Biomedical subjects

G Heinrich

Publications and source records attributed to G Heinrich.

At least 19 recordsLinked to original sources

PubMed1995

Prolonged action of a chimeric interleukin-2 receptor (CD25) monoclonal antibody used in cadaveric renal transplantation.

A high affinity chimeric CD25 mAb (chRFT5: SDZ CHI 621) blocking interleukin-2 binding to the interleukin-2 receptor alpha-chain was evaluated in a phase I/II study in human renal cadaveric transplantation. The chRFT5 was well tolerated with no immediate adverse effects during 6 spaced infusions (from before transplantation to day 24) in 24 patients escalating from 2.5- to 25-mg dosages. The chRFT5 had a long terminal half-life with a mean of 13.1 days. There was good correlation between the detection of chRFT5 in the serum by radioimmunoassay, the coating and suppression of CD25 on T cells, and antibody activity in patient serum samples. The chRFT5 activity persisted in vivo for up to 120 days. No antibody response to the chRFT5 was detected in any of the patients, even though two patients who required treatment with antithymocyte globulin or OKT3 developed xenogeneic antiglobulin responses while chRFT5 was still present in vivo. There was a 33% incidence of rejection and the first rejection episode always occurred during chRFT5 therapy. Patients who did not reject during therapy did not reject during the first year following transplantation. Equal numbers of patients received dual and triple immunosuppressive therapy together with chRFT5. Posttransplant lymphoproliferative disorder developed in 2 patients, both on triple therapy, at 9 months after transplantation. The disorder did not develop in any patient receiving dual therapy, and no further cases have been observed to a minimum of 2 years' follow-up. No other viral, fungal, or bacterial infectious complications were prevalent in patients treated with chRFT5.

Adult↗

PubMed1995

The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis.

The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors.

Animals↗

PubMed1995

Five Trk receptors in the zebrafish.

Using a homology-based cloning strategy we have identified five members of the Trk family in the zebrafish Danio rerio. They are homologous to the three mammalian Trk receptors in their conserved intracellular kinase regions and the organization of their extracellular regions. The five trk genes are differentially expressed in the developing brain, spinal cord, cranial ganglia, and retina. Full-length forms of three of the trk genes are expressed when neurons pioneer the major axon tracts, whereas the two other trk genes have a later onset of expression. Truncated transcripts and forms containing an extracellular juxtamembrane region insert were found. The degree of sequence variation and expression differences within the family suggest that each of the five zebrafish Trk receptors have a functionally distinct role. These findings demonstrate that the vertebrate Trk family is larger than previously appreciated.

Amino Acid Sequence↗

PubMed1995

Cultured human fibroblasts and not cultured human keratinocytes express a PTH/PTHrP receptor mRNA.

There is increasing evidence that parathyroid hormone (PTH) and PTH-related peptides (PTHrP) are involved in normal skin cell growth; therefore, we investigated whether the PTH/PTHrP receptor was expressed in cultured human keratinocytes and dermal fibroblasts. Northern analyses of poly (A)+ RNA isolated from cultured fibroblasts revealed two PTH/PTHrP receptor transcripts with one major band at 2.5 kb and one minor band at 2.3 kb. These transcripts were consistent with those found in human osteosarcoma cells, which are known to express PTH/PTHrP-R mRNAs. In contrast, after repeated Northern analyses no PTH/PTHrP receptor transcripts were found in poly (A)+ RNA isolated from cultured keratinocytes. Reverse-transcriptase/nested polymerase chain reaction analyses of total RNA isolated from cultured keratinocytes and fibroblasts confirmed the Northern analyses data that the PTH/PTHrP receptor was expressed in cultured fibroblasts but not in cultured keratinocytes. When cultured fibroblasts and keratinocytes were exposed to 10(-7) M PTH (1-34) there was a twofold increase in cAMP levels in the fibroblasts and no demonstrable increase was noted in keratinocytes. These results suggest that skin fibroblasts possess the classical PTH/PTHrP receptor and are target cells for PTH and PTHrP whereas keratinocytes do not have the receptor and are unresponsive to its N-terminal agonist in the stimulation of cAMP formation.

