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Biomedical subjects

B Christ

Publications and source records attributed to B Christ.

At least 19 recordsLinked to original sources
PubMed2006

Expression pattern of BMPs during chick limb development.

In vertebrates, BMPs (bone morphogenic proteins) play critical roles in establishing the basic embryonic body plan and are involved in the development of a large variety of organs and tissues. Here, we analyzed the expression pattern of various BMPs (2, 4, 5 and 7) by whole mount in situ hybridization during chick limb development. In limb, expression of BMPs suggests evolutionary conserved mechanisms of BMP-dependent differentiation between lower and higher vertebrates. During the early developmental stages, BMP-2 and BMP-7 are expressed in the posterior distal mesenchyme leaving a less prominent expression anteriorly. BMP-4 is initially expressed in the anterior mesenchyme and spreads later to the whole mesenchyme leaving a stronger expression at the anterior side. From HH-stage 25, expression of BMP-4 is observed in the anterior-posterior margins of the limb bud. The BMPs 2, 4 and 7 are expressed strongly in the AER, whereas BMP-5 is expressed as a weak signal in the distal mesoderm during the early stages of limb development. Later from HH-stage 25 onwards, BMP-5 is expressed in the dorsal and ventral muscular mass of the developing limb. As digits become identifiable, expression of BMPs are observed in the interdigital mesenchyme and can also be detected along the contours of the developing phalanges and at the distal tips of the digits. All these BMPs are found to be expressed in the developing feather buds from day 8 onwards.

Animals↗
PubMed2005

Glucagon-stimulated but not isoproterenol-stimulated glucose formation inhibition by interleukin-6 in primary cultured rat hepatocytes.

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.

Animals↗
PubMed2005

A microtiterplate-based screening assay to assess diverse effects on cytochrome P450 enzyme activities in primary rat hepatocytes by various compounds.

During the development of potential drugs it is useful to identify pharmacological and/or toxicological side effects of a compound as early as possible in order to exclude them from further development for reasons of time and cost. Activation or inactivation of members of the cytochrome P450-dependent monooxygenase system (CYP450) might indicate potential undesired effects of a given compound. However, results using CYP450 assay systems are often inconsistent because of different experimental settings. Therefore, it was the goal of the present study to optimize the CYP450 assay in primary rat hepatocytes with respect to the time point of addition of and duration of exposure to alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) as well as trans-resveratrol (RES), which have well-described stimulatory and inhibitory effects on CYP450 enzymes of the 1A and 2B family, respectively. Hepatocytes were also treated with putative lipoxygenase (LOX)/cyclooxygenase (COX) inhibitors with unknown impact on CYP450 enzyme activity in order to detect potential side effects. Cells were cultured for up to 7 days on 96-well microtiter plates, and enzyme activity was determined by a conventional fluorescence spectroscopy assay. ANF and BNF, given to the cells after 4 days of culture, stimulated CYP1A and 2B activities significantly in a concentration-dependent fashion after long-term exposure for at least 1 day. However, during short-term exposure for 1-6 h, CYP1A activity was inhibited, while CYP2B was increased weakly by ANF but not BNF. RES inhibited CYP1A activity during short- and long-term exposure without affecting CYP2B activity. From the results it was concluded that primary rat hepatocytes should be cultured for at least 3-4 days but no longer prior to the assay. The assay should be performed at two different time points of exposure, i.e., 6 h for short-term and 24 h for long-term exposure. The compounds under investigation should be applied at two different concentrations, e.g., at one time and 10 times higher concentrations, which should be oriented to the ED50, provided it is known for the respective substance. Under these assay conditions the LOX/COX inhibitors tested activated CYP1A enzyme activity in long-term but instead inhibited it in short-term experiments. CYP2B activity was stimulated during short- and long-term exposure. These results indicated drug side effects recommending exclusion of the compounds from the drug developmental process. Hence, in order to assess the pharmacological potential of novel compounds it is adequate to perform both short- and long-term experiments to concisely describe the effect of a compound on the CYP450 system.

Animals↗
PubMed2005

Development of lymphatic vessels: tumour lymphangiogenesis and lymphatic invasion.

