyte fibroblasts. GH stimulation of ALS promoter activity in H4-II-E cells was mediated by functional GH receptors; nonprimate (rat and bovine) GH gave identical stimulation to hGH, and stimulation by hGH occurred at physiological concentrations. Reverse transcriptase-PCR analysis indicated that GH receptor mRNA was present in H4-II-E cells at approximately 40% of the level seen in rat liver. GH also induced the expression of the endogenous c-fos gene, indicating that the signaling pathway necessary for the activation of gene expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a GH-responsive liver cell line that should provide a useful system in which to study the molecular mechanism of transcriptional regulation by GH of ALS and other hepatic genes.

Assessment of attenuated Salmonella typhimurium strains designed as potential carriers of foreign antigens for mucosal localization and immuno-genicity in a rabbit model.

Neither wild type nor attenuated S. typhimurium strains induced diarrheal illness in rabbits. All strains localized to the Peyer's patch at higher concentrations than in lumenal contents or adjacent ileum. Wild type S. typhimurium C5 induced a typhoid-like illness in rabbits with severe weight loss, bacteremia, persistent splenic colonization, and serum IgG response. Both attenuated strains were disseminated to spleen (day 3) but produced minimal systemic illness. They induced biliary IgA responses greater than the wild type (day 7), but minimal serum IgG responses. Both mutants of S. typhimurium are suitable for further development as live enteric vaccines to carry foreign antigens since they localize to Peyer's patch after oral inoculation, induce biliary antibody, and produce minimal systemic disease. The attenuated strains tested are systemically disseminated. It remains to be determined whether dissemination (determined by a large virulence plasmid) is necessary for the desired mucosal immune response or acceptable for an oral vaccine strain.

Rat PC12 pheochromocytoma cells synthesize insulin-like growth factor-binding protein-6.

The PC12 cell line established from a rat pheochromocytoma has been extensively studied as a model of neuronal differentiation. Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic for PC12 cells under serum-starved conditions. IGF activity is modulated by a family of six IGF-binding proteins (IGFBPs). It recently was reported that PC12 cells produced an IGFBP that had a marked preferential binding affinity for IGF-II over IGF-I. We now show that the main IGFBP produced by PC12 cells is rat IGFBP-6 and compare its properties with those of human IGFBP-6. The predominant IGFBP in medium conditioned by undifferentiated and differentiated PC12 cells migrated on sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis with an apparent molecular mass of 22.5-25 kilodaltons and was recognized by polyclonal antiserum to rat IGFBP-6 by immunoblotting. Rat IGFBP-6 mRNA (1.4 kilobases) was detected by Northern hybridization of total RNA extracted from PC12 cells using a rat IGFBP-6 cDNA probe. Rat IGFBP-6, like human IGFBP-6, is O-glycosylated; incubation with neuraminidase, fucosidase, and O-glycanase reduced its apparent molecular mass to 21 kilodaltons. Competitive binding studies of rat and human IGFBP-6 with [125I]IGF-II and unlabeled IGF-II or IGF-I demonstrated that both IGFBPs bound IGF-II with similar affinities (Ka, 1.5-1.8 x 10(11) M-1) and bound IGF-I with approximately 25- to 35-fold lower affinity than IGF-II. Thus, differences in amino acid sequence, such as deletion of nine amino-terminal residues (including two conserved cysteine residues) in rat IGFBP-6 compared with human IGFBP-6, do not alter its binding characteristics. PC12 cells should provide a useful system to define the regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF action.

