Biomedical subjects

B S Wulff

Publications and source records attributed to B S Wulff.

26 records · Page 2Linked to original sources

PubMed1994

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization.

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22-79, PHM, prepro-VIP 111-122, VIP and prepro-VIP 156-170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single- or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.

Adult↗

PubMed1994

Characterization of the effects of retinoic acid on vasoactive intestinal polypeptide gene expression in neuroblastoma cells.

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.

Aspartic Acid Endopeptidases↗

PubMed1993

Gene expression of insulin-like growth factor II in human intracranial meningioma.

BACKGROUND: Insulin-like growth factor II (IGF-II) is synthesized in the normal brain of adult humans predominantly in the choroid plexus and meninges and is secreted in the cerebrospinal fluid. The authors measured IGF-II transcripts and peptides in biopsy specimens from human intracranial tumors including astrocytomas, glioblastomas, and meningiomas. METHODS: The presence of IGF-II mRNA was analyzed in 12 human brain tumors by Northern analysis of total RNA extracted from tumor biopsies and by in situ hybridization of tissue sections. The amount of immunoreactive IGF-II was determined by radioimmunoassay of tumor extracts. RESULTS: Northern analysis of RNA from four meningiomas showed IGF-II mRNA of 6.0, 4.8, and 2.2 kb, and in situ hybridization revealed that meningioma tumor cells contained IGF-II mRNA. In contrast, biopsy specimens from four astrocytomas, one oligoastrocytoma, and four glioblastomas showed no IGF-II mRNA. Radioimmunoassay of IGF-II in tumor extracts showed that all tumors contained IGF-II (40-160 ng/g tissue). Two meningiomas contained the highest amounts of IGF-II (144 and 160 ng/g tissue). CONCLUSIONS: IGF-II mRNA is present in higher amounts in benign meningiomas than in malignant glioblastomas and astrocytomas, whereas the content of immunoreactive IGF-II is similar. On the basis of these findings, the authors believe that IGF-II may be involved in growth regulation of meningiomas.

Adult↗

PubMed1993

Structure-function relationship of the insulin-like growth factor-I receptor tyrosine kinase.

Insulin-like growth factor I (IGF-I) and insulin receptors are structurally similar with ligand-stimulated tyrosine kinase activity in their cytoplasmic domains. The function of the insulin receptor tyrosine kinase in signal transduction has been studied extensively in contrast to the IGF-I receptor tyrosine kinase. In the present study we have analyzed the regulatory function of the IGF-I receptor tyrosine kinase and carboxyl-terminal domains in mitogenic signaling by overexpression of mutant IGF-I receptors in mouse NIH-3T3 fibroblasts. A mutant IGF-I receptor, in which 3 tyrosines (Tyr1131, Tyr1135, and Tyr1136) analogous to the three major autophosphorylation sites in the insulin receptor kinase were replaced by phenylalanines, was devoid of kinase activity in vivo and in vitro and inactive with respect to IGF-I internalization and stimulation of thymidine incorporation. Another mutant IGF-I receptor, which lacks the 49 carboxyl-terminal amino acids (residues 1289-1337) of the beta-subunit, was fully active. Our data suggest that the structure-function relationship of the IGF-I receptor tyrosine kinase activation and signal transduction is similar to that of the insulin receptor.

3T3 Cells↗

PubMed1993

Processing of two homologous precursors, pro-neuropeptide Y and pro-pancreatic polypeptide, in transfected cell lines expressing different precursor convertases.

