Search

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.

The purpose of this study was to assess whether transperineal sonography is valid and accurate for discriminating the low (translevator) type of imperforate anus from the intermediate and high (supralevator) types. The distinction is critical in determination of the surgical approach.Fifty-six consecutively enrolled infants (0-90 days old) with imperforate anus underwent transperineal gray-scale sonography with a 12-MHz linear array transducer. The distance between the distal rectal pouch and the perineum was measured, and a cutoff distance for differentiating types of imperforate anus was identified. The sonographic findings were compared with the final diagnosis reached with radiographic and surgical findings.Transperineal sonography was feasible in all children without specific preparation. The mean distance between the distal rectal pouch and the perineum in the 22 infants with low imperforate anus was 10 +/- 4 (SD) mm compared with a mean of 24 +/- 6 mm in the 34 infants with intermediate or high anomalies (p0.001). The sensitivity of transperineal sonography was 100%; all 34 cases of intermediate or high imperforate anus were identified with a cutoff distance between the distal rectal pouch and the perineum of 15 mm. The specificity of sonography was 86% and the accuracy, 95%.Transperineal sonography is a valid and accurate noninvasive imaging technique for differentiating low and intermediate or high imperforate anus.

By flow cytometric experiments in Cyclamen persicum both with propidium iodide (PI) and DAPI (4′,6-Diamidino-2-phenylindol)-staining we were able to present (1) a new estimation for the absolute DNA content in the range of 3.17 pg DNA/2C and (2) ploidy abnormalities which were detected in the pathway of somatic embryogenesis. These aberrations might have arisen from ploidy mutations or abnormal polyploidization processes. Morphologically normal somatic embryos contained the smallest proportion of individuals with ploidy abnormalities, but also a regenerated plant in the green house displayed a significantly elevated DNA content. The results are discussed in the framework of future application of in vitro propagation techniques based on regeneration from fastly growing undifferentiated cells like long-term callus and suspension cultures.

There are only few pediatric surgical centers across the world with expertise for minimally invasive anatomical lung resections in children. Between September 2003 and September 2005, 67 children underwent thoracoscopic surgery at the Department of Pediatric Surgery, University Hospital of Tuebingen, Germany. In 19 of these cases a lung resection was carried out, 8 of them had an anatomical lung resection. All patients underwent general anesthesia without selective intubation for the procedure. Intrathoracic pressure with insufflation of carbon dioxide of 1.5 l / min was held at 3-5 mmHg. Two 5 mm ports for video and instruments and one 12 mm port for a stapling device were used. Resected lung specimens were removed from the thorax through an additional 2-3 cm long incision. A bronchoscopy was carried out during surgery in all patients. Median age at operation was 5.6 years (range 3 months-20 years). Median operation time was 150 minutes (range 94-250 min). Conversion to open surgery was performed in 3 cases. This was due to bleeding in one child, due to a stiff lung in another patient with cystic fibrosis and due to a vascular and bronchial malformation in a third child suffering from middle lobe syndrome. There were no postoperative complications. Our preliminary results show, that thoracoscopic lung resections in children can be performed without major complications and excellent cosmetic results. For the necessity of a conversion to open surgery possible reasons may be insufficient intrathoracic overview as well as congenital anomalies of the vascular and / or the bronchial tract. Co-morbidities such as rib-fusion, deformities of the thorax or scoliosis can be avoided using thoracoscopic procedures.

The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrified after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunhistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunhistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a complete eradication of the tumors. Chemotherapy seemed to upregulate the expression of resistance-associated genes in vitro and in vivo. The mechanism of multidrug resistance depends on the tumor subtype. Therefore, further investigations will be required to evaluate multidrug resistance in patients and to investigate new modalities for a reversal of multidrug resistance.

Macrophage migration inhibitory factor (MIF) is known to be involved in oncogenic transformation, tumour progression, and immunosuppression and is overexpressed in many solid tumours, including paediatric rhabdomyosarcoma (RMS). We investigated the function of MIF in RMS during treatment with cytotoxic drugs. RMS cell lines were analysed by flow cytometry, immunofluorescence staining, and ELISA. We demonstrated the overexpression of MIF in RMS cells and the enhanced expression and secretion after treatment with cytotoxic agents. Migration assays of RMS cells revealed that inhibitors of MIF (ISO-1, Ant.III 4-IPP, Ant.V, sulforaphane (SF)) and blocking antibodies caused reduced migration, indicating a role for MIF in metastatic invasion. Additionally, we investigated the function of MIF in immune escape. The development of a population containing immunosuppressive myeloid-derived suppressor cells was promoted by incubation in conditioned medium of RMS cells comprising MIF and was reversed by MIF inhibitors but not by antibodies. Although most inhibitors may restore immune activity, Ant.III and 10 µM SF disturbed T cell proliferation in a CFSE assay, whereas T cell proliferation was not reduced by 3 µM SF, ISO-1 or antibodies. However, the inhibition of MIF by blocking antibodies did not increase the killing activity of allogenic PBMCs co-cultured with RMS cells. Our results reveal that MIF may be involved in an immune escape mechanism and demonstrate the involvement of MIF in immunogenic cell death during treatment with cytotoxic drugs. Targeting MIF may contribute to the restoration of immune sensitivity and the control of migration and metastatic invasion.