Cycloheximide stabilizes insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and inhibits IGFBP-1 transcription in H4-II-E rat hepatoma cells.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of six proteins that modulate the biological activity of IGF-I and IGF-II and determine their bioavailability to tissues. One of the IGFBPs, IGFBP-1, is distinctive in the dynamic response of its levels in human plasma to metabolic changes. Parallel changes occur in IGFBP-1 mRNA and IGFBP-1 transcription in rat liver. Using the well differentiated H4-II-E rat hepatoma cell line as a model system, we demonstrated previously that IGFBP-1 transcription is positively regulated by dexamethasone and negatively regulated by insulin. We now examine the effect of the protein synthesis inhibitor, cycloheximide, on the hormonal regulation of IGFBP-1 gene expression. Preincubation of H4-II-E cells with 10.7 microM cycloheximide for 1.5 h did not prevent the induction of IGFBP-1 mRNA and IGFBP-1 transcription (determined in nuclear run-on assays) by dexamethasone. By contrast, cycloheximide treatment abolished the decrease in IGFBP-1 mRNA induced by insulin. Insulin rapidly decreased IGFBP-1 transcription in the absence of cycloheximide (> 50% inhibition in 20 min) and caused a similar decrease in cells pretreated with cycloheximide. Cycloheximide alone also decreased IGFBP-1 transcription. Similar results were observed with a second protein synthesis inhibitor, anisomycin, which also prevented the insulin-induced decrease in IGFBP-1 mRNA without abolishing the insulin-induced inhibition of IGFBP-1 transcription. These results suggest that although insulin decreases IGFBP-1 gene transcription in the presence of protein synthesis inhibitors, IGFBP-1 mRNA levels are maintained because of stabilization of the mRNA. Stabilization was demonstrated directly in actinomycin D-treated cells, where the t1/2 of IGFBP-1 mRNA increased from approximately 2 to approximately 20 h in the presence of cycloheximide; insulin did not affect IGFBP-1 mRNA turnover. Thus, cycloheximide-sensitive labile proteins contribute to the maintenance of basal IGFBP-1 promoter activity and the rapid turnover of IGFBP-1 mRNA, which determine the dynamic regulation of IGFBP-1 gene expression.

Preclinical evaluation of microencapsulated CFA/II oral vaccine against enterotoxigenic E. coli.

Colonization Factor Antigen (CFA/II) from enterotoxigenic Escherichia coli (ETEC) prepared under good manufacturing practices (GMP) was successfully incorporated into biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer microspheres (BPM) under GMP and found to be safe and immunogenic when administered intraduodenally to rabbits. Following vaccination, Peyer's patch cells responded by lymphocyte proliferation to in vitro challenge with CFA/II. Also, B cells secreting specific anti-CFA/II antibodies were found in spleens following vaccination. No pathological changes were found following total necropsies of ten rabbits vaccinated with CFA/II BPM. Sixty-three per cent of the CFA/II BPM were between 5 and 10 microns diameter by volume particle size distribution; 1.17% protein content; 2.15% moisture; < 0.01% acetonitrile; 1.6% heptane; 22 non-pathogenic bacteria and three fungi per 1 mg protein dose; and passed the general safety test. We conclude that the CFA/II BPM oral vaccine is immunogenic and safe to begin a Phase I clinical safety study following Investigational New Drug approval.

Post-transcriptional regulation of insulin-like growth factor binding protein-2 mRNA in diabetic rat liver.

IGFBP-1 and IGFBP-2 mRNAs are increased in the livers of streptozotocin-diabetic rats. A corresponding increase is observed in transcription of the IGFBP-1 but not the IGFBP-2 gene, indicating that the increase in steady-state levels of IGFBP-2 mRNA is a post-transcriptional effect. IGFBP-1 and IGFBP-2 mRNAs also differ in the rapidity of their response to insulin treatment: hepatic IGFBP-1 mRNA is normalized within 1 h, IGFBP-2 mRNA decreases more slowly. These differences suggest that IGFBP-2 may provide more chronic adaptation to metabolic change than IGFBP-1.

Insulin rapidly decreases insulin-like growth factor-binding protein-1 gene transcription in streptozotocin-diabetic rats.

Insulin-like growth factor-binding protein-1 (IGFBP-1) can inhibit or potentiate IGF action. The biological activity of IGFBP-1 is determined by many factors, including its abundance in tissues and plasma, posttranslational modifications, and localization. IGFBP-1 levels in human plasma are highly regulated. They are increased after acute fasting and in diabetes, and are rapidly reversed by refeeding and insulin treatment, respectively. Similarly, IGFBP-1 mRNA is increased in the liver of severely diabetic and ketotic rats and decreased after 4 days of insulin treatment. Insulin rapidly decreases IGFBP-1 mRNA and IGFBP-1 transcription in rat hepatoma cells. The present study asks whether the increase in IGFBP-1 mRNA in diabetic rat liver reflects increased gene transcription, whether insulin decreases IGFBP-1 mRNA through a transcriptional or posttranscriptional mechanism, and whether this decrease is sufficiently rapid to account for the dynamic fluctuations in plasma IGFBP-1. Rats were injected ip with 100 mg/kg streptozotocin and used 7 days later when they were hyperglycemic and failed to gain weight, but were not ketotic. Hepatic IGFBP-1 mRNA levels were 13.6 +/- 5.3-fold greater in diabetic than control liver and decreased to the low levels in nondiabetic controls within 1 h after insulin treatment. In run-on transcription assays, IGFBP-1 transcription was 12.6 +/- 1.5-fold greater in nuclei from diabetic than control liver and decreased to low control levels by 1 h after insulin injection. Normalization of hepatic IGFBP-1 mRNA in insulin-treated diabetic animals did not require restoration of euglycemia. IGFBP-1 mRNA and IGFBP-1 gene transcription also were increased in the kidney of diabetic ketotic rats. We propose that the dynamic regulation of IGFBP-1 gene transcription in diabetes and after insulin treatment, by determining the availability of IGFBP-1 in tissues and plasma, may be a critical factor in the modulation of IGF action.

