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W Rombauts

Publications and source records attributed to W Rombauts.

At least 55 records · Page 3Linked to original sources

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin.

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.

Amino Acid Sequence↗

Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein.

We demonstrate that the 204 bp intronic gene fragment of C3(1), which has a specific in vitro affinity for the androgen receptor, is able to confer androgen responsiveness to a heterologous promoter. This characteristic is completely destroyed by a single G----T substitution, affecting a 5'-TGTTCT-3' element that closely resembles the consensus sequence of the glucocorticoid and progesterone response elements (GRE/PRE). In fact we could show that this androgen response element (ARE) also acts as a similarly weak GRE or PRE in T-47D cells.

Androgen-Binding Protein↗

Maternal and fetal endocrine pancreas in the spontaneously diabetic BB rat.

The maternal and fetal endocrine pancreas were investigated in the diabetic BB rat on day 21 of pregnancy. The maternal pancreas of the diabetic rat contained practically no insulin-positive B cells. The A and D cell mass were also decreased, while plasma glucagon and somatostatin levels were normal or increased, confirming previous data. Six of 11 diabetic rats had B-cell-specific surface antibodies (ICSA), whereas only 1 of 10 nondiabetic rats was ICSA-positive. The volume density of insulin-positive cells was decreased in the fetal pancreas of diabetic BB rats compared to fetuses of nondiabetic rats, but the volume density of glucagon- and somatostatin-positive cells remained normal. The B cells of these fetuses were ultrastructurally less granulated and showed signs of increased cellular activity. Plasma insulin levels were decreased while plasma glucagon and somatostatin concentrations were normal. ICSA were not detected in fetuses of nondiabetic and diabetic rats. There were no differences in the histology of the spleen and thymus between both groups of fetuses. Metabolic characterization of the growth-retarded fetuses of diabetic rats revealed, besides lower plasma insulin concentrations, increased branched chain amino acid levels, and normal plasma Sm/IGF-I levels. The main conclusions from this study are: (1) Severe maternal diabetes decreases the pancreatic insulin-positive cell mass and plasma insulin levels in the fetus, but not the A and D cell mass and function; (2) ICSA are not detectable in fetal plasma; (3) the influence of maternal BB rat diabetes on fetal endocrine pancreas and metabolic environment resembles that of severe streptozotocin-induced diabetes.

Animals↗

Plasma amino acids in diabetic pregnant rats and in their fetal and adult offspring.

Amino acid profiles and total amino-acid concentrations are established in nonfasting plasma of pregnant control, mildly diabetic and severely diabetic rats, and of their fetal and adult offspring. In pregnant rats at day 20 of gestation plasma amino acids can be regarded as normal in mildly diabetic mothers, but are significantly decreased in severely diabetic mothers. In fetuses of control rats, amino acid levels are twice as high as in the mother (fetomaternal ratio 2.0); in the fetuses of mildly diabetic mothers they are significantly lower than normal (fetomaternal ratio 1.3); in the fetuses of severely diabetic mothers they are also significantly lower than normal but with a normal fetomaternal ratio (fetomaternal ratio 2.0). In adult offspring of mildly diabetic mothers the concentration of almost all amino acids as well as that of total amino acid pool is significantly lower than in the controls; in the offspring of severely diabetic mothers they can be regarded as normal. No specific amino acid or group of amino acids can be held responsible for any of these changes, since all differences with control values display an overall effect, involving all or almost all amino acids.

Amino Acids↗

The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5' part and upstream region of the C1 and C2 genes and analysis of their transcripts.

The complete gene encoding the polypeptide C1 of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5' part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position -150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, C1 and C2A, the only obvious conserved structural element is the homopurine stretch located at position -400, although sequence motifs resembling steroid hormone response elements are present at several locations.

Androgen-Binding Protein↗

A single 12.5-kilobase androgen-regulated mRNA encoding multiple proline-rich polypeptides in the ventral prostate of the rat.

Synthetic 32P-labeled oligonucleotides have been used to identify the prostatic proline-rich polypeptide (PRP) mRNA which has partially been characterized. The 14-mer d(G-G-T-T-C-T-G-C-A-T-A-A-T-G) complementary to the coding sequence for His-Tyr-Ala-Glu-Pro, a sequence element occurring in all 38-residue PRP variants, hybridizes specifically with a 12.5-kilobase mRNA which is clearly androgen-controlled. This oligonucleotide was used as an efficient primer for the construction of a PRP-specific lambda gt10 cDNA library. The nucleotide sequence of the inserts from several recombinant clones has been determined. This structural analysis revealed a PRP mRNA encoding a large precursor containing a number of tandemly repeated units. Each repeat codes for a sequence of 100 amino acids in which the highly conserved PRP sequence is embedded. From this polyprotein the large number of PRP variants must be generated by a post-translational processing mechanism which is still unknown. The high degree of conservation of both nucleotide and amino acid sequence in the entire unit also indicates that the PRP gene(s) likely evolved by multiplication of a 300-base pair ancestral DNA sequence. This has resulted in a noninterrupted repetitive DNA coding segment which is detected at the genomic level.