In children with hepatocellular carcinoma (pHCC) the 5-year overall survival rate is poor. Effects of cytostatic therapies such as cisplatin and doxorubicin are limited due to chemoresistance and tumor relapse. In adult HCC, several antitumor properties are described for the use of curcumin. Curcumin is one of the best-investigated phytochemicals in complementary oncology without relevant side effects. Its use is limited by low bioavailability. Little is known about the influence of curcumin on pediatric epithelial hepatic malignancies. We investigated the effects of curcumin in combination with cisplatin on two pediatric epithelial liver tumor cell lines. As mechanisms of action inhibition of NFkappaB, beta-catenin, and decrease of cyclin D were identified. Using a mouse xenograft model we could show a significant decrease of alpha-fetoprotein after combination therapy of oral micellar curcumin and cisplatin. Significant concentrations of curcuminoids were found in blood samples, organ lysates, and tumor tissue after oral micellar curcumin administration. Micellar curcumin in combination with cisplatin can be a promising strategy for treatment of pediatric HCC.

Background/Aims Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm. Procedure The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice. Results After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 × 10 6 ) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system. Conclusion This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine™ 2000 and FuGENE 6™; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61–86%) than the SeVV-GFP (4–24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7–43%) compared to Ad-GFP (4–16%). The Lipofectamine™ 2000 revealed a higher transfection efficiency than the FuGENE 6™ for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

Background/Purpose Enhanced green fluorescent protein (eGFP) is widely used as a marker in different biologic systems. After vector transfection, eGFP is expressed by eukaryotic cells and can be visualized using fluorescent microscopy. The aim of this study was to establish an eGFP-transfected human hepatoblastoma (HB) cell line as tool for further basic research studies. Methods The HB cell line HUH6 was transfected with the pEGFP-N1 vector by liposomal transfection. Enhanced green fluorescent protein–positive cells were sorted out by fluorescence-activated cell sort and selected using G418 resistance. Expression of eGFP-messenger RNA was assessed by single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Original and transfected cells were compared biologically and cytomorphologically. Results Vector transfection produced up to 15% eGFP-positive cells. After fluorescence-activated cell sort and G418 selection, a pure cell line was established with 100% eGFP-positive cells. Reverse transcriptase polymerase chain reaction revealed constant expression of eGFP-messenger RNA in fluorescending cells. Analysis of cell characteristics revealed no differences between transfected and original cells. Conclusions For the first time, the authors established an eGFP-transfected HB cell line. This cell line can serve as a promising tool for further studies investigating HB in vitro and in vivo. Our model might also be a basis for similar work on other pediatric solid tumors.

Purpose Three-dimensional visualization of solid tumors is possible because of high-resolution computed tomography and magnetic resonance imaging scans. However, additional preoperative information is often desirable in complex malignancies. For the first time, the authors present a model of preoperative 3-dimensional visualization and virtual resections in pediatric solid tumors. Methods Image analysis of various pediatric tumors was performed using the research software HepaVision2 (MeVis, Bremen). Organs, tumors, and the vascular system were extracted from multislice computed tomography scans. After hierarchical analysis of the vascular system, territories supplied or drained by the major vascular branches were calculated. Results were explored and virtual resections of organs were carried out using the research software InterventionPlanner (MeVis, Bremen). Data were correlated to intraoperative findings. Results Four hepatic malignancies, 4 renal tumors, and 3 other neoplasms were analyzed. The technique of 3-dimensional visualization was feasible for all investigated children (mean age 5 years and 9 months). Spatial relations between physiological and pathological structures were identified, and anatomical structures (vessels, tumor tissue, and organ parenchyma) were determined using colorimetric encoding. Virtual simulations of tumor resection were used successfully for planning of surgical procedures in the hepatic and renal tumors. Conclusions The technique of 3-dimensional tumor visualization and virtual simulation of tumor resections provides the basis for a successful planning of complex tumor resections in children. The efficiency of these techniques should be further analyzed in series with higher numbers and differentiations of tumors.

