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Biomedical subjects

P Hutzler

Publications and source records attributed to P Hutzler.

At least 19 recordsLinked to original sources
PubMed2001

Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Adenosine Triphosphate↗
PubMed2000

Characterization of Ggrb4, an adapter protein interacting with Bcr-Abl.

We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2-mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl-induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624) (Blood. 2000;96:618-624)

Adaptor Proteins, Signal Transducing↗
PubMed2000

Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study.

Large radiation doses cause postradiation vascular hyperpermeability by disrupting endothelia. The cumulative sequences of small doses (fractionated radiotherapy) standard in clinical practice cause it too, but not by endothelial disruption: the mechanisms are unknown. In this study, correlated fluorescent and ultrastructural localisation of a tracer revealed the architecture, fine structure and function of microvessels in mouse AT17 tumours, before and after 42 Gy fractionated radiation. Before irradiation, tumour vascular permeability lay in the normophysiological range defined by the gut and cerebral cortex. A double barrier regulated permeability: vesicular transport through the endothelial wall required approximately 2 h and then the basement membrane charge barrier trapped tracer for 2 h longer. Irradiation abolished the double barrier: tracer passed instantly through both endothelial wall and underlying basement membrane, forming diffusion haloes around microvessels within 2-5 min. Structurally, irradiated tumour microvessels were lined by a continuous and vital endothelium with closed interendothelial junctions; endothelial basement membranes were intact, though loosened. Irradiated endothelia exhibited extremely active membrane motility and intracellular vesicle trafficking. Radiation treatment raised vascular permeability by enhancing transendothelial transcytosis, and by altering the passive filter properties of the subendothelial basement membrane. This type of vascular hyperpermeability should be susceptible to pharmacological modulation.

Adenocarcinoma↗
PubMed2000

Molecular genetic changes in metastatic primary Barrett's adenocarcinoma and related lymph node metastases: comparison with nonmetastatic Barrett's adenocarcinoma.

Lymph node metastasis is one of the strongest negative prognostic factors for patients with Barrett's adenocarcinoma (BCA). However, despite the importance of the metastatic process in BCA, the molecular basis of it remains poorly understood. To search for cytogenetic events associated with metastasis in regional or distant lymph nodes in BCA, we investigated 8 primary BCA and their lymph node metastases and compared them with 18 nonmetastatic BCA. In metastatic primary BCA, we observed significantly more DNA gains on 3q (P = .013), 17q (P = .019), and 22q (P = .021) compared with nonmetastatic primary BCA. No statistically significant correlation could be observed between DNA copy number changes and the histopathologic stage, grade, or survival (P > .05). The most frequent alteration observed only in lymph node metastases but not in the related primary tumor was loss of 2q (5 of 8). Coamplification of 7p and chromosome 17 was found in 6 of 8 lymph node metastases. A comparison of DNA copy number changes between primary tumors and their corresponding metastases indicated a high degree of genetic heterogeneity. Fluorescence in situ hybridization analysis demonstrated the involvement of the Her-2/neu gene in primary BCA and its related lymph node metastases. Each of the investigated primary tumors and related lymph node metastases also showed striking heterogeneity with respect to Her-2/neu, with several areas displaying different levels of amplification. In summary, our data indicate that DNA copy number changes on 2q, 3q, 7p, 17q, and 22q may be involved in the metastatic process in BCA. Furthermore, the striking genetic heterogeneity that we found between primary BCA and its lymph node metastases may underlie BCA's poor responsiveness to therapy and could help explain why prognostic biomarkers measured exclusively in primary tumors give an incomplete view of the biologic potential of BCA.

Adenocarcinoma↗
PubMed2000

Increasing chromosome 1 copy number parallels histological progression in breast carcinogenesis.

Chromosome 1 copy number in the benign breast lesions hyperplasia and atypical duct hyperplasia (ADH) was investigated using fluorescence in situ hybridization on paraffin sections. Progression of chromosome 1 changes occurring in parallel with histological progression from normal through hyperplasia and ADH to ductal carcinoma in situ (DCIS) and invasive carcinoma was also assessed, both overall and within individual patients. The mean signal number for normal cells was 1.14, while that for hyperplasia was 1.56 and ADH was 1.5, while values for DCIS of 1.95 and invasive duct carcinoma of 1.79, were higher (P < 0.001). Six of the seven cases also showed a significant trend towards an increasing proportion of cells with greater than 2 signals per nucleus occurring with histological progression (P < 0.001). These results support the concept that benign proliferative breast disease is a biological precursor of in-situ and invasive ductal carcinoma, the early histological changes possibly indicating a field effect with further genetic changes required for the development of a malignant phenotype.

