[Diagnosis of the female urethra and examination technique with Doppler balloon urethrography].
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Endosonography↗
Biomedical subjects
G Heinrich
Publications and source records attributed to G Heinrich.
Identification of nuclear localization signals within the zinc fingers of the WT1 tumor suppressor gene product.
WT1 encodes a zinc finger protein with a key role in urogenital development that is inactivated in a subset of Wilms' tumors. This tumor suppressor gene product contains an amino-terminal dimerization domain required for trans-inhibition of wild-type WT1 activity by mutants defective for DNA binding. In the course of characterizing truncation mutants of WT1, we noted that the WT1 zinc fingers contain two functionally independent targeting signals required for nuclear localization of the protein. These novel signals lie within zinc fingers I and within zinc fingers II and III. We demonstrate that nuclear targeting of the WT1 homodimerization domain functionally antagonizes activity of the wild-type protein activity.
3T3 Cells↗
[Three-dimensional ultrasound study (3-D sonography) of the female breast].
Three-dimensional sonography of the mamma with a Voluson annular array transducer (10 MHz) (Kretztechnik, Austria) is a new method applicable in differential diagnosis of mamma foci. 50 patients (19 of them with breast cancer) were thus pre-surgically examined. Both sectional and stereoscopic representations were made use of. The suspected diagnoses and their correspondences with post-surgical findings were compared to the correspondences obtained through 2D-sonography. 3D-sonography produced 4 cases of the incorrectly positive diagnosis breast cancer, 2D-sonography 2 cases of incorrectly negative diagnoses. Other advantages of 3D-sonography over 2D-sonography include: better judgement of the conditional of focal environs (infiltration), existence and form of intracystic structures and of multifocal disease, short duration of examination, possible re-diagnosing of stored data.
Breast↗
Induction of vitamin D receptor mRNA expression in psoriatic plaques correlates with clinical response to 1,25-dihydroxyvitamin D3.
Psoriasis is a skin disorder characterized by hyperproliferation of epidermal keratinocytes. 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and its analogs have been shown to inhibit keratinocyte proliferation in vitro and to be therapeutically effective for the treatment of psoriasis. Some patients with psoriasis, however, do not have a favorable response to 1 alpha,25 (OH)2D3 therapy. To evaluate the differential responsiveness to 1 alpha (OH)2D3 treatment, we examined the expression of vitamin D receptor mRNA in psoriatic lesions by reverse transcription-polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as an internal control. In this double-blind clinical trial, we recruited 18 patients who received topical treatment of 1 alpha,25(OH)2D3 (15 microgram/g Vaseline) or placebo on separated psoriatic lesions for 8 weeks. In patients who showed >90% clinical improvements of their psoriatic lesions with 1 alpha,25(OH)2D3 (n=9), an increase of 130+/-37% in vitamin D receptor mRNA level was observed in 1 alpha,25(OH)2D3-treated lesions when compared with the corresponding placebo controls. There was no increase in vitamin D receptor mRNA level in the lesions treated with this drug in patients who did not respond to the treatment. These data suggest that the antiproliferative activity of 1 alpha,25(OH)2D3 is closely associated with the expression of its cognate receptor.
Base Sequence↗
Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.
There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.
Blotting, Western↗
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↗
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↗
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↗
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↗
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↗
Could abnormalities in insulin-like growth factors and their binding proteins during pregnancy result in gestational diabetes?
A number of dramatic changes have been documented in the insulin-like growth factors (IGFs-I and -II) and their binding proteins (IGFBPs) during pregnancy. In this study we have tested the hypothesis that a failure of the normal proteolytic modification of IGFBP-3 is responsible for gestational diabetes by examining serum samples taken in the third trimester from 29 women with uncomplicated pregnancies, 21 women with established Type 1 diabetes and 20 women with gestational diabetes. Analysis of IGFBP-3 by Western immunoblotting revealed that it was present in a modified form, migrating at around 29 kDa, in the circulation of all of the women investigated. Semiquantification of the activity of the protease which modifies the IGFBP-3 demonstrated considerable variation between individuals in their ability to fragment radiolabelled IGFBP-3 following a 45-min co-incubation. Surprisingly, in one individual (with gestational diabetes) there was no detectable protease activity even though her endogenous IGFBP-3 had been modified. However, overall there was no clear-cut difference in protease activity between the different groups. Radioimmunometric analysis of IGF-I revealed significantly higher levels in women with gestational diabetes than either of the other two groups (P < 0.05). Similarly IGFBP-3 levels were also increased in these same women (P < 0.05). In contrast, IGF-II levels did not alter between the three groups. In conclusion, our hypothesis was not supported by these data and gestational diabetes was found not to be associated with any reduction in the activity of the circulating IGFBP-3 protease which could have decreased the availability of the IGF nor with any alteration in IGFs which could explain the onset of diabetes in these women.
Adult↗
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↗
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↗
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↗
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↗
Dynamics of heterogeneous polymer networks.
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Journal Article↗
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↗
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↗