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Biomedical subjects

H Schipper

Publications and source records attributed to H Schipper.

At least 19 recordsLinked to original sources

Endurance exercise differentially stimulates heart and axial muscle development in zebrafish (Danio rerio).

Mechanical load is an important factor in the differentiation of cells and tissues. To investigate the effects of increased mechanical load on development of muscle and bone, zebrafish were subjected to endurance swim training for 6 h/day for 10 wk starting at 14 days after fertilization. During the first 3 wk of training, trained fish showed transiently increased growth compared with untrained (control) fish. Increased expression of proliferating cell nuclear antigen suggests that this growth is realized in part through increased cell proliferation. Red and white axial muscle fiber diameter was not affected. Total cross-sectional area of red fibers, however, was increased. An improvement in aerobic muscle performance was supported by an increase in myoglobin expression. At the end of 10 wk of training, heart and axial muscle showed increased expression of the muscle growth factor myogenin and proliferating cell nuclear antigen, but there were major differences between cardiac and axial muscle. In axial muscle, expression of the "slow" types of myosin and troponin C was increased, together with expression of erythropoietin and myoglobin, which enhance oxygen transport, indicating a shift toward a slow aerobic phenotype. In contrast, the heart muscle shifts to a faster phenotype but does not become more aerobic. This suggests that endurance training differentially affects heart and axial muscle.

Animals↗

Syringeal muscles fit the trill in ring doves (Streptopelia risoria L.).

In contrast to human phonation, the virtuoso vocalizations of most birds are modulated at the level of the sound generator, the syrinx. We address the hypothesis that syringeal muscles are physiologically capable of controlling the sound-generating syringeal membranes in the ring dove (Streptopelia risoria) syrinx. We establish the role of the tracheolateralis muscle and propose a new function for the sternotrachealis muscle. The tracheolateralis and sternotrachealis muscles have an antagonistic mechanical effect on the syringeal aperture. Here, we show that both syringeal muscles can dynamically control the full syringeal aperture. The tracheolateralis muscle is thought to directly alter position and tension of the vibrating syringeal membranes that determine the gating and the frequency of sound elements. Our measurements of the muscle's contractile properties, combined with existing electromyographic and endoscopic evidence, establish its modulating role during the dove's trill. The muscle delivers the highest power output at cycle frequencies that closely match the repetition rates of the fastest sound elements in the coo. We show that the two syringeal muscles share nearly identical contraction characteristics, and that sternotrachealis activity does not clearly modulate during the rapid trill. We propose that the sternotrachealis muscle acts as a damper that stabilizes longitudinal movements of the sound-generating system induced by tracheolateralis muscle contraction. The extreme performance of both syringeal muscles implies that they play an important role in fine-tuning membrane position and tension, which determines the quality of the sound for a conspecific mate.

Animals↗

Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns.

Genome and gene duplications are considered to be the impetus to generate new genes, as the presence of multiple copies of a gene allows for paralogues to adopt novel function. After at least two rounds of genome/gene duplication, the Runt gene family consists of three members in vertebrates, instead of one in invertebrates. One of the family members, Runx2, plays a key role in the development of bone, a tissue that first occurs in vertebrates. The family has thus gained new gene function in the course of evolution. Two Runx2 genes were cloned in the vertebrate model system the zebrafish (Danio rerio). The expression patterns of the two genes differ and their kinetics differ up to four fold. In addition, splice forms exist that are novel when compared with mammals. Together, these findings comprise opportunities for selection and retention of the paralogues towards divergent and possibly new function.

5' Untranslated Regions↗

Effects of decreased muscle activity on developing axial musculature in nicb107 mutant zebrafish (Danio rerio).

The present paper discusses the effects of decreased muscle activity (DMA) on embryonic development in the zebrafish. Wild-type zebrafish embryos become mobile around 18 h post-fertilisation, long before the axial musculature is fully differentiated. As a model for DMA, the nic(b107) mutant was used. In nic(b107) mutant embryos, muscle fibres are mechanically intact and able to contract, but neuronal signalling is defective and the fibres are not activated, rendering the embryos immobile. Despite the immobility, distinguished slow and fast muscle fibres developed at the correct location in the axial muscles, helical muscle fibre arrangements were detected and sarcomere architecture was generated. However, in nic(b107) mutant embryos the notochord is flatter and the cross-sectional body shape more rounded, also affecting muscle fibre orientation. The stacking of sarcomeres and myofibril arrangement show a less regular pattern. Finally, expression levels of several genes were changed. Together, these changes in expression indicate that muscle growth is not impeded and energy metabolism is not changed by the decrease in muscle activity but that the composition of muscle is altered. In addition, skin stiffness is affected. In conclusion, the lack of muscle fibre activity did not prevent the basal muscle components developing but influenced further organisation and differentiation of these components.

Animals↗

Characterization of intracellular aggregates using fluorescently-tagged polyglutamine-expanded androgen receptor.