Multidrug resistance (MDR) contributes to limited treatment results in human hepatoblastoma (HB). The MDR1 gene and its product P-glycoprotein (P-gP) has been identified as important factor in this development. In other tumors, P-gP modulation leads to a restored chemosensitivity of the cells. The aim of this study was to analyze the P-gP-modulating effects of PSC 833, a cyclosporine derivate, and verapamil on the chemotherapy of HB in vivo. HB from 2 patients were transplanted subcutaneously into nude mice NMRI (nu/nu). Animals were divided into 7 groups: Group 1 (Control); Group 2 (CDDP); Group 3 (DOXO); Group 4 (DOXO + verapamil); Group 5 (DOXO + PSC 833); Group 6 (CDDP + verapamil); and Group 7 (CDDP + PSC 833). If DOXO was administered (regardless of the combination), the dose was two times 60 mg/m2. If CDDP was administered, the dose was two times 27 mg/m2. When the chemosensitizers were administered, the doses for PSC 833 and for verapamil were four times 5 mg/kg body-weight. In the combined treatment groups the chemosensitizers were given ten minutes prior to CDDP and DOXO. Tumor volume developments and a-fetoprotein (AFP) alterations were assessed. Relative expression levels of the MDR1 gene after treatment were determined using a semiquantitative rT-PCR approach. In a mixed HB, both chemosensitizers combined with DOXO or CDDP produced a significant reduction of tumor growth (p = .0001-.00063) and AFP levels (p = .0006-.0128) compared to tumors treated with DOXO or CDDP only. Treatment results were identical to those in a less differentiated pure embryonal HB, but only in one case (DOXO + PSC 833, p = .031) significant. The chemosensitizers had no influence on the MDR1 gene expression. MDR1 modulators improve the efficiency of DOXO and CDDP treatment in xenotransplanted HB. They do not induce a further increase of drug resistance in the tumors. The data provide evidence that chemosensitizers might improve treatment results in patients with advanced or relapsed HB.

Differential painting of all five chromosome pairs of Arabidopsis thaliana revealed for the first time the interphase chromosome arrangement in a euploid plant. Side-by-side arrangement of heterologous chromosome territories and homologous association of chromosomes 1, 3 and 5 (on average in 35-50% of nuclei) are in accordance with the random frequency predicted by computer simulations. Only the nucleolus organizing region (NOR)-bearing chromosome 2 and 4 homologs associate more often than randomly, since NORs mostly attach to a single nucleolus. Somatic pairing of homologous approximately 100 kb segments occurs less frequently than homolog association, not significantly more often than expected at random and not simultaneously along the homologs. Thus, chromosome arrangement in Arabidopsis differs from that in Drosophila (characterized by somatic pairing of homologs), in spite of similar genome size, sequence organization and chromosome number. Nevertheless, in up to 31.5% of investigated Arabidopsis nuclei allelic sequences may share positions close enough for homologous recombination.

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

There are several reports of a closer-than-random colocalization of homologous chromosomes in the vegetative nuclei of diploid budding yeast. Here, we studied by fluorescence in situ hybridization (FISH) the nuclear distribution of chromosomes and found a slight tendency toward closer proximity between homologous (allelic) loci than between any nonhomologous chromosomal regions. We show that most of this preferential association is not due to vegetative (also known as somatic) pairing but is caused by the polar orientation of interphase chromosomes (Rabl orientation). We quantified the occasional loss of detectable fluorescence signals that is inherent to the FISH method. Signal loss leads to the occurrence of a single signal that may be misinterpreted as the close association of two homologous chromosomal sites. The nuclear distribution of homologous loci, when corrected for the influence of nuclear architecture and methodological faults, was not different or was only marginally different from a random relative positioning as predicted by computer simulation. We discuss here several possibilities for the residual homologous proximity that do not invoke homology-dependent vegetative pairing, and we conclude that, in diploid budding yeast, constitutive vegetative pairing is a negligible factor for the organization of the interphase nucleus.

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12Δ and slk19Δ mutants.

In Daucus carota L. subsp. sativus (Hoffm.) Arc. sixteen ideally extended mitotic pro‐metaphases with total genomic length variation of 9% were used for karyotype analysis after Giemsa C‐banding. T...

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved gamma-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

BACKGROUND The P-glyprotein (P-gp), which is a membrane channel encoded by the MDR1 gene, represents a possible explanation for multidrug resistance in human hepatoblastoma (HB). P-gp shows up-regulation in tumor cells after chemotherapy; however, to date, its exact role in HB has not been described. The authors investigated the role of the MDR1 gene in the clinical course of patients with HB and in an in vivo model of HB. They also studied the effects of the MDR1 antagonizer PSC 833 on chemotherapy in mice xenotransplanted with HB. METHODS Resected tumor specimens, including both primary tumors and recurrent tumors, from a child suffering from HB were investigated histologically. Cell suspensions from the originally removed tumor were incorporated subcutaneously into nude mice. Animals were treated with cisplatin (CDDP) plus PSC 833. MDR1 gene expression levels in the different resected tumors from the patient and in the xenotransplants after treatment were determined with polymerase chain reaction analysis. RESULTS MDR1 gene expression was increased in the patient's tumors after every course of chemotherapy from 30% to > 190%. In the xenotransplants, MDR1 gene expression was enhanced significantly after chemotherapy (PCDDP = 0.008; PCDDP+PSC = 0.002). Tumor volumes (P < 0.001) and serum α-fetoprotein levels (P = 0.0002) were significantly lower in the animals that were treated with CDDP + PSC compared with the animals that were treated with CDDP alone. CONCLUSIONS The current results suggest that MDR1 gene expression and P-gp are a potential mechanism of drug resistance in HB. The chemosensitizer PSC 833 significantly improved the effects of chemotherapy in animals xenotransplanted with HB. These data encourage further studies concerning the role of chemosensitizers in overcoming multidrug resistance in patients with HB. Cancer 2002;95:1795–801. © 2002 American Cancer Society. DOI 10.1002/cncr.10858

