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Biomedical subjects

M Jansen

Publications and source records attributed to M Jansen.

At least 145 records · Page 8Linked to original sources
PubMed1995

Translational control of gene expression.

Translational regulation of mRNA is an important step in the control of gene expression. In a general way, the efficiency of the translational apparatus can be influenced either positively or negatively by changing the level or the activity of rate-limiting protein factors taking part in the process of translation. But translational control can also be very specific, affecting only a single mRNA or class of mRNA molecules. In most of these cases regulation takes place at the level of initiation of translation, which is often attributable to structural peculiarities of the mRNA in question, especially of the 5'-untranslated region or leader. This review summarizes the mechanisms which lie at the root of translational control. A better understanding of these mechanisms will eventually provide us with new drugs and antisense oligonucleotide technology, aimed at influencing the level of expression of single proteins. These developments are of interest to basic researchers and clinicians alike, because they may profoundly change the ways in which we treat, e.g. viral infections and malignancies in the future.

Animals↗
PubMed1995

Overexpression of human insulin-like growth factor-II in transgenic mice causes increased growth of the thymus.

In order to determine the effects of IGF-II overexpression on growth of mice, transgenic mice were produced carrying one of three different H-2Kb human IGF-II minigenes in which different non-coding exons (exon 5, truncated exon 5 or exon 6) preceded the coding exons 7, 8 and 9. These were spaced by truncated introns and for proper polyadenylation an SV40 polyadenylation signal was incorporated. The highest levels of IGF-II minigene mRNA expression were found in lines containing the truncated exon 5 construct (II5'). Those containing exon 6 (II6) had less expression and 5 constructs (II5) gave only moderate levels of mRNA expression. In general mRNA expression was highest in thymus and spleen, low in liver and kidney and absent in the brain. In addition, one II5' line showed expression in the brain. Serum IGF-II levels at 8 weeks of age were increased 7- to 8-fold in homozygous transgenic lines with construct II5' without brain expression and 2- to 3-fold in the one that showed expression in the brain; serum IGF-I levels were unchanged. Serum IGFs in the lines containing the constructs II5 and II6 were not different from those of the controls. In all cases body length and weight as well as the weight of several organs such as brain, liver, kidneys, heart and spleen when expressed as a function of age did not differ from controls. Only the thymus showed a significant increase in weight in the transgenics II5'. Inbreeding of 2 lines containing construct II5' with pituitary deficient Snell dwarf mice did not influence body length or weight despite increased serum IGF-II levels. Again the thymus showed a marked increase in growth. The biological activity of the IGF-II peptide was further demonstrated by increased serum IGF-binding protein-3 in the transgenic dwarf mice, as shown by Western ligand blotting. In summary, overexpression of IGF-II in transgenic normal and dwarf mice does not affect overall body growth, but causes increased growth of the thymus. This suggests a role for IGF-II in thymic development by paracrine/autocrine action.

Animals↗
PubMed1995

Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.

The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.

Bacterial Proteins↗
PubMed1995

Regulation of oxytocin expression in the bovine corpus luteum. Orphan receptors and the oxytocin promoter.

The bovine oxytocin gene is massively up-regulated during the early development of the corpus luteum. Oxytocin transcription is induced in a highly synchronous fashion in the granulosa cells of the dominant follicle at the time of ovulation. The possibility to isolate large numbers of differentiating granulosa-luteal cells from exactly defined stages of development allows the investigation of the factors controlling oxytocin expression in vivo by molecular and cell biology methods. Using primary cultures of bovine granulosa cells the synergistic activation of oxytocin transcription by the cAMP pathway and stimulation of IGF-I or insulin receptors could be established. Analysis of transcription factors isolated from the nuclei of bovine granulosa cells and corpus luteum led to the identification of the tissue-specific orphan receptor SF-1 binding to the promoter of the actively transcribed oxytocin gene. The luteinizing bovine granulosa cells provide the only easily accessible experimental system established so far in which the endogenous oxytocin gene is expressed. Although the link between increased cAMP level and receptor tyrosine kinase activation on the one hand and the induction of oxytocin transcription on the other has not been established yet, these experiments constitute one of the few direct approaches to investigate the complexity of events that regulate oxytocin expression in vivo.