Amino Acid Sequence↗

Mapping of rat prostatic binding protein genes C1, C2, and C3 to rat chromosome 5 by in situ hybridization.

Rat prostatic binding protein genes C1, C2, and C3 were mapped on rat chromosome 5 by in situ hybridization on rat peripheral blood chromosome preparations using three different cDNA probes. Of the grains detected, 15.9%, 25.2%, and 19.6%, respectively, mapped to chromosome 5. For each probe, the label was predominantly located on 5q31, but for C2 and C3 an additional site on 5q21 was found. The results suggest that three genes coding for the different polypeptide chains of rat prostatic binding protein map to the same chromosome and presumably to the same chromosome band.

Androgen-Binding Protein↗

Comparison of the 5' upstream putative regulatory sequences of three members of the alpha 2u-globulin gene family.

We have isolated and characterized seven members of the alpha 2u-globulin gene family from a rat genomic library. The 5' upstream region (up to 1250 base pairs starting from the EcoRI site in exon 2) of three clones was sequenced. The major transcriptional start points were located 25 base pairs downstream from the 'TATA' box. A very high degree of homology was observed over the entire studied region. Two of the examined genes displayed structural features which suggest that their expression may be impeded. A high degree of homology was observed between the promotor regions of alpha 2u-globulin and those of the major urinary protein (MUP) multigene family of the mouse. A remarkable feature is the variable length of an A-rich region between the putative 'CAAT' and the 'TATA' consensus sequences. The size of this region differs markedly between MUP and alpha 2u-globulin and between different members of the alpha 2u-globulin gene family. Comparison of the alpha 2u-globulin promotor with the corresponding region of other androgen-dependent genes (the C1, C2 and C3 subunits of prostatic steroid binding protein) reveals the presence of an A-rich region of homology located approximately 378 base pairs upstream from the cap site in the alpha 2u-globulin genes. This region compares well with a sequence of putative enhancer function previously demonstrated in the alpha-fetoprotein promotor and in the immunoglobulin heavy chain promoter.

Alpha-Globulins↗

Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions.

The complete sequence (2879 bp) of the androgen-controlled rat prostatic binding protein C2 gene and 1023 bp of the 5'- and 2127 bp of the 3'-flanking regions have been determined. The gene contains three exons (93, 203 and 147 bp) and two introns (1630 and 806 bp). It is flanked by two homopurine-homopyrimidine stretches of 55 and 131 nucleotides respectively, located at positions -405 and 4151. These sequences are remarkably sensitive towards S1-nuclease, indicating an altered DNA conformation under superhelical stress. Several palindromes and dyad structures are observed in the 5'-upstream region of the gene and at position -457, and 80% homology to the consensus sequence of a glucocorticoid receptor binding site is found.

Amino Acid Sequence↗

Determination of areal densities of blood vessel wall components in histological sections by means of image analysis.

A technique is described which allows quantification of specifically stained, intermingled vessel wall components in paraffin-embedded tissue. The dissociation of elastic, muscular and connective tissue and the determination of their areal densities in the superior vena cava and in the ascending aorta of the dog, was performed by automated image analysis. The percentages of elastic+connective tissue, determined by this technique, correlated significantly with the data derived from biochemical measurements. It was further found that the structure of the vena cava wall was a function of the distance from the right atrium.

Animals↗

Influence of castration and androgen treatment on the synthesis of prostatic binding protein and the concentration of its mRNA in the rat ventral prostate.

The regulation by androgens of the synthesis of prostatic binding protein (PBP), an abundant secretory protein of the rat ventral prostate, was studied by measuring the rate of incorporation of [3H]leucine into total protein and into PBP by prostatic tissue in vitro. PBP-specific synthesis dropped to 30% of the intact level after 3 days and to 5.5% after 7 days. Administration of androgens to 7 day-castrated rats reversed these changes: 36% of the control value was reached after 3 days of treatment and 70% after 5 days. The mRNAs of PBP were measured under similar conditions by means of dot hybridization, using C1-, C2- and C3-specific cDNA-clones. The 3 mRNAs followed a parallel course. They decreased to about 18% on the third day after castration and to about 2% after 7 days. Androgen treatment of 7-day castrated rats produced a small increase of these levels after 1 day. After 3 days 15% of the intact level was reached. The general similarity of the time course of PBP synthesis and PBP mRNA levels in castrated and androgen treated rats supports a transcriptional regulation mechanism, although there also is some evidence for an influence on protein synthesis in general.