Pediatric hepatoblastoma (HB) is commonly treated by neoadjuvant chemotherapy and surgical tumor resection according to international multicenter trial protocols. Complete tumor resection is essential and survival rates up to 95% have now been achieved in those tumors classified as standard-risk HB. Drug resistance and occurrence of metastases remain the major challenges in the treatment of HB, especially in high-risk tumors. These conditions urgently require the development of alternative therapeutic strategies. One of those alternatives is the modulation of apoptosis in HB cells. HBs regularly overexpress anti-apoptotic proteins of the Bcl-family in comparison to healthy liver tissue. This fact may contribute to the development of chemoresistance of HB cells. Synthetic small inhibitory molecules with BH3-mimetic effects, such as ABT-737 and obatoclax, enhance the susceptibility of tumor cells to different cytotoxic drugs and thereby affect initiator proteins of the apoptosis cascade via the intrinsic pathway. Besides additive effects on HB cell viability when used in combination with cytotoxic drugs, BH3-mimetics also play a role in preventing metastasation by reducing adhesion and inhibiting cell migration abilities. Presumably, including additive BH3-mimetic drugs into existing therapeutic regimens in HB patients might allow dose reduction of established cytotoxic drugs and thereby associated immanent side effects, while maintaining the antitumor activity. Furthermore, reduction of tumor growth and inhibition of tumor cell dissemination may facilitate complete surgical tumor resection, which is mandatory in this tumor type resulting in improved survival rates in high-risk HB. Currently, there are phase I and phase II clinical trials in several cancer entities using this potential target. This paper reviews the available literature regarding the use of BH3-mimetic drugs as single agents or in combination with chemotherapy in various malignancies and focuses on results in HB cells.

Laparoscopic procedures for children and adults already provide many advantages in two-dimensional (2D) vision. Only limited experiences exist for laparoscopic three-dimensional (3D) procedures in vivo. The aim of this prospective trial was to identify indications and limitations of the 3D-system in laparoscopic minimally invasive procedures in children and adults.In a prospective quality assurance for laparoscopic 3D evaluation in children and adults, a total of 53 consecutive patients (22 children, 31 adults) were included. Laparoscopic transabdominal, retroperitoneal and thoracoscopic procedures were performed. For laparoscopic 3D imaging a Camera Control Unit (CCU), 3D monitor and 3D-TIPCAM® were used. Patient data, operative procedures and image quality of the 3D system were assessed.Of 53 patients, 22/53 were children and 31/53 adults with a mean age of 7.6 years (range, 10 months to 15 years) and 51.5 years (range, 18 to 79 years), respectively. 8/22 children were two years old or younger. No relevant difficulties occurred with nausea, fatigue, vertigo, eye blurring or double vision, burning eyes, visual fatigue, inconvenience of visual adaptation of 3D to 2D, or medical discomforts for the surgeons in both children and adults. Difficulties were mainly addressed to the small distance of the video endoscope and the organ tissue in small children and affected mainly image definition, resolution and eye focusing.Advantages of 3D over 2D were mainly considered to be of relevant benefit in adults. Subjective advantages were seen in children and adults for stereoscopic depth perception, better visualization of anatomical structures and understanding of the anatomy, as well as for complex maneuvers such as suturing.

The organization of centromeric chromatin of diploid barley (Hordeum vulgare) encoding two (α and β) CENH3 variants was analysed by super-resolution microscopy. Antibody staining revealed that both CENH3 variants are organized in distinct but intermingled subdomains in interphase, mitotic and meiotic centromeres. Artificially extended chromatin fibres illustrate that these subdomains are formed by polynucleosome clusters. Thus, a CENH3 variant-specific loading followed by the arrangement into specific intermingling subdomains forming the centromere region appears. The CENH3 composition and transcription vary among different tissues. In young embryos, most interphase centromeres are composed of both CENH3 variants, while in meristematic root cells, a high number of nuclei contain βCENH3 mainly dispersed within the nucleoplasm. A similar distribution and no preferential arrangement of the two CENH3 variants in relationship to the spindle poles suggest that both homologs meet the same function in metaphase cells.

More than half of all congenital deformities can be detected in utero. The initial surgical correction is of paramount importance for the achievement of good long-term results with low surgical morbidity and mortality.Selective literature review and expert opinion.Congenital deformities are rare, and no controlled trials have been performed to determine their optimal treatment. In this article, we present the prenatal assessment, treatment, and long-term results of selected types of congenital deformity. Congenital diaphragmatic hernia (CDH) affects one in 3500 live-born infants, while esophageal atresia affects one in 3000 and small-bowel atresia one in 5000 to 10,000. If a congenital deformity is detected and its prognosis can be reliably inferred from a prenatal assessment, the child should be delivered at a specialized center (level 1 perinatal center). The associated survival rates are 60-80% after treatment for CDH and well over 90% after treatment for esophageal or small-bowel atresia. Despite improvements in surgical correction over the years, complications and comorbidities still affect 20-40% of the treated children. These are not limited to surgical complications in the narrow sense, such as recurrence, postoperative adhesions and obstruction, stenoses, strictures, and recurrent fistulae, but also include pulmonary problems (chronic lung disease, obstructive and restrictive pulmonary dysfunction), gastrointestinal problems (dysphagia, gastro-esophageal reflux, impaired intestinal motility), and failure to thrive. Moreover, the affected children can develop emotional and behavioral disturbances. Minimally invasive surgery in experienced hands yields results as good as those of conventional surgery, as long as proper selection criteria are observed.Congenital deformities should be treated in recognized centers with highly experienced interdisciplinary teams. As no randomized trials of surgery for congenital deformities are available, longitudinal studies and registries will be very important in the future.