Spinal bulbar muscular atrophy (SBMA) is a classic CAG-repeat neurodegenerative disease. It is caused by expansion of a polyglutamine (polyGln) tract in the androgen receptor (AR). Recent evidence has indicated a potential role for nuclear and cytoplasmic inclusions in the pathogenesis of these diseases. We have used blue and green fluorescently-tagged AR to show that both wild-type (WT) and poly-Gln-expanded full-length AR can form aggregates and that aggregation is not related to cytotoxicity. Twenty to thirty-five percent of all cell types transfected into COS cells showed aggregation containing both amino- and carboxy-terminal fluorescent tags. The aggregates reacted with (F39.4.1), an anti-AR antibody and with IC2, an expanded polyGln tract antibody. Western analysis of protein extracts revealed little evidence of proteolysis although some cleavage of the fusion proteins was seen. The general caspase inhibitor, Z-DEVD-FMK, did not affect aggregation in either wild type or polyGln-expanded GFP-AR transfected cells. Surprisingly, addition of Mibolerone a synthetic androgen significantly decreased inclusion formation in both WT and polyGln-expanded AR-transfected cells. Overall, we show that both WT and polyGln expanded full-length AR are found in aggregates and that proteolysis is not a requirement for aggregation. Our results also suggest that toxicity is not related to intracellular aggregation of polyGln expanded AR.

Journal Article↗

TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes.

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.

Animals↗

Regulation of interleukin 1 beta RNA expression in the common carp, Cyprinus carpio L.

The intron-exon organisation of the carp IL-1beta gene consists of 2455bp and comprises seven exons. Three IL-1beta RNA transcripts have been found in carp: (1) a fully spliced product; (2) exon 1-7 with introns 5 and 6; and (3) exon 1-7 with intron 5 only. The intron-containing products probably represent partially spliced transcripts. IL-1beta mRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. Constitutive expression of the IL-1beta mRNA was found in these organs with a predominant expression in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1beta producing cells was shown by in situ hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA), lipopolysaccharide (LPS) or retinoic acid (RA), to phagocytes isolated from the head kidney, resulted in expression of IL-1beta intron-containing transcripts. Of these, only PMA and LPS were stimulators that induced the fully spliced transcript. A role for the nuclear factor (NF)-kappaB pathway in carp IL-1beta expression was shown with suppression of the LPS-induced IL-1beta expression by NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). Cortisol was able to inhibit in vitro constitutive expression of IL-1beta transcripts. Addition of cortisol simultaneously with LPS could not substantially inhibit transcription.

Animals↗

Comparison of anterior-posterior development in the porcine versus chicken embryo, using goosecoid expression as a marker.

During early embryonic development, pig and chicken embryos share striking morphological similarities. In the present study, the timing and location of expression of mRNA for goosecoid (gsc), a gene classically expressed in the nodal region of developing embryos, was examined and compared in preprimitive streak and gastrulating pig and chicken embryos. The expression of gsc appeared first in the hypoblast and second in the hypoblast of pig and chicken embryos. Because gsc expression in these tissues was not symmetrical, gsc appears to be a useful marker for the onset of embryonic polarity. During gastrulation in both species, gsc expression became confined to cells in and around the node, in the epiblast and mesoderm layers. The only significant species-related difference in the distribution of gsc expression at these stages of development was the presence of gsc expression in the gut endoderm of chicken but not pig embryos. Certainly, our results suggest that the molecular mechanisms that control anterior-posterior development in different classes of vertebrates are remarkably similar. In addition, we were able to demonstrate that the pattern of gsc expression appears to provide a more sensitive and accurate means of determining the developmental stage of early porcine embryos than the more commonly used trophoblast or embryoblast size. Using gsc expression and accompanying embryo morphometric changes, we were able to develop a four-point scale that may offer a more accurate means of quantifying early embryo development in pigs.

Animals↗

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

Adult↗

Workshop I: Parkinson's disease and sleep--results of the group discussion.

The group agreed on the facts that unwanted sleep onset has been observed after non-ergot as well as ergot dopamine agonists, that patients on these drugs need to be warned, that patients who have experienced sleepiness already must not drive a car unless the dosage is lowered and sleepiness has vanished, that a genetic predisposition for narcoleptic cataplexy has to be ruled out, that predictors of so-called sleep attacks need to be explored individually with the help of sleepiness scales and collectively in a careful study, respectively.

Causality↗

Zebrafish CTH1, a C3H zinc finger protein, is expressed in ovarian oocytes and embryos.

The Zfcth1 gene is, as the previously cloned carp cth1 gene, related to the mammalian TIS 11 family of primary response genes and encodes a protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during oogenesis and early embryogenesis up to gastrulation in the zebrafish (Danio rerio). Maternal cth1 message is present in the ovary of 1-month-old fish and of adult fish in oocytes at all stages of maturation. In the youngest oocytes the message is localized in the cytoplasm all around the nucleus, in larger oocytes the message becomes restricted to the future animal pole of the embryo, and in mature oocytes the expression is sharply localized in the cortical layer under the micropyle. After ovulation the cth1 messenger spreads over the cytoplasmic cap and is distributed over the blastomeres during subsequent cleavages. In subsequent stages maternal expression of cth1 gradually disappears. From early epiboly stages onward embryonic cth1 expression is localized to the germ ring and the hypoblast cells in the central part of the embryonic shield. In the shield, cth1 expression largely overlaps with the area of gooscoid expression in the first involuting cells. In stages after 70% of epiboly cth1 expression diminishes and soon can no longer be detected in the embryo. Next to a developmental role in cell fate determination we propose a function for cth1 during oocyte maturation.