Transcription of the insulin-like growth factor-binding protein-2 gene is increased in neonatal and fasted adult rat liver.

The insulin-like growth factor-binding proteins (IGFBPs) are a family of proteins that specifically bind IGF-I and IGF-II, determine their bioavailability to tissues, and modulate their actions in target tissues. Levels of IGFBPs in plasma and IGFBP mRNAs in liver are highly regulated with developmental age and metabolic status. We now demonstrate that the increase in IGFBP-2 mRNA in fasted adult rat liver and in the liver of normal neonatal rats reflects an increased rate of transcription. When adult rats were fasted for 2-3 days, IGFBP-2 mRNA was increased in liver, but not in brain or kidney. The increase in hepatic IGFBP-2 mRNA was observed after only 1 day of fasting. Levels decreased by half after 6 h of refeeding and returned to their low starting values after 2 days of refeeding. Transcription-elongation experiments indicated that transcription of the IGFBP-2 gene was increased in fasted liver. The rate of transcription increased 9.2- +/- 3.5-fold for transcripts labeled in exon 1 and 6.6- +/- 2.4-fold for transcripts labeled in exons 2, 3, and 4, suggesting that fasting causes a uniform increase in the number of RNA polymerase II molecules along the length of the IGFBP-2 gene. We infer from these results that the regulation of IGFBP-2 gene transcription in fasting occurs at the level of initiation rather than elongation. IGFBP-2 gene transcription also was increased 3.8- +/- 1.2-fold (exon 1) and 2.9- +/- 0.9-fold (exons 2, 3, and 4) in nuclei from 2-day postnatal rat liver compared with adult rat liver, consistent with the greater abundance of IGFBP-2 mRNA in neonatal rat liver.

[The effect of nitrous oxide anesthesia combined with low dose alfentanil for minor surgery of short duration].

This study was conducted in 30 adult patients with ASA class I-II physical status who received minor operations. Anesthetic induction was achieved by injecting thiopental 4 mg/kg intravenously in addition to N2O/O2 (4L/2L) delivered via Ventri Mask, followed by alfentanil 7 micrograms/kg intravenously 3 min later. Maintenance of anesthesia was accomplished by N2O/O2 in conjunction with alfentanil 0.25-2.5 micrograms/kg/min, delivered intravenously by a syringe pump. Our result showed that in an operation of average duration around 40.2 +/- 10.5 min., the average dose of alfentanil used was 0.62 +/- 0.15 micrograms/kg/min. The respiration rate fell from 13.4 +/- 0.4 cpm to 8.4 +/- 1.1 cpm 2 min later following alfentanil injection, which was statistically significant. SaO2 fell from 97.9 +/- 0.4% to 94.0 +/- 0.8% 3 min after alfentanil injection, which was statistically significant. End-tidal carbon dioxide partial pressure elevated from 39.4 +/- 0.6 mmHg to a peak of 45.3 +/- 1.2 mmHg 5 min after alfentanil injection which was also statistically significant. Temporary apnea was noted in 3 cases, but they all resumed spontaneous respiration after a short period of assisted ventilation. Changes in systolic and diastolic pressure during anesthesia were not marked. Pulse rate was noted to decrease from 80.3 +/- 2.7 bpm to 70.5 +/- 2.0 bpm 1 min after alfentanil injection, which was statistically significant (p less than 0.05). After discontinuation of N2O, the time required to regain the ability to follow orders of "open your eyes," "show your thumb" and "say your name" in sequence was 72.5 +/- 10.6s, 88.2 +/- 11.6s, 128.1 +/- 23.0s, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin rapidly inhibits insulin-like growth factor-binding protein-1 gene expression in H4-II-E rat hepatoma cells.