During recent years, minimally invasive surgery (MIS) has become the standard approach for various operations in infants and children. This also holds true for surgery in children with solid tumors. Meanwhile, more and more oncological biopsies and resections are being performed laparoscopically or thoracoscopically. Despite its increasing role in pediatric tumor surgery, the different national and international multicenter trial groups have not yet implemented MIS within guidelines and recommendations in most of the current treatment protocols. An increasing number of reports describe a potential role of MIS in the different entities of pediatric surgical oncology. Over the time, there has been a diverse development of this approach with regard to the different neoplasms. The aim of this article is to give an overview and to describe the current state of the art of MIS in pediatric solid tumors.

Response (tumour volume reduction) to induction chemotherapy has been used to stratify secondary local and systemic treatment of Intergroup Rhabdomyosarcoma Study Group III (IRSG-III) embryonal rhabdomyosarcoma (RME) in consecutive CWS-trials. To evaluate its actual impact we studied response-related treatment and outcomes.Patients with IRSG-III RME21 years and non-response (NR,33% volume reduction) in five consecutive CWS-trials were analysed and compared with partial responders (PAR, ≥ 33% reduction). The NR was reviewed and sub-classified as Objective Response (OR,0%-33% reduction) or Stable/Progressive Disease (SPD).Fifty-nine of 529 patients had NR (n = 34 OR, n = 25 SPD). Primary risk-factors including age, tumour size, and TN-classification did not differ between NR and PAR groups but NR had more patients with unfavourable sites comparatively (P = 0.04). There were no differences in primary risk-factors between OR and SPD. Significant factors associated with poor outcome in multivariate analysis were NR, TN-classification, age10 years, tumour size5 cm and therapy in older trials. After response assessment n = 24 NR continued to receive induction chemotherapy, n = 32 received other combinations and n = 3 no further chemotherapy. Forty-two non-responders were irradiated, and the tumours were completely resected in n = 20. After a median follow-up of 8 years, 34 NR are alive. Seventeen of 21 failures leading to disease-related deaths were locoregional. The five-year overall survival rate (OS) was 76 ± 4% for PAR, 79 ± 14% for OR, but only 40 ± 19% for SPD (P0.001).Response to induction chemotherapy appears to be an important surrogate marker of poor outcome in patients with SPD largely due to ineffective local control.

Total nephrectomy (TN) remains the standard treatment of unilateral Wilms tumors (uWT). The SIOP WT-2001 protocol allowed Nephron Sparing Surgery (NSS) for polar or peripherally non-infiltrating tumors.Inventory of the current SIOP NSS-experience.2,800 patients with a unilateral, localized or metastatic and an unequivocal surgical technique recorded were included. All had neo-adjuvant chemotherapy and delayed surgery. In 91 (3%) NSS was performed and in 2709 TN. Data was retrieved from the SIOP WT 2001 database.NSS group contained 65% stage I tumours and the TN group 48%. Tumor volume (at diagnosis and surgery) was significantly smaller in the NSS group. Within stage III, after NSS, 7/12 (58%) had positive margins (M +), 5 with tumor negative lymph nodes (LN-). After TN, 355/712 (55%) had M + , 182 were LN-. Treatment of M+ in the NSS group resulted in two conversions to TN (one combined with radiotherapy), three patients had radiotherapy only and in two patients local therapy, if given, was not recorded. After NSS, four recurrences occurred. For localized disease the 5-year overall (OS) and event free survival (EFS) in NSS group was 100 and 94.8 (95% CI:89.9-99.9), respectively, while OS and EFS in the TN group were 94.4 (95% CI: 93.2-95.5, log-rank test P = 0.06) and 86.5 (95% CI:85.0-88.1, log-rank test P = 0.06), respectively.NSS was only performed in 3% of patients with uWT. Despite excellent survival with few relapses, the gain of nephrons needs to be weighed against the risk to induce stage III with intensified therapy.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications., Spirodela, or duckweed, is a basal monocotyledonous plant with both pharmaceutical and commercial value. Here, the authors sequence the genome of Spirodela polyrhiza, suggesting its genome has evolved by neotenous reduction and clonal propagation, and provide a platform for future comparative genomic studies in angiosperms.

