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Biomedical subjects

A Kowalski

Publications and source records attributed to A Kowalski.

At least 37 records · Page 2Linked to original sources
PubMed1991

Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle.

Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.

Animals↗
PubMed1991

Expression of IGF-I and insulin receptor genes in the rat central nervous system: a developmental, regional, and cellular analysis.

Using a solution-hybridization assay and specific oligonucleotidic probes, we have studied IGF-I and insulin receptor mRNAs in the rat central nervous system during development. The expression of mRNAs was maximal at embryonic day 15 and 20 for IGF-I receptors, and at embryonic day 20 and the day of birth for insulin receptors. After birth, the expression of both receptor transcripts decreased and reached minimal levels in the adult. At the time at which these transcripts were maximally expressed (embryonic day 20), the regional analysis indicated that IGF-I receptor transcripts were widely distributed in the brain. In contrast, insulin receptor transcripts were restricted to certain areas in which they were coexpressed with the IGF-I receptor transcripts. We next analyzed which cells at embryonic day 20 expressed those receptor transcripts. Late embryonic neurons, astrocytes, and neonatal progenitors of oligodendrocytes synthesized both IGF-I and insulin receptor mRNAs after a short time in culture. However, astrocytes expressed preferentially IGF-I receptor transcripts, while young progenitors for oligodendrocytes expressed high levels of insulin receptor transcripts. As a whole, our data indicate that during rat CNS development expression of IGF-I and insulin receptors appears to be stage- and cell-specific. The differences observed between the expression of both receptors might point to a specific, but coordinative role of IGF-I and insulin and their receptors during that time.

Animals↗
PubMed1991

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.

Animals↗
PubMed1991

Effects of insulin, insulin-like growth factor-I (IGF-I) and progesterone on glucose and amino acid uptake in Xenopus laevis oocytes.

In Xenopus oocytes, insulin or IGF-I activated glucose transport and maturation, but not amino acid transport, as measured by the uptake of alanine. Glucose transporter identical or closely related to the mammalian erythroid/brain transporter (Glut-1/EGT) were found in oocyte membranes. The EC50 for stimulation of glucose uptake and of maturation were similar (1-5 x 10(-8) M for insulin and 2-8 x 10(-10) M for IGF-I), confirming that these effects were mediated through IGF-I receptors. Other agents, such as phorbol 12-myristate 13-acetate (TPA) (0.5 microM) and vanadate (2 mM) evoked only part of the insulin effect on glucose uptake (50% and 65%, respectively), without being additive to insulin. In contrast, progesterone at 1 microM, a potent inducer of maturation, inhibited at least partially the insulin-induced glucose uptake. Uptake of alanine and glucose was decreased after prolonged incubations (3-6 h) with agents that trigger maturation, and was dramatically reduced in oocytes that have undergone maturation (unfertilized eggs). Maturation is thus accompanied by a reduction in glucose and amino acid transports. These result further document the validity of Xenopus oocytes as a model to study insulin and IGF-I signalling.

Alanine↗
PubMed1991

Insulin-like effects of vanadate on glucose uptake and on maturation in Xenopus laevis oocytes.

Vanadate, an inhibitor of phosphotyrosyl phosphatases that exerts insulin-like effects in intact cells, stimulated both maturation and glucose uptake in isolated Xenopus laevis oocytes. Vanadate enhanced the effects of insulin/IGF-I and progesterone on maturation in a dose-dependent manner, with an effective concentration of 750 microM and a maximum at 2 mM, whereas, in the absence of hormone, activation of maturation was seen at 10 mM vanadate. Further, vanadate at 2 mM increased glucose uptake, but this effect was not additive to that of the hormone. In cell-free systems, vanadate caused a 12-fold stimulation of autophosphorylation of the oocyte IGF-I receptor in the absence, but not in the presence, of IGF-I and inhibited largely, but not totally, receptor dephosphorylation induced by an extract of oocytes rich in phosphotyrosyl phosphatase activities. These effects were dose dependent, with effective concentrations of 50-100 microM and maxima at 2 mM. Moreover, using an acellular assay to study the effect of vanadate on the activation of maturation promoting factor (MPF), we found that vanadate at 2 mM stimulated the activation of the MPF H1 kinase. This suggests that vanadate did not prevent dephosphorylation of p34cdc2 on tyrosine residues. Vanadate thus exerted insulin-like effects in oocytes, including stimulation of maturation. These effects might result from a direct or indirect action of vanadate on the IGF-I receptor kinase and on MPF activity.