Amino Acid Sequence↗

Chlorambucil induction of HsRad51 in B-cell chronic lymphocytic leukemia.

Our previous studies with B-cell chronic lymphocytic leukemia (B-CLL) have suggested that one of the mechanisms of nitrogen mustard (NM) drug resistance is increased repair of drug-induced damage. We have postulated that recombination may play a crucial role in this process. The human homologue of Rad51, (HsRad51), has homology to the RecA protein in Escherichia coli, which is implicated in recombination repair and induction of DNA repair enzymes. In this report, we have examined the expression and distribution of HsRad51 protein in lymphocytes from patients with B-CLL to see whether the expression of HsRad51 is associated with NM damage to the malignant B lymphocytes, specifically chlorambucil (CLB), which is the standard alkylating agent used to treat patients with B-CLL. We have analyzed the intracellular distribution of HsRad51 protein in these lymphocytes before and after treatment with CLB by immunofluorescence. In vitro CLB treatment induces Rad51 expression, as measured by increased immunopositive staining in all CLL samples. In the CLB-resistant CLL lymphocytes, there was a linear correlation between induction of Rad51 protein at 5.4 microM CLB and the in vitro LD50 dose of CLB. Surprisingly, although it has been reported that Rad51 is induced in S phase and only 10% of cells from cell lines expressed positive immunostaining for Rad51, our CLL lymphocytes, which were not subjected to in vitro drug exposure, were 90% positive for Rad51, despite their nonproliferative state, which suggests that there is chronic activation of the protein. Our results suggest that CLB activates HsRad51-directed recombination repair and that this process may be important in NM drug-induced cytotoxicity.

Antineoplastic Agents, Alkylating↗

The carp homeobox gene Ovx1 shows early expression during gastrulation and subsequently in the vagal lobe, the facial lobe and the ventral telencephalon.

The homeobox gene Carp-Ovx1 shows similarity to vertebrate and invertebrate Ovx genes and to Drosophila unplugged. Its expression pattern was studied by in situ hybridization in carp embryos and juveniles. During segmentation, expression becomes gradually limited to the neural tube. In juveniles up to 9 weeks old, cells in the ventral telencephalon, the facial lobe and the vagal lobe show Ovx1 expression, confining expression to parts with chemosensory projections.

Amino Acid Sequence↗

Blastomeres and cells with mesendodermal fates of carp embryos express cth1, a member of the TIS11 family of primary response genes.

The carp cth1 gene, related to the mammalian TIS11 family of primary response genes, encodes a novel fish protein with two putative CCCH zinc fingers. This report describes the RNA expression of this gene during cleavage, blastula and gastrula stages of carp embryos. Cth1 mRNA is present in all cleavage stage blastomeres as a maternal message. After the late blastula stage, the maternal expression decreases, revealing a spot of higher expression at the margin of the blastoderm of the dome stage embryo. Further decrease of the maternal message reveals a ring of cth1 expressing cells at the blastoderm margin from the stage of 40% epiboly onwards. By alpha-amanitin treatment we established that this local cth1 expression is of zygotic origin. At the onset of gastrulation the cells of the cth1 ring involute, starting with those in the shield region, and at approximately 60% epiboly the ring is fully involuted and occupies the hypoblast layer. All cth1 transcripts have disappeared at completion of epiboly. We discuss a possible role for the putative cth1 protein during cleavage and gastrulation.

Amino Acid Sequence↗

Precocious onset of spermatogenesis in juvenile carp (Cyprinus carpio L., Teleostei) following treatment with low doses of L-thyroxine.

Two strains of carp (Cyprinus carpio L.), a heterogenic and an all male isogeneic, were used as experimental models to assess the effects of low doses of L-thyroxine (T4) on testicular development in juvenile fish. In four experiments (two with each strain) precocious onset of spermatogenesis was induced following exposure to 15 micrograms T4 l-1, but not to 8 micrograms or 30 micrograms T4 l-1. No significant increase in body weight and testis diameter (week 16 excepted) or numbers of primary spermatogonia was observed. Total T4, measured in the isogeneic strain, increased significantly in the serum of experimental specimens within two weeks after the onset of exposure, evidencing a readily absorption of the exogeneous T4. The contents of free T4 and total triiodothyronine (T3) in the serum were measured too, showing different levels in the two experiments, as also was observed with total T4. The content of free T3, which is formed in the cells of peripheral tissues by deiodination of T4 and which supposedly exerted the observed effect on spermatogenesis, could not be determined by the used standard Amerlite assays. It is hypothesized that T3 enhances in males the activity of gonadotropic hormones, which in turn will stimulate steroid production, similar to findings in females.

Age Factors↗

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains.

Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.

Antibodies, Viral↗