Background Human hepatoblastoma is an infrequent liver tumor in children. Although many hepatoblastomas can be treated adequately with well-defined treatment regimens, problems still persist with advanced and non-resectable tumors; in these cases, an effective chemotherapy is necessary to improve the patients' prognosis. This underlines the need for alternative anti-tumor agents in the treatment of human hepatoblastoma. The aim of this study was to investigate the therapeutic effects of topotecan, a water-soluble camptothecin analog (topoisomerase-I-antagonist), in an in vivo model of three human hepatoblastomas xenografted subcutaneously into nude mice. Procedure Hepatoblastoma cell suspensions from three children were transplanted subcutaneously into nude mice NMRI (nu/nu). Treatment with topotecan was initiated when the tumors reached a volume between 50 and 80 mm3. A dose of 6.6 mg/kg of topotecan were given intraperitoneally every 4 days on four occasions. The tumor volume development and α-fetoprotein alterations were measured and statistically analyzed. After the treatment, the tumors were investigated histologically and by immunhistochemistry. Results There was a significant reduction of tumor growth in all treated tumor xenografts vs. untreated control groups (mean relative volume 3.1 vs. 47.4; P = 0,0015–0,0079). Serum α-fetoprotein levels were reduced in all three cell lines, in two of them significantly (mean 44,535 kU/l vs. 228,883 kU/l; P = 0.005–0.246). Histologically, the tumor necrosis rates were higher and immunhistochemistry showed lower proliferation activities in the treated tumor xenografts vs. the control groups. Conclusion The data show that topotecan is an effective agent in the treatment of human hepatoblastoma xenografts. From these results, treatment with topotecan appears to be a promising alternative in the pre- and postoperative therapy of patients suffering from human hepatoblastoma. Med Pediatr Oncol 2001;37:449–454. © 2001 Wiley-Liss, Inc.

The current 'state-of-art' as to chromosome painting in plants is reviewed. We define different situations described as painting so far: i) Genomic in situ hybridisation (GISH) with total genomic DNA to distinguish alien chromosomes on the basis of divergent dispersed repeats, ii) 'Chromosomal in situ suppression' (CISS) hybridisation with chromosome-derived DNA probes and blocking of interchromosomally dispersed repeats by total genomic or C0t-1 DNA in excess, iii) exceptional cases of single chromosome painting by probes containing chromosome-specific dispersed repeats, and iv) Fluorescence in situ hybridisation (FISH) with extended contigs of large insert clones for painting of those chromosomes of a euploid complement which harbour the cloned sequences. While GISH was successfully applied in most plant hybrids and/or their derivatives, painting of individual chromosomes by CISS hybridisations of chromosome-specific DNA probes have so far not revealed convincing results in plants. The reason for this failure and the use of possible alternative approaches are discussed. At least for small plant genomes, painting by large insert single sequence clones provides a promising alternative tool to solve cytogenetic questions, which up to now could not be tackled otherwise. An example of such a painting is described in detail for Arabidopsis thaliana.

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.

Background/Purpose: Hepatoblastoma (HB) is the most common primary malignant liver tumor affecting infants and young children. The α-fetoprotein level is elevated in 95% of all children with hepatoblastoma. Therefore, it is of interest to assess targeting of the HB marker α-fetoprotein by antibody imaging. In this pilot study, the authors investigated the radioimmunoscintigraphy of xenotransplanted HB in nude mice utilizing an anti-α-fetoprotein antibody. Methods: HB cell suspensions from tumors of 3 children were transplanted subcutaneously into nude mice NMRI (nunu). A total of 200 μg of intact anti-α-fetoprotein antibody was injected intravenously into 8 animals from each HB. Before injection, the monoclonal antibody was labeled with iodine (I) 131 (specific activity of 75 MBq/mg, labeling yield of 95%) using the conventional iodogen method. Planar scintigraphic images of anesthetized mice in posterior views were acquired with a gamma camera immediately after injection, and after 1, 2, 3, 7, and 14 days. The biodistribution data were obtained by killing and dissecting animals, and the activity in the tissues was measured in a gamma counter. The α-fetoprotein levels in the animals' sera were recorded 15 days after imaging and were compared with the control group. Results: A total of 66% of the hepatoblastomas could be detected by scintigraphy. Within 24 hours, the mean specific tumor uptake in nude mice hepatoblastomas with a volume of over 1,000 mm 3 , was 14% per injected dose (±3.9%). The biological half-life of the labeled antibody complex in the tumor was 3.86 (±0.84) days. Thyroid uptake of free I-131 was 2.85% per injected dose (±1.5%) reflecting the deiodination of the labeled antibody complex. Conclusions: The results show the possibility of imaging xenotransplanted hepatoblastoma with 131 I-labeled anti-α-fetoprotein and may, in the future, determine tumor recurrence and extension, and thereby improve the prognosis of advanced HBs.