Base Sequence↗

PubMed1995

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.

Base Sequence↗

PubMed1994

Ozone formation in the greater Cairo area.

Ozone formation in the greater Cairo area was studied in 1990 in a 3-week measurement period performed at three sites (Shoubra El-Kheima, Mokattam Hill, Helwan), covering a north-south direction of 27 km, and in 1991, from the beginning of April until the end of October, by measurement of the seasonal variation of ozone at one site at El-Kobba. The sinusoidal shape in the diurnal volume fraction plots with peak values of 120 ppb and daily mean value of 50 ppb throughout the year indicate a substantial contribution of photochemistry to the ozone content of the atmosphere. Ozone is produced predominantly over the industrial area in the north and in the centre of Cairo and transported southward by the prevailing northerly winds. Contrary to many urban areas in Europe and in North America, fairly high average ozone levels of 40 ppb are observed during the night throughout the spring and the summer. This may imply that health hazards and crop damage are higher in the greater Cairo area than in Central Europe.

Air↗

PubMed1994

The development of neurotrophin receptor Trk immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The purpose of this study was to examine the cellular distribution of the Trk family of neurotrophin receptors in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Semithin sections from zebrafish retinae were examined immunohistochemically for the presence of Trk polypeptides using commercially available antisera that cross-react with the fish. Cross-reactivity was confirmed by Western blot. Trk polypeptides were detected at about 1 day of age on the surfaces of retinal neuroblasts and faint Trk immunoreactivity was observed in the primordial optic nerve at 1.5 days. By 2 days the optic nerve was clearly positive for Trk and at 2.5 days Trk immunoreactivity was found in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers, as well as in the optic nerve. At 3 days and 4 days the location of Trk immunoreactivity was unchanged but by 4 days it had diminished in intensity. In the adult zebrafish retina Trk immunoreactivity was found in the same locations as in the embryonic fish, as well as in a population of cells in the middle of the inner nuclear layer and in photoreceptors. We conclude that Trk neurotrophin receptors are present in the zebrafish eye during development and that their persistence in the adult may support the continuous neural reorganization that accompanies the growth of the eye in the fish.

Animals↗

PubMed1994

The development of GABA immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The goal of this study was to determine the pattern of gamma-aminobutyric acid (GABA) expression in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Zebrafish embryos were fixed at intervals between 1 and 4 days postfertilization, and semithin plastic sections were prepared for postembedding immunocytochemistry with antisera against GABA. Sections were also prepared from several adult zebrafish eyes for comparison. GABA immunoreactivity first appeared in the optic nerve at 2 days postfertilization, and by 2.5 days the inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer, and optic nerve were all positive for GABA. The GABA expression in the retinal ganglion cell layer and optic nerve was transient, however, and these structures were largely unlabeled by 4 days postfertilization. The pattern of GABA immunoreactivity at 4 days resembled that seen in the adult zebrafish: A large population of presumptive amacrine cells was labeled at the base of the INL, and the IPL was positive for GABA, as were occasional cells in the ganglion cell layer. Horizontal cells, particularly at the retinal margins, were also GABA positive beginning at about 3 days postfertilization. The transient expression of GABA in retinal ganglion cells and their axons during the period when synaptic contacts are being established both within the retina and between the retina and central targets suggests that GABA may have a role in the development of this system, in addition to serving as a classical neurotransmitter.

Animals↗

PubMed1994

Expression of neurotrophin genes in human fibroblasts: differential regulation of the brain-derived neurotrophic factor gene.

Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. We previously reported that BDNF and NGF gene expression are differentially regulated in mouse L929 fibroblasts. Here we examine expression of these three neurotrophins in human fibroblasts. Northern blots detected BDNF and NT-3 mRNAs in fibroblasts derived from lung (WI-38), calvarium and foreskin. WI-38 cells and foreskin fibroblasts expressed 1.6 kb as well as 4 kb BDNF mRNAs whereas only the smaller BDNF mRNA was detected in calvarium fibroblasts. NGF mRNA was present in foreskin and calvarium but not lung fibroblasts. In WI-38 cells serum treatment increased levels of BDNF mRNA within 2 hr. Cycloheximide did not inhibit the increase. Treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) transiently suppressed BDNF mRNA. Treatment with both serum and TPA first stimulated and then transiently suppressed BDNF mRNA. TPA and/or serum did not significantly affect BDNF mRNA in calvarium fibroblasts. These results show that human fibroblasts derived from different tissues express and regulate neurotrophin genes differentially.