In human solid cancer, the lymph node status is the most important prognostic indicator for the clinical outcome of patients. Follow-up data has shown that about 80% of metastasis follows an orderly pattern of progression via the lymphatic network while about 20% systemic metastasis occurs, bypassing the lymphatic system. Over the past few years, advances have been made in understanding the cellular and molecular aspects of physiological lymphangiogenesis and tumour-induced lymphangiogenesis, and the majority of studies point out to a positive correlation between tumour-induced lymphangiogenesis and lymphatic metastasis. However, the impact of intra- and peritumoural lymphatics on the tumour biology and the first steps of lymphatic metastasis, i.e. the invasion of tumour cells into the lymphatic vessels, are not well understood. We will give an outline of i. the physiological process of lymphangiogenesis, ii. tumour-induced lymphangiogenesis and lymphatic metastasis, iii. lymphatic invasion and the common pathways of tumour-lymphangiogenesis and lymphatic invasion. The growing interest in this topic has brought up a number of new molecular players in the field, which may provide the basis for a rational therapy against the process of lymphatic dissemination of tumour cells.

Animals↗
PubMed2004

The role of Emx2 during scapula formation.

The scapula is subdivided into head, collum, and blade. Due to the expression pattern of Emx2 and the absence of the scapula blade in Emx2 knockout mice, it has been suggested that Emx2 is involved in the formation of the scapula. Micromanipulation experiments revealed that ectoderm ablation over the somites does not affect Emx2 expression but inhibits the formation of the scapula blade indicating that Emx2 is not sufficient to induce scapula blade formation. Furthermore, we show that the formation of the scapula head is dependent, scapula blade formation independent of FGFR-1-mediated signaling. Overexpression of Emx2 does not influence scapula blade formation but leads to the development of an additional posterior digit in the anterior border of the limb. Taken together, the data presented implicate that Emx2 expression is necessary but not sufficient for the development of the scapula blade. It is not a marker for scapula development but rather provides positional information along the proximodistal and anterior-posterior limb axes, whereas the specificity of the developing skeletal elements is determined by the concerted interaction of Emx2 with other factors.

Animals↗
PubMed2004

Control of the temporal and spatial Uncx4.1 expression in the paraxial mesoderm of avian embryos.

Uncx4.1 is a homeobox containing transcription factor that determines the development of the pedicles of the neural arches, transverse processes and proximal ribs. In this paper we characterize the expression pattern of Uncx4.1 during chick embryogenesis with special focus on its expression in the paraxial mesoderm. In the presomitic mesoderm, Uncx4.1 is expressed in the caudal halves of the somites I and II. In the newly formed somites, Uncx4.1 expression remains in the caudal somite halves but becomes restricted to the somitocoele and the ventral epithelial wall. After somite compartmentalization, Uncx4.1 is expressed in the caudal half of the sclerotome in a well defined spatial and temporal pattern. Micromanipulations revealed that Uncx4.1 expression in the presomitic mesoderm is independent of signals from the axial structures and presumably induced by the intrinsic Notch/Delta driven oscillator activity that determines craniocaudal somite polarity. In contrast, in the maturing somite Uncx4.1 expression depends on signals from the axial structures. The notochord-floor plate complex is essential for maintaining Uncx4.1 expression in the caudal somite half. The neural tube is necessary for providing sufficient Uncx4.1 positive sclerotomal material to enable development of pedicles of the neural arches and transverse processes.

Animals↗
PubMed2004

The homeobox transcription factor Prox1 is highly conserved in embryonic hepatoblasts and in adult and transformed hepatocytes, but is absent from bile duct epithelium.