Introduction In children with hepatocellular carcinoma (pHCC) overall survival is poor. In adult HCC several antitumor properties are described in in vitro models for the use of curcumin (CUR). Methods Hepatoma cell lines (HuH6, HepT1, HepG2, HC-AFW1) were treated with CUR or, and Cisplatin (PDDT), cultures were either kept in the dark or exposed to blue light (PDT; 480 nm, 300W, 10 seconds), MTT-tests were performed. ROS production was measured. Reduction of cancer stem cells (CSC) was investigated with FACS analyses. Orthotopic growth of the pediatric hepatocellular carcinoma cell line HC-AWF1 in NSG mice was induced. By the increase serum alpha fetoprotein AFP >5 U/mL mice were treated with micellar CUR, PDDT, or CUR PDDT and compared to controls. Curcuminoid levels in serum and organ lysates as well as AFP serum levels were investigated. Results In all cell lines IC50 were significantly lower after blue light exposure than after CUR alone (p Conclusion These data prove the potential of micellar curcumin as a complementary agent in pediatric oncology to enhance the overall survival of patients with pediatric liver tumors.

A 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library by Jiang et al. (1996, Proc. Natl Acad. Sci. USA, 93, 14210-14213). We have amplified a partially homologous 809 bp sequence from barely genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescent in situ hybridization (FISH). Sequence analysis showed this barley homolog of pSau3A9 to have high similarity to the integrase region of the polyprotein gene of Ty3/gypsy group retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described by Aragon-Alcaide et al. (1996, Chromosoma, 105, 261-268) and may represent all or part of the long-terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.

The resolution of the chromosomal positions of six high- and one low-copy sequences by oligonucleotide-primed in situ (PRINS) labelling was compared with corresponding data obtained after fluorescent in situ hybridization (FISH) on field-bean and barley chromosomes. While PRINS proved to be suitable for the rapid detection of high-copy tandem repeats at the same loci as those revealed by FISH, no clear PRINS signal was obtained for the low-copy family of vicilin genes at their locus on field-bean chromosome II. This indicates that localization of short target sequences by primer extension via Taq polymerase in situ does not yet provide a resolution equal, or superior, to FISH on plant chromosomes. Therefore, the use of a cocktail of chromosome-specific single-copy sequences as primers for PRINS is no alternative for the not as yet feasible chromosome painting in plants.

Penile agenesis is a rare congenital anomaly in which early gender reassignment is recommended. In the past, multiple operations were carried out to form feminized external genitalia. The authors performed a definite genital reconstruction in a neonate using the posterior sagittal approach. Preoperatively, a magnetic resonance imaging (MRI) scan demonstrated erectile tissue, which was preserved and incorporated into the genital plasty. The operation included bilateral orchiectomy, urethral reconstruction, sigmoid vaginal replacement, and formation of the labia. The authors discuss the controversy around gender assessment. They emphasize the urethral hypotrophy to be a main challenge in this anomaly and conclude that immediate complete reconstruction is possible and should be performed in patients with this delicate condition.

A total of 307 central venous access devices were implanted by open surgery in 268 pediatric oncology patients. Their data were retrospectively evaluated from a surgical point of view. The cumulative duration of implantation of 116 subcutaneous tunneled Catheter (STC) was 12,206 days and 45,524 days for 191 portsystems. According to the duration in each group, ports were superior to STC when infection, complication and occlusion were taken into consideration. Infection of the ports was more frequent in children under 3 years than in older patients. Conversely, in the STC-group, children over 10 years presented with the highest rate of infection episodes. The number of complications was high in young children for both types of catheter, decreasing with age. Occlusion rates were similar for both systems and were not age related. However, thrombolysis was more successful in portsystems and few catheters had to be removed for this reason. The age-related data of the presented study are discussed with regard to the surgical procedure. Recommendations are made concerning the adequate choice of catheter system, the surgical technique and postoperative management. Additionally, the medicotechnical aspect of different catheter types is discussed and some suggestions and/or requests are directed to the industrial developing laboratories.

A comprehensive survey of the molecular-cytogenetic features of the Vicia faba chromosome complement (2n = 12) is given. It includes previous as well as new original data. Various Giemsa, restriction endonuclease and fluorochrome banding patterns, azacytidine-mediated segment extension, replication patterns, lateral A/T asymmetry and sequence localization data for tandemly arranged simple sequence repeats, dispersed repeats and coding sequences as well as histone acetylation patterns are considered. This allows not only to distinguish and characterize telomeres, subtelomeres, centromeres and the NOR, but also the structure of the 5S rRNA gene loci and two main types of interstitial heterochromatin. Additionally, it offers physical landmarks within euchromatic areas. Thus, the field bean genome, exemplified by the reconstructed karyotype ACB, belongs to the cytogenetically best investigated plant genomes.