Animals↗
PubMed1994

Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes.

The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.

Base Sequence↗
PubMed1994

Differential polysomal localization of human insulin-like-growth-factor-2 mRNAs in cell lines and foetal liver.

Examination of the association of insulin-like-growth-factor-2 mRNAs with polyribosomes in five cell lines revealed that greater than 50% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin-like-growth-factor-2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20-30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin-like-growth-factor-2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin-like-growth-factor-2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin-like-growth-factor-2 mRNAs.

Cell Line↗
PubMed1994

Effect of purine synthesis inhibition on WiDr spheroids in vitro or on WiDr or colon 38 tumors in vivo. Complete growth inhibition but not regression.

Clinical responses for anticancer agents are based upon tumor regression. We have investigated the potential of glycineamide ribonucleotide transformylase (GAR TFase) inhibitors to produce regressions in multiple preclinical models of colon carcinoma. The growth of multicellular tumor spheroids of WiDr human colon carcinoma was inhibited by the GAR TFase inhibitors 5-deazaacyclotetrahydrofolate (5-DACTHF), its 2'-fluoro, 3'-fluoro, 10-deaza, and 10-thia analogs as well as 5,10-dideazatetrahydrofolate, but none of the compounds caused spheroid regressions. By contrast, complete spheroid disruption was observed with exposure to etoposide, m-AMSA (amsacrine), piritrexim, or 2-desamino-2-methyl-10-propargyl-5,8-dideazafolate (DMPDDF). Light microscopy of the spheroids treated with either 5-DACTHF or DMPDDF suggested that the reason for the difference is extensive cell kill throughout the spheroid in the presence of DMPDDF compared with little or no kill, over that found in controls, with 5-DACTHF. Treatment of spheroids with 5-DACTHF in the presence of 1 microM hypoxanthine resulted in no significant reversal of growth inhibition; 50% reversal required 10 microM hypoxanthine. The spheroid studies were extended to in vivo studies examining the effects of 5-DACTHF on established WiDr and colon 38 tumors. The results showed that, in contrast to melphalan, which produced cures and tumor regressions, 5-DACTHF produced reversible growth inhibition with no significant regression of tumors. The results predict that clinical response, typically measured by tumor regression, may be rare following single agent therapy with inhibitors of de novo purine biosynthesis.

Acyltransferases↗
PubMed1994

Potent gastrin-releasing peptide (GRP) antagonists derived from GRP (19-27) with a C-terminal DPro psi [CH2NH]Phe-NH2 and N-terminal aromatic residues.

We have previously reported that octapeptides with a -DPro psi[CH2NH]Phe- NH2 C-terminus are potent GRP antagonists and have greatly enhanced in vivo stability. Now we report the detailed syntheses of such peptides and additional attempts to further increase metabolic stability. Replacement of the -DPro psi[CH2NH]Phe-NH2 with a "-DPro-statine"-Phe-NH2 led to less potent antagonistic activity. The introduction of ThiAla and BzthAla, to replace His and Trp, respectively, did not increase activity. A series of analogs having different aromatic residues at the N-terminal, other than 3-phenylpropionic acid, are equally potent. These residues show increased activity when hydrophilic substitutions are added to the aromatic ring. Replacement of the C-terminal Phe by DPhe and D2Nal is tolerated. Even though none of these peptides have higher activity than the original lead peptide, they are potentially more metabolically stable.

Amino Acid Sequence↗
PubMed1994

Two orphan receptors binding to a common site are involved in the regulation of the oxytocin gene in the bovine ovary.