Blotting, Northern↗

PubMed1994

The fates of the blastomeres of the 16-cell zebrafish embryo.

We present a fate map for the 16-cell-stage blastomeres of the zebrafish embryo Brachydanio rerio. We injected high molecular weight fluorescent dextran into cleavage-stage cells to observe the contributions of the descendants of the first 16 cells to the adult. The patterns derived from these early cells are similar, but not identical among different embryos. Furthermore, two-color injections showed that sister blastomeres at the 16-cell stage regularly contribute to different sets of adult structures. A few of the scored tissues could not be mapped in a manner consistent with the predicted axes. Other tissues were mapped to several of the 16 blastomeres. Many of the tissues map with high probability to a few 16-cell-stage blastomeres. Thus, based on 112 injections in 56 different embryos, we have constructed a fate map by assigning probabilities for the contribution of each blastomere to each of 31 tissues in the 26-hour embryo.

Animals↗

PubMed1994

Expression of 25-hydroxyvitamin D3-24-hydroxylase mRNA in cultured human keratinocytes.

It is well documented that 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3), the most active vitamin D metabolite, inhibits epidermal keratinocyte proliferation and promotes differentiation. 1 alpha,25(OH)2D3 can be produced in keratinocytes from 25-hydroxyvitamin D3 by the enzyme 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase). Hydroxylation of 1 alpha,25(OH)2D3 by 25-hydroxyvitamin D3-24-hydroxylase (24-OHase), the first step in the catabolic pathway of 1 alpha,25(OH)2D3 could significantly reduce the intracellular concentration of 1 alpha,25(OH)2D3. Therefore, the expression of 24-OHase could have a critical regulatory role in 1 alpha,25(OH)2D3-dependent gene expression. As a first step to examine this possibility, the steady state level of 24-OHase mRNA in cultured human keratinocytes (CHK) was investigated. 24-OHase mRNA was not detected in control CHK. 1 alpha,25(OH)2D3 caused a dose- and time-dependent increase in 24-OHase mRNA level. The highest accumulation of 24-OHase mRNA was observed in CHK treated with 0.1-1 microM 1 alpha,25(OH)2D3. The level of 24-OHase mRNA reached a plateau 12-24 hr after 1 alpha,25(OH)2D3 treatment. 1 beta,25-dihydroxyvitamin D3, the stereoisomer of 1 alpha,25(OH)2D3, failed to induce 24-OHase mRNA expression significantly. In addition to 24-OHase mRNA, a 1.0-kb mRNA hybridized strongly with both rat and human 24-OHase cDNA probes. The origin of this 1.0-kb message is unknown at present, however, it was regulated by 1 alpha,25(OH)2D3. These results demonstrate that 1 alpha,25(OH)2D3 up-regulates the expression of 24-OHase mRNA, and this may be an important first step in the initiation of catabolism of 1 alpha,25(OH)2D3 in human keratinocytes.

Calcitriol↗

PubMed1993

Cloning and characterization of the gene encoding murine insulin-like growth factor-binding protein-2, mIGFBP-2.

We present a characterization of the single-copy gene, mIGFBP-2, encoding the murine insulin-like growth factor-binding protein-2 (mIGFBP-2). It consists of four exons with sizes of 470 +/- 2, 227, 141 and > 475 nucleotides (nt). The first intron spans 23 kb of genomic sequence, and the complete gene extends to more than 28 kb. Two kb of the 5'-flanking region were sequenced. This region has no TATA or CAAT boxes but is G+G-rich and contains several potential regulatory sequence motifs. A total of five GC boxes, which may serve as potential binding sites for a transcription factor, Sp1, are present immediately upstream of the transcription start point (tsp). By primer extension, we identified a single tsp at nt position -85 +/- 2. The murine IGFBP-2 locus was mapped to the proximal region of mouse chromosome 1, to a region of conserved synteny with human chromosome 2q. A comparison of the deduced amino acid sequences of mouse, rat and human IGFBP-2 reveals a high degree of homology between all three species.