Animals↗
PubMed1989

Effect of retinyl acetate on nuclear proteins in rabbit liver.

1. Retinyl acetate injected intraperitoneally to adult rabbits fed on standard diet caused detectable changes in the polyacrylamide gel patterns of liver nucleoplasmic and 0.35 M NaCl-soluble chromatin proteins. 2. Both histones and non-histone proteins soluble in 5 M urea were not affected in vitamin A-treated animals. 3. It seems that variations in liver nuclear proteins from retinyl acetate-administered rabbits may reflect retinol-dependent alterations in structure and function of their chromatins.

Animals↗
PubMed1989

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action.

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.

Amino Acids↗
PubMed1989

Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies.

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Expression of two types of receptor for insulin-like growth factors in human malignant glioma.

Two types of receptors for insulin-like growth factors (IGFs) were characterized in glioma cell lines established from different human brain tumors of glial origin (astrocytoma grades III and IV) by competitive binding assay, affinity labeling, and protein phosphorylation. Type I IGF receptor is a heterodimer composed of alpha-subunits (Mr 130,000), which bind IGF I and II with equal affinity, and of beta-subunits (Mr 98,000), which show tyrosine kinase activity and autophosphorylation stimulated by IGF I and II with equal potency. The type II IGF binding site is a monomer (Mr 250,000) which binds IGF II with 10 times higher affinity than IGF I. The cellular concentration of type II IGF binding site is about 2- to 5-fold higher than the amount of type I IGF receptor. The characteristics of the two types of IGF receptors in human glioma cell lines are similar to those described recently in fetal rat astrocytes. In contrast the type I IGF receptor in glioma cells is different from that studied previously in normal adult brain regarding the equal affinity for IGF I and II, and the higher molecular size of the alpha-subunit (130,000 versus 115,000). It is suggested that glioma cells may represent a fetal cell type in tumor development of adult human brain. A role of IGF in malignant glioma has not yet been determined, but the presence of IGF receptors is a prerequisite for cellular actions of IGF.

Brain Neoplasms↗
PubMed1988

Receptors for insulin-like growth factors in the central nervous system: structure and function.

Insulin-like growth factors (IGFs) I and II are homologous peptides, which stimulate growth of several vertebrate tissues. Expression of IGF I and IGF II genes and production of IGFs have recently been demonstrated in rat and human brain. In search for the function of IGF I and IGF II in the central nervous system, we have studied IGF receptors in fetal and adult mammalian brain and growth effects of IGFs on primary cultures of fetal rat astrocytes. Two types of IGF receptor are present on adult rat brain cortical plasma membranes, on fetal rat astrocytes and on human glioma cells. Type I IGF receptor is composed of 2 types of subunits: alpha-subunits which bind IGF I and IGF II with high affinity and insulin weakly, and beta-subunits which show tyrosine kinase activity and autophosphorylation stimulated by IGF I and IGF II with almost similar potency. The molecular size of the type I IGF receptor alpha-subunit is larger in cultured fetal rat astrocytes and human glioma cells than in normal adult brain (Mr 130,000 versus 115,000), whereas the beta-subunit has the same electrophoretic mobility (Mr 94,000). The type II IGF receptor is a monomeric protein (Mr 250,000), which binds IGF II 5 times better than IGF I, and does not recognize insulin. The amounts of type II IGF receptor are significantly higher in fetal and malignant cells than in adult brain. Based on these findings we suggest that IGF receptors in brain undergo changes during fetal development and malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Insulin-like growth factor I in cultured rat astrocytes: expression of the gene, and receptor tyrosine kinase.

Gene expression, receptor binding and growth-promoting activity of insulin-like growth factor I (IGF I) was studied in cultured astrocytes from developing rat brain. Northern blot analysis of poly(A)+ RNAs from astrocytes revealed an IGF I mRNA of 1.9 kb. Competitive binding and receptor labelling techniques revealed two types of IGF receptor in astroglial cells. Type I IGF receptors consist of alpha-subunits (Mr 130,000) which bind IGF I with significantly higher affinity than IGF II, and beta-subunits (Mr 94,000) which show IGF I-sensitive tyrosine kinase activity. Type II IGF receptors are monomers (Mr 250,000) which bind IGF II with three times higher affinity than IGF I. Both types of IGF receptor recognize insulin weakly. DNA synthesis measured by cellular thymidine incorporation was stimulated 2-fold by IGF I and IGF II. IGF I was more potent than IGF II, and both were significantly more potent than insulin. Our findings suggest that IGF I is synthesized in fetal rat astrocytes and acts as a growth promoter for the same cells by activation of the type I IGF receptor tyrosine kinase. We propose that IGF I acts through autocrine or paracrine mechanisms to stimulate astroglial cell growth during normal brain development.