The karyotype of H. annuus was analysed by computer-aided image processing with respect to the chromosome length, arm ratio, occurrence and chromosomal position of intercalary heterochromatin and the location of 18S/25S and 5S ribosomal RNA genes. The karyotype was subdivided into a group of four acrocentric chromosome pairs, of which two were distinguishable by HKG (HCl, KOH, Giemsa) banding and a group of 13 meta- to submetacentric pairs. The latter could be subdivided into seven pairs with one and six pairs with two HKG bands. Three pairs of submetacentric satellite chromosomes revealed 18S/25S rDNA loci after fluorescence in situ hybridization (FISH) and silver staining. A fourth, smaller and possibly inactive, locus occurred in the terminal position on a metacentric pair. One submetacentric satellite chromosome pair revealed a 5S rRNA gene locus in the pericentromeric position; a second locus marked a submetacentric pair with one HKG band. The C-banding technique marks exclusively centromeric heterochromatin. Measurements of chromosomes in combination with Giemsa banding and FISH enabled the discrimination of most chromosome pairs of the sunflower.

Mouse embryonic stem (ES) cells were used as an experimental model to study the effects of electromagnetic fields (EMF). ES-derived nestin-positive neural progenitor cells were exposed to extremely low frequency EMF simulating power line magnetic fields at 50 Hz (ELF-EMF) and to radiofrequency EMF simulating the Global System for Mobile Communication (GSM) signals at 1.71 GHz (RF-EMF). Following EMF exposure, cells were analyzed for transcript levels of cell cycle regulatory, apoptosis-related, and neural-specific genes and proteins; changes in proliferation; apoptosis; and cytogenetic effects. Quantitative RT-PCR analysis revealed that ELF-EMF exposure to ES-derived neural cells significantly affected transcript levels of the apoptosis-related bcl-2, bax, and cell cycle regulatory "growth arrest DNA damage inducible" GADD45 genes, whereas mRNA levels of neural-specific genes were not affected. RF-EMF exposure of neural progenitor cells resulted in down-regulation of neural-specific Nurr1 and in up-regulation of bax and GADD45 mRNA levels. Short-term RF-EMF exposure for 6 h, but not for 48 h, resulted in a low and transient increase of DNA double-strand breaks. No effects of ELF- and RF-EMF on mitochondrial function, nuclear apoptosis, cell proliferation, and chromosomal alterations were observed. We may conclude that EMF exposure of ES-derived neural progenitor cells transiently affects the transcript level of genes related to apoptosis and cell cycle control. However, these responses are not associated with detectable changes of cell physiology, suggesting compensatory mechanisms at the translational and posttranslational level.

Drug resistance and metastasis remain major challenges in the treatment of high-risk hepatoblastoma (HB) and require the development of alternative therapeutic strategies. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We investigated the impact of apoptosis sensitisers, BH3-mimetics, on the interaction between the host and HB to reduce tumour growth and dissemination while enhancing immunity. BH3-mimetics, such as obatoclax and ABT-737, enhanced the apoptosis-inducing effect of TRAIL and TNF-α resistant HB cells (HepT1 and HUH6). Tumour cell migration was inhibited by ABT-737 and more markedly by obatoclax. In an orthotopic model of HB, tumour uptake was reduced when the cells were pretreated with low concentrations of obatoclax. Only 1 of 7 mice developed HB in the liver, compared with an incidence of 0.8 in the control group. In summary, our study showed that apoptosis sensitisers had broader effects on HB cells than expected including migration and susceptibility to cytokines in addition to the known effects on drug sensitization. Sensitising HB to apoptosis may also allow resistant HB to be targeted by immune cells and prevent tumour cell dissemination.

We present novel filter elements with an asymmetric angle dependent transmission based on high-contrast gratings. Asymmetric means a different efficiency for positive and negative incidence angles. Our approach provides the realization of asymmetric direction selective filters by using blaze-like grating structures combined with subwavelength high contrast gratings respectively grating periods in the resonance domain. We also discuss the influence of the effective medium theory on the transmission function depending on the angle of the incident light. For realization of those high contrast gratings Silicon is chosen as material with high refractive index and adequate compatibility with semiconductor fabrication.

In order to test crossability of Lolium and Dactylis, a total of 4126 florets of six different varieties and ploidy levels of Lolium multiflorum Lam. and L. perenne L. were pollinated using a blend of pollen from two cultivars and three ecotypes of Dactylis glomerata L. Additionally, reciprocal pollinations were carried out on 363 florets of two D. glomerata cultivars with pollen of L. multiflorum. Both pre- and post-zygotic cross barriers were strong. Auxin application was effective in overcoming post-zygotic barriers. One viable hybrid plant arose from 16 embryos. The hybrid between L. multiflorum and D. glomerata showed characteristics of both parents. The symmetric character of the hybrid was confirmed by genomic in situ hybridization. Backcross pollinations with pollen of both parents yielded two plants from five embryos with L. multiflorum only. Both the production of allopolyploid hybrids having characters of both species and the transfer of useful characters of D. glomerata into L. multiflorum should be possible.

In situ localization of short low- or single-copy sequences is still difficult in plants. One solution to this problem could be the use of large yeast artificial chromosomes (YACs) for fluorescence in situ hybridization. Two YACs specific for a single copy marker on the long arm of the NOR-chromosome 2 of tomato (Lycopersicon esculentum) were selected. Both probes hybridized exclusively to this chromosome, although one produced a slightly dispersed hybridization signal. Hybridization of these YACs onto potato chromosome showed a clear single locus on the homoeologous potato chromosome in both cases.