Prox1 is a transcription factor with two highly conserved domains, a homeobox and a prospero domain. It has been shown that Prox1 knock-out mice die during early embryonic stages and display a rudimentary liver. We have studied the expression of Prox1 at RNA and protein levels in chick, rat, mouse and human liver and in transformed and non-transformed hepatic cell lines. Prox1 is expressed in early embryonic hepatoblasts and is still expressed in adult hepatocytes. Prox1 protein is located in the nuclei of hepatoblasts, which grow into the neighboring embryonic mesenchyme. The expression pattern in chick, mouse, rat and human embryos is highly conserved. Besides albumin and alpha-fetal protein, Prox1 belongs to the earliest markers of the developing liver. In adult liver, Prox1 is expressed in hepatocytes but is absent from bile duct epithelial and non-parenchymal cells (Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells and myofibroblasts). Isolated primary hepatocytes and hepatoma cell lines (HepG2, Hep3B) are Prox1 positive, whereas the immortalized murine liver cell-line MMH, which constitutively expresses the receptor c-met, is Prox1 negative. Transfection of MMH with Prox1 cDNA increases the expression level significantly as compared to control transfectants. In HepG2 and Hep3B, the Prox1 levels are even up to 100 times higher. Our studies show that Prox1 is a highly conserved transcription factor, expressed in hepatocytes from the earliest stages of development into adulthood and over-expressed in hepatoma cell lines. Its absence from bile duct epithelial cells suggests a function for the specification of hepatoblasts into hepatocytes. The genes controlled by Prox1 need to be studied in the future.

Animals↗
PubMed2004

Expression pattern of VEGFR-2 (Quek1) during quail development.

The growth and maintenance of the blood and lymphatic vascular systems is to a large extent controlled by members of the vascular endothelial growth factor (VEGF) family via the tyrosine kinase receptors (VEGFRs) expressed in angioblastic cells. Here, we analyzed the Quek1 (VEGFR-2) expression pattern by whole mount in situ hybridization during quail development. During early embryogenesis, Quek1 expression was detected at the caudal end of the blastoderm and primitive streak and in the head paraxial mesoderm. In somites, expression was observed from HH-stage 9 onwards in the dorsolateral region of both the forming and recently formed somites. During somite maturation, expression persists in the lateral portion of the somitic compartments, the dermomyotome and the sclerotome. Additionally, a second expression domain in the maturing somite was observed in the medial part of the sclerotome adjacent to the neural tube. This expression domain extended medially and dorsally and surrounded the neural tube during later stages. In the notochord, expression was observed from HH-stage 23 onwards. In the limb bud, expression was initiated in the mesenchyme at HH-stage 17. During organogenesis, expression was detected in the pharyngeal arches and in the anlagen of the esophagus, trachea, stomach, lungs, liver, heart and gut. Expression was also seen in feather buds from day 7 onwards. Our results confirm the angiogenic potential of the mesoderm and suggest that VEGFR-2 expressing cells represent multiple pools of mesodermal precursors of the hematopoietic and angiopoietic lineages.

Animals↗
PubMed2004

Expanding hepatocytes in vitro before cell transplantation: donor age-dependent proliferative capacity of cultured human hepatocytes.

BACKGROUND: For hepatocyte transplantation as well as experimental purposes, it would be advantageous to be able to expand human hepatocytes in vitro. However, under serum-free conditions, even with supplements of HGF (hepatic growth factor) and EGF (epidermal growth factor), proliferation of human hepatocytes is hampered. The aim of this study was to identify differences in the proliferative capacity of cultured primary human hepatocytes related to the age of the liver donors. METHODS: Proliferation was determined by BrdU-uptake, ploidy was measured using propidium iodide staining and flow cytometry, and the expression of cell cycle related proteins was determined by Western blotting. RESULTS: During the initial culture, juvenile hepatocytes proliferated better than adult hepatocytes. The proliferation rate declined to barely detectable levels after 8 days in culture in both juvenile and adult hepatocytes. The higher proliferative capacity of juvenile hepatocytes was associated with a larger fraction of diploid cells and a higher viability. The expression of regulatory cell cycle related proteins was higher in juvenile than in adult hepatocytes. CONCLUSIONS: The proliferation of human hepatocytes in vitro is critically related to a large fraction of diploid hepatocytes. The expression of regulatory cell cycle proteins reflects the proliferative capacity of cultured human hepatocytes. Juvenile as compared to adult human hepatocytes may be better suited for expansion in culture and could have a stronger repopulation capacity in vivo.

Age Factors↗
PubMed2004

Assessment of the biological performance of the needle-free injector INJEX using the isolated porcine forelimb.

