Biomedical subjects

H J Dubbink

Publications and source records attributed to H J Dubbink.

7 recordsLinked to original sources

PubMed1999

An Sp1 binding site is essential for basal activity of the human prostate-specific transglutaminase gene (TGM4) promoter.

Human prostate-specific transglutaminase (hTG(P)) is a cross-linking enzyme encoded by the TGM4 gene. The TGM4 gene promoter was characterized by deletion mapping and mutational analysis. Promoter constructs, containing the minimal promoter requirements, could efficiently drive transcription in the prostate cancer cell lines PC346C and LNCaP and the hepatic cancer cell line Hep3B. The region between positions -113 and -61 was demonstrated to be essential for core promoter activity. Further analysis revealed the functional importance of an Sp1 binding motif, 5'-ACCCCGCCCC-3', at positions -96 to -87. This sequence is a binding site of the ubiquitous transcription factors Sp1 and Sp3.

Base Sequence↗

PubMed1999

Human prostate-specific transglutaminase: a new prostatic marker with a unique distribution pattern.

Human prostate-specific transglutaminase (hTGp) is a cross-linking enzyme, the physiologic function of which has not been established unequivocally yet. To gain insight into its distribution, we raised antisera against hTGp. By using Western blotting analysis, we found that these antisera specifically recognize a 77-kDa protein in prostatic fluids, seminal plasmas, and prostatic tissues. The concentrations of hTGp in these fluids and tissues were found to be highly variable among individuals. Immunohistochemical examination of several formalin-fixed paraffin-embedded human tissues revealed an exclusive expression in the prostate. The histologic localization and distribution of hTGp within the prostate was assessed by studying multiple sections from tumor-containing prostatectomy specimens and needle biopsies. hTGp expression was entirely restricted to luminal epithelial cells. No basal epithelial cells or stromal cells were stained. Within the prostate, large areas without any hTGp-positive cells were seen. Immunopositive cells were present either in a scattered pattern or concentrated in single or multiple glands in which all luminal epithelial cells expressed hTGp. The latter staining pattern occurred frequently, but not exclusively, in the peripheral zone, whereas scattered expression was most often observed in the transitional zone. Expression of the hTGp protein could occasionally be observed in high-grade prostatic intraepithelial neoplasia, but was not detected in prostate carcinoma cells. The expression pattern as observed for hTGp has not been found thus far for any other prostate-specific marker.

Androgen-Binding Protein↗

PubMed1998

The human prostate-specific transglutaminase gene (TGM4): genomic organization, tissue-specific expression, and promoter characterization.

Human prostate-specific transglutaminase (hTGP) is a cross-linking enzyme secreted by the prostate. In this study, we performed dot blot analysis of 50 normal human tissues to demonstrate unambiguously the prostate-specific expression of hTGP. Furthermore, we elucidated the genomic organization of the TGM4 gene, the gene encoding hTGP. The structure of this gene displays striking similarity to that of other transglutaminase (TGase) genes. The TGM4 gene spans approximately 35 kb of genomic DNA and consists of 13 exons and 12 introns. The main transcription initiation site is located 52 bp upstream of the translational start codon. A hTGP splice variant of intron 1 was detected. This splice variant contains an in-frame antisense Alu element insertion. The TGM4 promoter was analyzed by sequencing and transfection experiments. At positions -1276 to -563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA. Deletion mapping of the TGM4 promoter in the transiently transfected human prostate cancer cell line PC346C showed comparable activity of 2.1-, 1.5-, and 0.5-kb promoter fragments.

Amino Acid Sequence↗

PubMed1998

Expression of prostatic factors measured by reverse transcription polymerase chain reaction in human papillomavirus type 18 deoxyribonucleic acid immortalized prostate cell lines.