The processing of two homologous precursors, pro-neuropeptide Y (pro-NPY) and pro-pancreatic poly-peptide (pro-PP), was studied in four neuroendocrine cell lines after transfection: CA-77 medullary thyroid carcinoma cells, AtT-20 corticotrope pituitary cells, RIN2A-19 pancreatic endocrine cells, and NB1 neuroblastoma cells. Northern blot analysis indicated that the AtT-20 cells only expressed precursor convertase 3; in contrast, NB1 cells only expressed precursor convertase 2, whereas the RIN2A-19 and CA-77 cells expressed both enzymes. Despite these differences in expression pattern of precursor convertases the four cell lines were, surprisingly, indistinguishable in respect to their processing of pro-PP and pro-NPY. In all four cell lines, pro-NPY was almost completely converted to NPY, and, in all four cell lines, only around 50% of the PP precursor was converted to PP. The relatively poor processing efficiency of pro-PP was rather similar to the processing efficiency of the endogenously produced precursors in the respective cell lines, pro-calcitonin (CA-77), proopiomelanocortin (AtT-20), proinsulin (RIN2A-19), and pro-vasoactive intestinal polypeptide (NB1). At least in the CA-77 cells, NPY and PP were apparently sorted to the regulated secretory pathway, as upon stimulation with secretagogue the release of the transfected peptides increased in parallel with the endogenously expressed peptide, calcitonin gene-related peptide. Mutagenesis studies showed that on the N-terminal side of the di-basic processing site, the otherwise important difference in structure between PP and NPY, a proline for glutamine in position 34, was not responsible for the difference in processing efficiency. On the C-terminal side of the processing site, the efficient processing of pro-NPY could not be transferred to pro-PP by exchanging the whole C-terminal domains of the precursors. It is concluded that pro-NPY is processed more efficiently than pro-PP in all neuroendocrine cell lines tested independent on their expression of the two main precursor convertases and that mutagenesis data indicate that the structural element responsible for the efficient processing of pro-NPY is not located on the N-terminal side of the dibasic processing site.

Amino Acid Sequence↗

PubMed1993

Efficient amidation of C-peptide deleted NPY precursors by non-endocrine cells is affected by the presence of Lys-Arg at the C-terminus.

Post-translational processing of peptide precursors producing amidated, biologically active peptides generally occurs in specially differentiated endocrine or neural cells. However, we have previously shown that a C-peptide-deleted precursor of neuropeptide Y (NPY1-39) in which the precursor terminates in the sequence Gly-Lys-Arg was partially amidated by the non-endocrine cell line, CHO. In the present study we show that two other non-endocrine cell lines, NIH 3T3 and BHK, also possess amidating activities and that the NPY1-39 precursor was completely converted to NPY1-36 amide by the NIH 3T3 cell line. The role of the two basic residues (Lys-Arg) in the C-terminus was studied by transfection of a construct encoding a NPY precursor terminating with glycine alone. Both the CHO and NIH 3T3 cell lines, transfected with this construct, secreted a significantly smaller fraction of NPY reactive material as amidated NPY compared to the fraction of amidated NPY secreted by the cells transfected with the NPY1-39 precursor. It is concluded that the capacity to perform C-terminal amidation appears to be a universal feature of eukaryotic cells and that the carboxypeptidase E-like enzyme influences the amidation process, beyond its known ability to remove the C-terminal basic residues.

3T3 Cells↗

PubMed1991

CHO cells synthesize amidated neuropeptide Y from a C-peptide deleted form of the precursor.

Post-translational processing of peptide precursors producing amidated, biologically active peptides is generally believed to occur only in specially differentiated endocrine or neural cells. Previously it has been shown that endoproteolytic processing of peptide precursors is very inefficient in non-endocrine cells like CHO cells. We have studied the processing of a C-peptide-deleted precursor of neuropeptide Y (NPY) in which the precursor terminates in the sequence Gly-Lys-Arg and does not require any dibasic specific endoproteolytic processing. Following transfection of CHO cells with an expression plasmid encoding this mutated NPY precursor, between 50 and 80 percent of the synthesized NPY was secreted from stable transfectants as authentic amidated NPY as assessed by both a C-terminal amide specific radioimmunoassay and by isoelectric focusing. It is concluded that amidated peptides can be produced in non-endocrine cells provided they are presented with a precursor which does not have to be endoproteolytically processed.

Amino Acid Sequence↗

PubMed1990

Partial processing of the neuropeptide Y precursor in transfected CHO cells.

The activation of regulatory peptides by post-translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non-neuroendocrine cell line, which were transfected with a eukaryotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro-NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPY immunoreactivity was even correctly amidated. Extracellular degradation of pro-NPY in the tissue culture medium generated immunoreactivity which corresponded in size to NPY. It is concluded that precursor processing can occur in non-neuroendocrine cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture medium.

Animals↗