The peptide hormone oxytocin is highly expressed in the hypothalamus within only a small number of magnocellular neurons. However, it is also expressed in a much larger number of cells in the bovine corpus luteum at high levels in an estrous cycle-dependent manner. By using nuclear extracts from this tissue for in vitro binding studies, two protein complexes have been shown to bind to a common site in the bovine oxytocin promoter. One of these proteins has been identified as the bovine homologue of the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The second protein is here characterized as the bovine homologue of a tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The relative expression of these two factors during luteal development correlates with the level of luteal oxytocin gene expression, with SF-1 being the factor binding to the promoter of the oxytocin gene when this promoter is activated. Cotransfection experiments using the murine testicular cell line TM4 show that SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly by transducing a stimulatory signal to the RNA polymerase.

Animals↗
PubMed1993

Three Hsp70 genes are located in the C4-H-2D region: possible candidates for the Orch-1 locus.

The central region of the mouse MHC harbors a recombinational hot spot area. Most recombinations in this part of the complex take place between the Hsp70.1 gene and the G7 gene. This interval is of interest since structurally indistinguishable recombinant haplotypes do differ in functional behavior. Susceptibility to experimental allergic orchitis, which is controlled by the Orch-1 locus, is one example. We have analyzed the hot spot region at the molecular level in order to understand the molecular organization of this chromosomal segment. From a C57BL genomic library we constructed a cosmid contig bridging the interval between Hsp70.1 and G7. The Orch-1 gene maps to a 60-kb segment of DNA in which we found a new Hsp70 homologue, Hsp70.3. Thus, as in the human MHC, the central region of the mouse MHC harbors a cluster of three Hsp70 genes; Hsp70.1, Hsp70.3, and Hsc70t. Two other genes are located in this critical interval (G7b and G7a/Bat-6), and there might still be other undetected genes present in the region. Heat shock proteins play an important role in a large number of physiological processes and it is tempting to speculate that Hsc70t, which exhibits testis-specific expression, may be identical to Orch-1.

Animals↗
PubMed1993

Cosegregation of missense mutations of the luteinizing hormone receptor gene with familial male-limited precocious puberty.

Familial male-limited precocious puberty is a male-limited autosomal dominant condition. It is characterized by increased testosterone synthesis in the absence of testicular stimulation by luteinizing hormone (LH). We hypothesised that an abnormal configuration of the LH receptor might autonomously activate G protein coupling, and thereby cause the overproduction of testosterone in this condition. To test this hypothesis, we screened for mutations in a part of the LH receptor gene that is important for G protein binding. DNA sequence variation was detected in 2 out of 5 families with male-limited precocious puberty by the single strand conformation polymorphism technique. Direct sequencing demonstrated different single nucleotide substitutions in the sixth transmembrane region of the LH receptor gene. The mutations cosegregated with the disorder in both families (lod score 5.76 without recombination). Both mutations cause an amino acid substitution in the sixth transmembrane domain, close to the C-terminal portion of the third cytoplasmatic loop, a region which is important for the binding of G proteins. We conclude that familial male-limited precocious puberty cosegregates with missense mutations in the LH receptor gene. These findings support the hypothesis that increased activity of the LH receptor is the pathogenetic mechanism that causes the abnormal pubertal development in this condition.

Amino Acid Sequence↗
PubMed1993

Macrophages and multicellular tumor spheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cell proliferation and migration.

In a new co-culture model involving multicellular tumor spheroids and different phenotypes of human macrophages, we studied the effects of the latter on migration and proliferation of the human colon carcinoma cell line, HRT-18. The macrophage phenotypes are detectable with monoclonal antibodies and are inducible in culture. 12-O-tetradecanoyl-phorbol-13-acetate-activated macrophages are associated with the phenotype 27E10, which is an acute inflammatory macrophage. The glucocorticoid-induced macrophage phenotype RM3/1 is associated with the down-regulation of inflammation. The phenotype resembling the mature resident macrophage termed 25F9 arises spontaneously in prolonged culture. It could be shown that inflammatory macrophages are localized at invasive areas of the tumor-host interface of colorectal carcinoma, whereas resident and anti-inflammatory macrophages were found in the central tumor region or at well-bordered areas of the tumor-host interface. The results obtained with this co-culture model show that 27E10-associated macrophages stimulate tumor cell migration and inhibit tumor cell proliferation. RM3/1 had only a slight inhibiting effect on proliferation and a slight promoting effect on migration. The 25F9-positive macrophage-stimulated tumor cell proliferation and inhibited migration completely. This investigation indicates that this in vitro system is useful for studying different macrophage effects on tumor cells and that indeed proliferation and migration of tumor cells could be influenced in an opposite manner by different types of macrophages.