BACKGROUND: The development and utilization of novel needle-free injection devices in order to minimize needle stick injuries make increasing demands for suitable assay systems, which reflect the physiological situation in humans as close as possible. OBJECTIVES: It was therefore the goal of the present study to test the biological performance of a needle-free injector (INJEX) by the use of porcine skin as a model with a high predictive value for the feasibility in humans because of its close similarity to human skin. METHODS: In order to use porcine skin in the context of the underlying tissues, the isolated porcine forelimb was chosen as an assay model for use with the INJEX injector. Ink or the fluorescent dye fluorescein-isothiocyanate was injected and the penetration depth was determined metrically and dye distribution histologically. To assess the resorption of heparin, needle injection was compared with needle-free injection in a perfused limb model. RESULTS: Increasing amounts of ink increasingly penetrated into subcutaneous tissue layers in a cone-shaped manner mainly following lead structures. Penetration was hampered by skin thickness and by the deep muscle fascia, which served as a penetration barrier. Resorption of heparin was similar irrespective of injection by the use of a needle or the INJEX device. CONCLUSIONS: The isolated porcine forelimb serves as a versatile tool for the assessment of the biological performance of needle-free injection devices such as INJEX. Further studies are necessary to correlate the model for drug delivery in humans.

Absorption↗
PubMed2004

Investigation on interaction between tacrolimus and sildenafil in kidney-transplanted patients with erectile dysfunction.

OBJECTIVE: Sildenafil may provide an effective treatment for erectile dysfunction, frequently observed in uremic patients and after kidney transplantation. Pharmacokinetic interactions between sildenafil and tacrolimus are to be expected due to a common elimination pathway via cytochrome P450 3A4. Therefore, the pharmacokinetics during combined use of these agents were studied over 9 days. MATERIAL AND METHODS: Nine male patients (age 29-52 years) were included, who had previously participated in a recent interaction study with sildenafil given as a single dose. Comedication remained unchanged in order to avoid introducing confounding factors. In the previous study in the patients, tacrolimus blood levels with and without sildenafil were measured for pharmacokinetic analysis. In the present study, 25 mg sildenafil were coadministered daily over 9 days and tacrolimus levels were assessed at sampling times optimized using simulation. In addition, laboratory parameters and blood pressure changes were measured and adverse effects monitored. RESULTS: Terminal half-lives of tacrolimus did not differ significantly and trough levels did not change when sildenafil was coadministered daily over 9 days. Mean arterial blood pressure was lower after sildenafil intake. Two patients had to reduce their antihypertensive treatment, 6 patients reported mild side effects. In 1 case, there was an asymptomatic, temporary increase in the serum concentration of gamma-GT. CONCLUSIONS: There was no evidence obtained for a change in elimination half-life or average concentration of tacrolimus during repeated coadministration of sildenafil. Since blood pressure decreased, a starting dose of 25 mg sildenafil and, if necessary, adjustment of the dose of antihypertensive drugs on days when sildenafil is given has to be considered. With respect to the observed blood pressure changes, pharmacokinetic/pharmacodynamic interaction studies with other antihypertensive drugs are of critical importance in these patients.

Adult↗
PubMed2003

Temporal and spatial protease nexin 1 expression during chick development.

Protease nexin 1 (Pn-1) or glia derived nexin is a secreted protease inhibitor. By screening a chick embryonic cDNA library, we isolated Pn-1 cDNA and analyzed its expression pattern during development by in situ hybridization. Pn-1 was first observed at HH-stage 3 in the primitive pit. At HH-stage 7, expression was observed in the medial part of the neural folds and asymmetrically in the right lateral plate mesoderm and at the left side of Hensen's node. At HH-stage 10-11, Pn-1 was expressed in the closing neural tube, lateral plate mesoderm and paraxial head mesoderm. From HH-stage 12 onwards, expression was observed caudally in the lateral plate mesoderm and cranially in the Wolffian duct. At the level of the compartmentalized somite, expression was seen in the sclerotome. Pn-1 was also expressed in the anterior wall of the pharynx and still in the paraxial head mesoderm. At HH-stage 15, the expression in the Wolffian duct remained caudally while the expression in the sclerotome extended along the whole body axis. A stronger expression was observed in the cranial four somites. From HH-stage 17-18 onwards, expression became visible in the mesenchyme of the developing limb buds. At these stages, expression was no longer observed in the Wolffian duct. At HH-stage 36, Pn-1 was expressed in the vertebral bodies, in the neural tube, and in the metanephros.