The insulin-like growth factor-binding proteins (IGFBPs) are thought to determine the distribution of IGF-I and IGF-II between the blood and tissue compartments and to modulate their biological activities. A dynamic metabolic role for one of the IGFBPs, IGFBP-1, is suggested by the fact that plasma IGFBP-1 was increased after fasting and diabetes and rapidly decreased by refeeding or insulin treatment, respectively. IGFBP-1 mRNA also is increased in the livers of diabetic rats and decreased by insulin treatment. To understand the molecular basis for this regulation, we have examined the effects of insulin on IGFBP-1 and IGFBP-1 mRNA in the H4-II-E cell line derived from the well differentiated H35 rat hepatoma. IGFBP-1, identified by ligand blotting and immunoblotting, is the major IGFBP in H4-II-E cells. Incubation of H4-II-E cells with insulin for 24 h decreased IGFBP-1 in the culture medium by approximately 50%. Inhibition was observed at physiological concentrations of insulin (ED50, less than 0.5 nM), but not at higher concentrations of IGF-II. These results, together with the fact that H4-II-E cells do not possess IGF-I receptors with which insulin might cross-react, suggest that insulin acts via the insulin receptor. Insulin inhibited IGFBP-1 in the medium by 80% in the absence of glucose, suggesting that the inhibition is a direct effect of insulin; glucose exerted a smaller independent effect in the absence of insulin. Insulin decreased IGFBP-1 mRNA in H4-II-E cells by 50% within 1 h and by 90% after 2-12 h of incubation. Nuclear run-on transcription assays indicated a corresponding decrease in the rate of IGFBP-1 gene transcription. Pretreatment of H4-II-E cells with dexamethasone stimulated IGFBP-1 transcription and increased steady state IGFBP-1 mRNA; stimulation was abolished by insulin treatment, indicating that inhibition by insulin was dominant over induction by dexamethasone. Thus, insulin, acting through the insulin receptor, rapidly decreases the abundance of IGFBP-1 mRNA in H4-II-E cells. Regulation occurs at least in part at the level of gene transcription. We propose that regulation of IGFBP-1 synthesis is an important component of the regulation of IGFBP-1 by insulin in vivo.

Tissue, developmental, and metabolic regulation of messenger ribonucleic acid encoding a rat insulin-like growth factor-binding protein.

Insulin-like growth factor-II (IGF-II) is the predominant insulin-like growth factor in fetal and neonatal rat serum and tissues. In serum, it occurs complexed to a 30-kDa nonglycosylated IGF-binding protein (IGFBP) that is immunologically related to the IGFBP in BRL-3A rat liver cells (rIGFBP-2). Levels of rIGFBP-2 and IGF-II decrease in rat serum after birth. Using a recently isolated cDNA clone for rIGFBP-2 as hybridization probe, we now compare the expression of rIGFBP-2 and IGF-II in fetal tissues and the effects of hypophysectomy and fasting on the abundance of these mRNAs in adult rat liver. rIGFBP-2 mRNA is expressed at high levels in term gestation liver and at lower levels in other tissues. The ratio of rIGFBP-2 to IGF-II mRNAs in stomach, kidney, and lung is similar to that seen in liver, whereas IGF-II mRNA is more abundant than rIGFBP-2 mRNA in muscle, intestine, heart, and skin. Both mRNAs are more abundant in fetal tissues than in the corresponding tissues from adult rats. Dexamethasone treatment of 4-day-old rats for 4 days caused a greater (90%) decrease in hepatic IGF-II mRNA than in rIGFBP-2 mRNA (50%), suggesting subtle differences in the developmental regulation of the two mRNAs. Even more striking differences were observed in the regulation of the two mRNAs in adult rats after hypophysectomy or fasting. Hepatic rIGFBP-2 mRNA was increased 10- to 20-fold compared to age-matched control rats, whereas IGF-II mRNA was not increased. A parallel increase in serum rIGFBP-2 was observed, suggesting that this regulation may result at least in part from the increased abundance of rIGFBP-2 mRNA. Thus, in addition to modulating the stimulation of growth and differentiation by IGF-II in fetal tissues, rIGFBP-2 may play a homeostatic role during catabolic states in the adult rat.

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

The fetal rat binding protein for insulin-like growth factors is expressed in the choroid plexus and cerebrospinal fluid of adult rats.

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.