Carcinoma↗
PubMed1992

Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia.

A point mutation in the POU-specific portion of the human gene that encodes the tissue-specific POU-domain transcription factor, Pit-1, results in hypopituitarism, with deficiencies of growth hormone, prolactin, and thyroid-stimulating hormone. In two unrelated Dutch families, a mutation in Pit-1 that altered an alanine in the first putative alpha helix of the POU-specific domain to proline was observed. This mutation generated a protein capable of binding to DNA response elements but unable to effectively activate its known target genes, growth hormone and prolactin. The phenotype of the affected individuals suggests that the mutant Pit-1 protein is competent to initiate other programs of gene activation required for normal proliferation of somatotrope, lactotrope, and thyrotrope cell types. Thus, a mutation in the POU-specific domain of Pit-1 has a selective effect on a subset of Pit-1 target genes.

Animals↗
PubMed1991

Testicular dysgenesis and mental retardation in two incompletely masculinized XY-siblings.

Aberrations of fetal sexual development were studied in two retarded phenotypic female 46,XY dysgonadal sisters from a consanguineous marriage. Endocrine evaluation revealed an inadequate response of plasma-testosterone to human chorionic gonadotropin (hCG) stimulation and a normal response to adrenocorticotropic hormone (ACTH) administration. At exploratory laparotomy dysgenetic testes and remnants of the Müllerian and of the Wolffian duct were found. Loss of testicular function, resulting in male pseudohermaphroditism (MPH), can occur at different times during intrauterine development, resulting in a variety of clinical manifestations. A thorough evaluation is warranted in all patients in order to reach a correct diagnosis which is of importance for appropriate gender assignment and genetic counseling.

Adolescent↗
PubMed1990

Molecular mechanisms of glial cell swelling in acidosis.

The mechanism of glial swelling in acidosis were investigated in an in vitro model. C6 glial cells were exposed to pH levels between 7.4 and 6.2. The cell volume response was determined by flow cytometry. Cell swelling of 110% to 115% was observed if pH was reduced to 6.8 or below. This effect was independent of the length of exposure to acidosis. Swelling induced by pH could be attenuated by (a) inhibition of the Na+/H+ antiporter by amiloride, (b) replacement of bicarbonate by HEPES, and (c) inhibition of carbonic anhydrase by acetazolamide. Absence of Na+ ions from the incubation medium completely prevented acidosis-induced glial swelling. Inhibition of the Cl-/HCO3- antiporter reduced swelling only in its first phase. The results suggest that glial swelling in acidosis may be from an activation of anion and cation antiporters as an attempt to maintain a normal intracellular pH. It is concluded that swelling of glial cells in the ischemic penumbra zone evolves along similar mechanisms.

Acetazolamide↗
PubMed1989

Transcripts of the insulin-like growth factors I and II in human hepatoma.

Using complementary DNAs of human insulin-like growth factors as probes, expressions of the insulin-like growth factors I and II mRNA were examined in seven human hepatoma tissues and their adjacent nontumorous livers. The level of insulin-like growth factor I mRNA in hepatoma was lower than that in the nontumorous liver control. This phenomenon was probably caused by the low expression of human growth hormone receptor in hepatoma tissues. The levels of insulin-like growth factor II mRNA vary among hepatomas. Some show elevated expression; some have diminished expression compared to their nontumorous liver counterparts. In four of the seven hepatomas, expression of fetal forms of insulin-like growth factor II transcripts was observed and may represent dedifferentiation of insulin-like growth factor II expression during hepatocarcinogenesis.

Carcinoma, Hepatocellular↗