OBJECTIVES: To investigate expression of the prostatic markers prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM), and the androgen receptor (AR) after human papillomavirus (HPV) type 18 deoxyribonucleic acid (DNA) transfection and subsequent immortalization of human prostate epithelial cells. METHODS: Recently, two human prostate epithelial cell lines were established by HPV transformation: PZ-HPV-7, derived from normal peripheral zone (PZ) tissue, and CA-HPV-10, derived from high Gleason grade adenocarcinoma. Expression of PSA was studied by the reverse transcription polymerase chain reaction (RT-PCR), because in preliminary studies using immunocytochemistry and Northern blotting, no PSA expression was found. PSM was analyzed by RT-PCR and nested RT-PCR. These analyses included primary human prostate cell strains. Furthermore, androgen-supplemented methylthiazol tetrazolium (MTT) growth assays were performed and expression of AR was studied by immunocytochemistry. Prostate carcinoma cell lines LNCaP and PC-346C were included as positive controls and breast carcinoma cell line MCF-7 as a negative control. RESULTS: Both cell lines exhibited low levels of RNA for PSA and PSM in comparison with cell lines LNCaP and PC-346C. AR expression by immunocytochemistry was negative using monoclonal antibody F39.4 and polyclonal antibody SP-197. In an androgen-supplemented environment, growth rates of both HPV immortalized cell lines were not stimulated in contrast to LNCaP. CONCLUSIONS: RNA transcripts of PSA and PSM were detected by RT-PCR in HPV immortalized prostate epithelial cell lines PZ-HPV-7 and CA-HPV-10. The expression of prostate-specific markers may further validate the utility of this stepwise transformation model of human prostate carcinogenesis.

Antigens, Surface↗

PubMed1996

The aroA gene of Campylobacter jejuni.

The gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aroA) cloned from Campylobacter jejuni (Cj) strain 81116 was identified by complementation of an Escherichia coli (Ec) auxotrophic aroA mutant. The Cj aroA gene has been sequenced. It encodes an enzyme of 428 amino acids (aa), that is homologous to other bacterial EPSP synthases, especially that of Bacillus subtilis with which it has a 39% aa identity. The transcriptional start point was mapped. It is present in an upstream open reading frame (ORF) that has a strong homology to the gene encoding phenylalanine tRNA synthetase (pheS). Downstream from aroA another ORF is present which is homologous to the lytB gene of Ec. The stop codon of the aroA gene overlaps the start codon of lytB.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Tissue specific and androgen-regulated expression of human prostate-specific transglutaminase.

Transglutaminases (TGases) are calcium-dependent enzymes catalysing the post-translational cross-linking of proteins. In the prostate at least two TGases are present, the ubiquitously expressed tissue-type TGase (TGC), and a prostate-restricted TGase (TGP). This paper deals with the molecular cloning and characterization of the cDNA encoding the human prostate TGase (hTGP). For this purpose we have screened a human prostate cDNA library with a probe from the active-site region of TGC. The largest isolated cDNA contained an open reading frame encoding a protein of 684 amino acids with a predicted molecular mass of 77 kDa as confirmed by in vitro transcription-translation and subsequent SDS/PAGE. The hTGP gene was tissue-specifically expressed in the prostate, yielding an mRNA of approx. 3.5 kb. Furthermore, a 3-fold androgen-induced upregulation of hTGP mRNA expression has been demonstrated in the recently developed human prostate cancer cell line, PC346C. Other well established human prostate cancer cell lines, LNCaP and PC-3, showed no detectable hTGP mRNA expression on a Northern bolt. The gene coding for prostate TGase was assigned to chromosome 3.

Amino Acid Sequence↗

PubMed1993

Single point mutations in domain II of the yeast mitochondrial release factor mRF-1 affect ribosome binding.

We have recently described two yeast strains that are mutated in the MRF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational termination. In the present study we report the cloning, sequencing, as well as an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located one amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartment. Over-expression of the mutant alleles in wild type and mrf1 mutant yeast strains produces a phenotype consistent with a reduced affinity of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E. coli small ribosomal protein S4 can be reversed by a slight over-expression of the MRF1 gene.

Alleles↗