Animals↗

PubMed1992

Differential regulation of the nerve growth factor and brain-derived neurotrophic factor genes in L929 mouse fibroblasts.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are structurally related survival and differentiation factors for distinct sets of peripheral and central neurons. The regulation of NGF gene expression has been extensively studied in L929 mouse fibroblasts. L929 cells also express the BDNF gene. Northern blot hybridization analysis revealed 4 discrete BDNF mRNA species in L929 cells and rat hippocampus after induction of seizures with kainic acid. Serum as well as 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated NGF and all 4 BDNF mRNAs in L929 cells. Treatment with both agents induced NGF mRNA to a much larger extent than the BDNF mRNAs. The induction of the BDNF mRNAs was rapid, with nearly maximal levels by 1 hr. In contrast, NGF mRNA induction occurred later and peaked at 4-6 hr. Both NGF and BDNF mRNA induction were inhibited by actinomycin D. Cycloheximide, on the other hand, inhibited only NGF but not BDNF mRNA induction. Corticosterone rapidly decreased NGF mRNA but not the BDNF mRNAs, and had no effect on seizure-induced NGF or BDNF mRNAs. Forskolin did not stimulate NGF or BDNF mRNAs. In contrast to NGF mRNA, forskolin did not interfere with the serum induction of BDNF mRNAs. These results demonstrate that 2 genes which encode closely related neurotrophic factors are differentially regulated in L929 cells. The molecular mechanisms which bring about this differential regulation remain to be elucidated.

Animals↗

PubMed1992

Uptake and transfer factors of 137Cs by mushrooms.

The 137Cs content of 118 species (668 samples) of higher fungi collected in the period from August 1984 to October 1989 at three different locations in Styria, Austria, was determined by gamma-spectrometry. The Cs-content of most mushrooms has been increasing since September 1986. In order to find out which factors determine the 137Cs-contamination of mushrooms and the transfer-value soil to mushroom, the concentration of total and plant-available radiocesium in soils as well as the pH-value, the content of humus, clay, silt, sand, exchangeable cations, the composition of the clay minerals, and the particle size distribution of the soils of two different locations were examined. The higher the 137Cs contamination of the soil, the thicker the layer of humus and the higher the content of humus, the lower the pH-value, and the lower the amount of essential cations, especially of K+, the higher the amount of 137Cs plant-available will be. Therefore, the contamination of the mushrooms in the coniferous forest of Koralpenblick (1000 m) is higher than in the mixed forest at the Rosenberg around Graz at approx. 500 m height. Of 26 different species of mushrooms measured at both sites, only 61% show the highest TF-values soil to mushrooms also at the Koralpenblick. In the spruce forest at Koralpenblick there are many species of mushrooms with high 137Cs-contamination which were not found at the Rosenberg. However, the properties of the species to which a mushroom belongs are more important than environmental conditions and soil properties. The transfer values of 40K stay within narrow bounds, whereas those of 137Cs differ widely.

Accidents↗

PubMed1992

The human nerve growth factor gene: structure of the promoter region and expression in L929 fibroblasts.

We previously studied the transcriptional mechanisms involved in expression of the murine nerve growth factor (NGF) gene. To investigate the regulation of transcription of the human NGF gene, the promoter region was cloned. The nucleotide sequences of the human and mouse genes are greater than 90% similar near their promoters. The cloned human promoter was transcriptionally active in mouse L929 fibroblasts. 5' Deletion analyses indicated that the -85 to -45 region stimulates basal transcription 6-fold. This segment is greater than 80% identical in human and mouse genes except for an AP-1 consensus sequence found only in the human gene. A second AP-1 consensus sequence at +34, previously shown to function as a regulatory element in the mouse gene, is identical in both genes. Gel shift analyses of L929 cell extracts revealed binding of protein to oligonucleotide probes spanning each of the two AP-1 consensus sequences of the human gene. The gel shift patterns differed, suggesting interaction of different proteins with the two probes. Our results demonstrate that the human NGF gene promoter is transcriptionally active in mouse fibroblasts, and implicate an upstream region in basal transcription.