The 34 kilodalton insulin-like growth factor binding proteins in human cerebrospinal fluid and the A673 rhabdomyosarcoma cell line are human homologues of the rat BRL-3A binding protein.

Three members of a family of insulin-like growth factor binding proteins have been identified by nucleotide sequencing of cDNA clones: the binding subunit of the 150 kDa IGF-binding protein complex in human serum, the 30 kDa IGF binding protein in human amniotic fluid, and a 30 kDa binding protein (BP-3A) isolated from the rat BRL-3A cell line. The present study demonstrates by molecular hybridization and immunoreactivity that the human counterpart of rat BP-3A is a 34 kDa IGF binding protein that is present in human cerebrospinal fluid and is synthesized and secreted by the A673 human rhabdomyosarcoma cell line.

Rat C6 glial cells synthesize insulin-like growth factor I (IGF-I) and express IGF-I receptors and IGF-II/mannose 6-phosphate receptors.

We have used the rat C6 glial cell line as a model system to study the role of insulin-like growth factors (IGF) in neuroglial cells of the central nervous system (CNS). Northern blot analysis of C6 RNA demonstrated the presence of IGF-I mRNA and undetectable IGF-II mRNA. IGF-I and IGF-binding protein(s), but not IGF-II, were detected in C6 glial cell-conditioned medium. The level of IGF-I was 1-4 ng/ml in conditioned medium based on a human IGF-I standard. The immunoreactive IGF-I inhibited [125I]IGF-I binding to the IGF-I receptor on chick embryo fibroblasts and stimulated [3H]thymidine incorporation into chick embryo fibroblast DNA. Competitive binding and affinity cross-linking experiments using [125]IGF-I and [125I]IGF-II demonstrated the presence of IGF-I receptors (type I) and IGF-II/mannose 6-phosphate receptors (type II) on C6 glial cell membranes. An immunoglobulin (no. 3637) directed against the rat IGF-II receptor blocked the degradation of [125I]IGF-II added to C6 glial cells, presumably by blocking receptor-mediated internalization. We were unable to demonstrate an autocrine role for IGF in the C6 glial cell line, since [3H]thymidine incorporation into DNA was stimulated equally well by IGF-I-deficient rat serum and normal serum, and added IGF did not stimulate [3H]thymidine incorporation into DNA when tested alone or when added to IGF-I-deficient serum. We propose that neuroglial cell-derived IGF-I may serve as a paracrine growth stimulus in the central nervous system.

Synthesis of insulin-like growth factor II (IGF-II) in fetal rat tissues: translation of IGF-II ribonucleic acid and processing of pre-pro-IGF-II.

The single insulin-like growth factor II (IGF-II) gene is transcribed into multiple RNA species in most fetal and neonatal rat tissues. For IGF-II to serve as a local growth factor in fetal tissues, IGF-II RNA must be translated into pre-pro-rat (r) IGF-II, and the biosynthetic precursor processed to smaller biologically active forms. IGF-II RNA extracted from fetal rat liver, muscle, intestine, lung, and stomach, from rat placenta, and from fetal or neonatal mouse liver and lung directed the synthesis of 22,000 mol wt pre-pro-IGF-II in a reticulocyte lysate cell-free translation system. A biosynthetic precursor of this size had been observed previously in translation of RNA from BRL-3A rat liver cells and is predicted by the nucleotide sequence of cDNA clones encoding rIGF-II. Consistent with the developmental pattern of expression of IGF-II RNA observed in hybridization studies, RNA from adult rat liver, muscle, and intestine did not direct the synthesis of pre-pro-rIGF-II. To determine whether the IGF-II biosynthetic precursor was processed to smaller biologically active IGF-II, term fetal rat tissues were extracted with acid-ethanol, the extracts were fractionated by acid gel filtration, and the IGF pools were examined in a RIA specific for IGF-II. Levels of 1-2 micrograms/g were observed in liver, limb, lung, intestine, and brain; lower levels were observed in heart and kidney. In general, the levels of immunoreactive IGF-II corresponded to the levels of IGF-II mRNA. These results suggest that IGF-II mRNA is translated, and pre-pro-IGF-II processed to mature IGF-II in different fetal rat tissues. In contrast to IGF-I, in which alternative RNA splicing generates possible precursor molecules containing different COOH-terminal propeptide segments, we find no evidence for an IGF-II precursor in rat tissues other than 22,000 mol wt pre-pro-rIGF-II.