Androgen-Binding Protein↗

Correlation between mechanical properties and wall composition of the canine superior vena cava.

The mechanical properties (modulus of elasticity and stress-relaxation) of different venous segments of the canine superior vena cava were determined as well as the composition of the vessel wall by means of physical, biochemical and histological methods. It was found that the wall of the vena cava was structurally and mechanically a function of the metric position with respect to the right heart: the modulus of elasticity increased, the stress-relaxation decreased, the concentration of hydroxyproline, collagen and elastin increased and the amount of muscle fibres decreased with increasing distal distance from the right heart. A significant linear correlation coefficient was observed between the modulus of elasticity and the structural wall components. The data presented show the axial heterogeneity and the dependency of the mechanical properties upon the venous vessel wall composition.

Animals↗

Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli.

cDNA clones of the mRNA coding for tissue-type plasminogen activator (t-PA) have been obtained and their nucleotide sequences compared to those reported previously. A gene coding for t-PA has been reconstructed and inserted into vectors for expression in prokaryotic cells. Relatively high levels of t-PA accumulated in inclusion bodies in Escherichia coli containing an optimized expression plasmid, but only a small proportion of the insoluble protein was recovered as active enzyme using a variety of solubilization procedures.

Amino Acid Sequence↗

Proline-rich polypeptides bound to rat prostatic binding protein. The primary structure of the two main components, proline-rich polypeptides IV and V.

The complete primary structures of the two main forms, PRP-IV and PRP-V, of a proline-rich polypeptide bound in vivo to rat prostatic binding protein has been determined. Their sequences were established using manual Edman degradation of the native polypeptide and of purified fragments derived from trypsin and thermolysin digestions. Both polypeptides contain 38 amino acid residues (Mr = 4397 and 4339); cysteine, methionine, and serine are missing. In spite of the high proline content (21%), no polyproline stretches were detected. PRP-IV and PRP-V show an extensive structural homology and differ only by three substitutions. These amino acid replacements are located in the NH2-terminal part of the molecule at positions 6 (His leads to Pro), 10 (Pro leads to His), and 11 (Asp leads to Gly). Moreover, each component displays a microheterogeneity at several positions in the sequence which indicates that multiple structural variants exist for PRP-IV and PRP-V. These data not only suggest the existence in rat ventral prostate of a multigene family coding for the proline-rich polypeptides but also the occurrence of a pronounced genetic polymorphism for these components. In addition, a remarkable sequence homology is observed between the PRP components and the region of the B chain in the precursor of mouse renin.

Amino Acid Sequence↗

Different forms of alpha 2u-globulin in male and female rat urine.

alpha 2u-Globulin is usually considered to be present only in male rat urine. This study demonstrates that a very similar protein exists in female rat urine and compares its properties with those of the male form. Isoelectric focusing followed by immunofixation reveals considerable microheterogeneity of alpha 2u-globulin in male and female rat urine. Important sex differences are noted in the banding pattern. The isoelectric point of the major male component (pI approximately equal to 5.3) is considerably higher than that of the major female components (pI approximately equal to 4.6). In addition, the female form of alpha 2u-globulin has a somewhat higher mobility on sodium dodecyl sulphate/polyacrylamide gel electrophoresis than its male counterpart. These sex differences are preserved after purification of alpha 2u-globulin from male and female rat urine by affinity chromatography and enrichment of the major male and female components by ion-exchange chromatography. Immunologically no differences are observed between these purified components and their amino acid composition reveals only minor differences. A slightly higher carbohydrate content is observed in the major female component than in the major male component. Finally evidence is presented that oestrogen treatment suppresses the male forms of alpha 2u-globulin but has no effect on the female forms. The observed differences between the male and female forms and their different hormonal control suggest that they are encoded by different genes.

Alpha-Globulins↗

The nucleotide sequence of cDNA complementary to the C1 component of rat prostatic binding protein.

The mRNA for component C1 of rat prostatic binding protein has been cloned and characterized. A partially purified mRNA fraction for this complex protein was reverse-transcribed into double-stranded cDNA and cloned into the PstI site of plasmid pBR 322. The 426-base-pair insert of the recombinant plasmid pC1A75 was completely sequenced. The coding region corresponds precisely to the 88 amino acid residues of C1 and in addition contains the information of a signal peptide of 23 residues. The 5' non-coding region counts only 19 nucleotides and is incomplete but the 3'-terminal non-coding part of 60 nucleotides extends into the poly(A) tail. Sequence analysis of other C1-positive clones indicates the presence of sequence rearrangements which must have occurred during the cloning procedure. Possible mechanisms for the generation of these cloning artefacts are discussed.

Androgen-Binding Protein↗

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S.

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.

Amino Acid Sequence↗