Amino Acid Sequence↗
PubMed2001

Arterial identity of endothelial cells is controlled by local cues.

The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

Animals↗
PubMed2001

Upregulation of the cochaperone Mdg1 in endothelial cells is induced by stress and during in vitro angiogenesis.

Angiogenesis research has focused on receptors and ligands mediating endothelial cell proliferation and migration. Little is known about the molecular mechanisms that are involved in converting endothelial cells from a proliferative to a differentiated state. Microvascular differentiation gene 1 (Mdg1) has been isolated from differentiating microvascular endothelial cells that had been cultured in collagen type I gels (3D culture). In adult human tissue Mdg1 is expressed in endothelial and epithelial cells. Sequence analysis of the full-length cDNA revealed that the N-terminal region of the putative Mdg1-protein exhibits a high sequence similarity to the J-domain of Hsp40 chaperones. We show that this region functions as a bona fide J-domain as it can replace the J-domain of Escherichia coli DnaJ-protein. Mdg1 is also upregulated in primary endothelial and mesangial cells when subjected to various stress stimuli. GFP-Mdg1 fusion constructs showed the Mdg1-protein to be localized within the cytoplasm under control conditions. Stress induces the translocation of Mdg1 into the nucleus, where it accumulates in nucleoli. Costaining with Hdj1, Hdj2, Hsp70, and Hsc70 revealed that Mdg1 colocalizes with Hsp70 and Hdj1 in control and stressed HeLa cells. These data suggest that Mdg1 is involved in the control of cell cycle arrest taking place during terminal cell differentiation and under stress conditions.

Amino Acid Sequence↗
PubMed2001

Embryonic central nervous system angiogenesis does not involve blood-borne endothelial progenitors.

We asked, whether, in the blood of avian embryos, endothelial precursor cells circulate that actually contribute to the growing vascular system in and around the central nervous system (CNS). We compared the morphology and distribution of QH1-positive cells after transplantation of quail paraxial mesoderm, after blood transfusion, in quail-chick parabiosis, or after quail bone-marrow transplantation. After head mesoderm transplantation from quail to chick, we observed sprouting endothelial cells (ECs), capillary tube formation, and chimeric endothelial lining of large arteries in the host brain. These QH1-positive quail cells showed EC morphologies that demonstrated three different aspects of CNS angiogenesis: invasion by means of filopodia, clonal proliferation and tube formation, and integration into preexisting EC layers. After blood transfusion or in chick-quail parabiosis, blood-borne QH1+ cells were found in the lumen of but not integrated into the wall of the host vascular system. Neither were QH1+ cells observed in the capillary walls of parabiotic chick chorioallantoic membranes. In both cases, the quail cells showed typical macrophage morphology. In chicks that had received quail bone marrow transplants onto their chorioallantoic membranes, QH1+ cells with macrophage, but not EC shape were occasionally seen near the inoculation site. We conclude that (1) blood-borne cells do not become ECs or directly contribute to angiogenesis inside, or in vascular plexuses around the CNS during embryonic development; (2) blood-borne cells do not contribute to the intraneural macrophage population of the embryonic CNS.

Afferent Pathways↗
PubMed2001

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs.

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.

Amino Acid Sequence↗
PubMed2001

Regulation of Epha4 expression in paraxial and lateral plate mesoderm by ectoderm-derived signals.

Somitogenesis in all vertebrates involves a mesenchymal to epithelial transition of segmental plate cells. Such a transition involves cells altering their morphology and their adhesive properties. The Eph family of receptor tyrosine kinases has been postulated to regulate cytoskeletal organization. In this study, we show that a receptor belonging to this family, EphA4, is expressed in the segmental plate in a region where cells are undergoing changes in cell shape as a prelude to epithelialization. We have identified the ectoderm covering the somites and the midline ectoderm as sources of signals capable of inducing EphA4. Loss of EphA4 results in cells of irregular morphology and somites fail to form. We also show that when somites fail to develop, expression of EphA4 in the lateral plate is also lost. We suggest that signaling occurs between the somites and the lateral plate mesoderm and provide evidence that retinoic acid is involved in this communication.

Animals↗