Animals↗

PubMed1992

Structure of the human insulin-like growth factor binding protein-2 gene.

Insulin-like growth factors (IGFs) are polypeptide hormones with structural homology to proinsulin. IGFs circulate in blood bound to specific IGF binding proteins (IGFBPs). cDNA sequences of six members of a family of human and rat IGFBPs have been published. Here we present a partial characterization of the human IGFBP-2 gene. This single copy gene is located on chromosome 2 and spans a total of more than 32 kilobases (kb) of genomic sequence. It is organized in four exons with sizes of more than 568, 220, 141, and 496 nucleotides. The intron between exon one and exon two contributes 27 kb to the size of the IGFBP-2 gene. The second and the third introns comprise 1.1 kb and 1.95 kb, respectively. When the structure of the IGFBP-2 gene is compared to that of the IGFBP-1 and IGFBP-3 genes, the exon boundaries are found to be conserved in these three genes. A single transcriptional start site was localized to 113 +/- 2 nucleotides 5' of the ATG start codon of IGFBP-2 translation. Furthermore, the region between nucleotides -635 and -2 upstream of the ATG was demonstrated to exhibit promoter activity in human Jurkat K16 cells. This region is devoid of TATA or CAAT consensus sequence motifs and has a high content of dC and dG nucleotides. In this respect the putative IGFBP-2 promoter region resembles the promoters which are often associated with housekeeping genes.

Amino Acid Sequence↗

PubMed1991

Insulin-like growth factor binding protein expression in human small cell lung cancer cell lines.

Insulin-like growth factor binding proteins (IGF-BP) are secreted by several human small cell lung cancer cell lines (SCLC). In order to identify the IGF-BPs from SCLC cell lines the RNA from 10 different SCLC cell lines was analyzed by Northern blot analysis with the probes for three different IGF-BPs, IGFBP-1, IGFBP-2, and IGFBP-3. No hybridization signal could be detected with the probes encoding for IGFBP-1 and IGFBP-3. The hybridization with different IGFBP-2-specific oligodeoxynucleotide probes and with the corresponding full-length cDNA showed that all SCLC cell lines which secreted IGF-BPs express IGFBP-2.

Base Sequence↗

PubMed1991

Structural and functional identification of regulatory regions and cis elements surrounding the nerve growth factor gene promoter.

The transcriptional mechanisms which contribute to the regulation of nerve growth factor (NGF) production are still largely unknown. We previously expressed the NGF promoter region in transgenic mice to localize cis regulatory elements to within 5 kb of the promoter. To further map these elements, and to begin to study the corresponding transacting factors, we here assayed the effects of 5' deletions and point mutations and examined the binding of nuclear factors to the NGF promoter region using L929 cell fibroblasts. Sequential deletions delineated regions upstream from the promoter which stimulated and inhibited transcription. DNAse-1 footprinting experiments identified four upstream segments, designated F2, F4, F6 and F8, which bound L929 cell nuclear proteins. F2 and F4 mapped to stimulatory and F6 and F8 to inhibitory regions. Competition experiments using a heptanucleotide present in both F2 and F4 segments suggested that they may be bound by related factors. Gel shift assays showed that the F8 binding proteins are less abundant in L929 cells than in NIH 3T3 fibroblasts and B16 melanoma cells. In addition to the upstream segments, a downstream AP-1 consensus sequence bound L929 nuclear proteins. Mutation of the AP-1 consensus sequence eliminated binding of nuclear proteins and reduced transcriptional activity. Our results indicate that transcriptional activator as well as suppressor regions surround the NGF gene promoter. The regulation of NGF production is likely to involve cis elements within these regions and transacting factors that bind to them.

Animals↗