Animals↗
PubMed1987

Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes.

We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.

Amino Acids↗
PubMed1986

Presence of an insulin-stimulated serine kinase in cell extracts from IM-9 cells.

Insulin responsive protein kinase activities of wheat germ purified glycoproteins were examined. Glycoproteins were first incubated without or with insulin, and then exposed to a serum containing antibodies to insulin receptor. Thereafter, both immunoprecipitates and supernatants were studied for their kinase activity toward histone. Incubation with anti receptor antibodies promoted insulin receptor beta subunit and histone phosphorylation. More important insulin receptor depleted extract contained a kinase activity toward histone, that was increased by preincubation with insulin. This stimulation was observed only when insulin was added before the immunoprecipitation of insulin receptors. Alkali treatment and phosphoamino acids analysis revealed that the kinase activity remaining in the supernatant is serine specific. These findings suggest, that a serine kinase activity is associated with the insulin receptor, that it can be separated from the insulin receptor with anti receptor antibodies, that the serine kinase is activated by the hormone-receptor complex.

Cell Line↗
PubMed1985

Relation between raised concentrations of fucose, sialic acid, and acute phase proteins in serum from patients with cancer: choosing suitable serum glycoprotein markers.

Serum concentrations of fucose, sialic acid, and eight acute phase proteins were measured in single specimens from patients with cancer in order to determine whether the raised concentrations of protein bound sugars commonly found in cancer correlate with increased concentrations of the acute phase proteins. Strong positive correlations were found only with alpha 1-acid glycoprotein, alpha 1-antitrypsin, and haptoglobins. Changes in protein bound sugars and acute phase proteins were also examined in relation to patients' disease states. Serum fucose was raised more often in patients with advanced disease than in those in whom the spread of the tumour was more restricted; increased sialic acid concentrations, however, were found with a similar frequency in both these groups. Combined use of fucose and sialic acid values gave a high degree of marker positivity which could be only slightly improved on by including measurement of acute phase proteins. The combined use of serum fucose and sialic acid concentrations may have value in monitoring patients with cancer: the sialic acid provides an index of the acute phase response and the fucose a measure of the tumour spread.

Acute-Phase Proteins↗
PubMed1985

The sexual side-effects of antidepressant medication: a double-blind comparison of two antidepressants in a non-psychiatric population.

Clinical reports have suggested that antidepressant medication may contribute to the sexual dysfunction experienced by some depressed patients. A double-blind trial in a non-psychiatric male population compared amitriptyline (tricyclic), mianserin (tetracyclic) and placebo for their effects on nocturnal sexual arousal. In a three-way crossover design active drug or placebo were taken for two weeks preceding measurement of the frequency, amplitude and duration of nocturnal penile tumescence and synchronous sleep indices. Both active compounds significantly decreased the amplitude and the total duration of nocturnal erections. The effects on sleep indices were as previously reported. Few differences were found between the tricyclic and tetracyclic drugs. Some implications of these findings are considered.

Adolescent↗
PubMed1984

Insulin receptors in rat brain: insulin stimulates phosphorylation of its receptor beta-subunit.

In rat brain cortex synaptosomes insulin stimulated the phosphorylation of its own receptor beta-subunit (94 kDa) as identified by immunoprecipitation with anti-insulin or anti-receptor antiserum. The receptor alpha-subunit (115 kDa) was characterized by specific labeling with 125I-labeled photoreactive insulin. These observations indicate that: (i) insulin receptors in brain are composed of alpha-subunits which bind insulin, and beta-subunits, the phosphorylation of which can be stimulated by insulin; (ii) the size of alpha-subunits in brain is significantly smaller than in other tissues (115 vs 130 kDa), whereas beta-subunits (94 kDa) are identical. We suggest that brain insulin receptors represent a subtype regarding their binding function, whereas their enzyme function is more conserved.

Affinity Labels↗