Breast Neoplasms↗
PubMed2000

Evaluation of c-erbB-2 overexpression and Her-2/neu gene copy number heterogeneity in Barrett's adenocarcinoma.

Amplification of the Her-2/neu gene is accompanied by overexpression of its cell surface receptor product, c-erbB-2 protein. To investigate the degree of intratumoural heterogeneity we applied immunohistochemistry in primary Barrett's adenocarcinoma (BCA) (n = 6) and dysplasia adjacent to the carcinoma (n = 4). In addition, fluorescence in situ hybridisation (FISH) was performed in primary BCA (n = 5) and dysplastic areas (n = 4). For an objective evaluation digital image analysis and laser scanning microscopy were used. Five of six BCA showed a marked intratumoral heterogeneous staining pattern ranging from areas in which the tumour cells were negative or faintly positive to tumour areas with a strong staining of the entire membrane. Among the two dysplastic areas also a heterogeneous staining pattern was observed. FISH analysis revealed marked heterogeneity of intratumoral gene copy number changes in all BCA showing populations with different fractions of cells with polysomy, low level amplification and high level amplification. One dysplasia showed a minor population with Her-2/neu signal clusters. In conclusion, we observed marked intratumoural heterogeneity of c-erbB-2 protein overexpression and Her-2/neu gene copy number in the majority of the primary BCA analyzed. Digital image analysis and laser scanning microscopy were helpful in quantifying the variations in protein expression and DNA copy number in individual tumour cells. The observed heterogeneity could hamper the exact diagnostic determination of the c-erbB-2 status in small biopsies and possibly influence the effectiveness of a potential c-erbB-2 targeting therapy. Figures on http://www.esacp.org/acp/2000/20-1/walch.htm+ ++.

Adenocarcinoma↗
PubMed2000

Tumor-derived mutated E-cadherin influences beta-catenin localization and increases susceptibility to actin cytoskeletal changes induced by pervanadate.

E-cadherin participates in homophilic cell-to-cell adhesion and is localized to intercellular junctions of the adherens type. In the present study, we investigated the localization of adherens junction components in cells expressing mutant E-cadherin derivatives which had been previously cloned from diffuse-type gastric carcinoma. The mutations are in frame deletions of exons 8 or 9 and a point mutation in exon 8 and affect the extracellular domain of E-cadherin. Our findings indicate that E-cadherin mutated in exon 8 causes beta-catenin staining at lateral cell-to-cell contact sites and, in addition, abnormally located beta-catenin in the perinuclear region. Moreover, the various mutant E-cadherin derivatives increased the steady-state levels of alpha- and beta-catenin and were found in association with these catenins even after induction of tyrosine phosphorylation by pervanadate. Sustained pervanadate treatment led, however, to rounding-up of cells and induction of filopodia, changes which were first detectable in cells expressing E-cadherin mutated in exon 8. The deterioration of the cell contact was not accompanied with disassembly of the E-cadherin-catenin complex. Based on these observations, we propose a model whereby in the presence of mutant E-cadherin tyrosine phoshorylation of components of the cell adhesion complex triggers loss of cell-to-cell contact and actin cytoskeletal changes which are not caused by the disruption of the E-cadherin-catenin complex per se, but instead might be due to phosphorylation of other signaling molecules or activation of proteins involved in the regulation of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1999

Tumour-associated E-cadherin mutations alter cellular morphology, decrease cellular adhesion and increase cellular motility.

A major function of the cell-to-cell adhesion molecule E-cadherin is the maintenance of cell adhesion and tissue integrity. E-cadherin deficiency in tumours leads to changes in cell morphology and motility, so that E-cadherin is considered to be a suppressor of invasion. In this study we investigated the functional consequences of three tumour-associated gene mutations that affect the extracellular portion of E-cadherin: in-frame deletions of exons 8 or 9 and a point mutation in exon 8, as they were found in human gastric carcinomas. Human MDA-MB-435S breast carcinoma cells and mouse L fibroblasts were stably transfected with the wild-type and mutant cDNAs, and the resulting changes in localization of E-cadherin, cell morphology, strength of calcium-dependent aggregation as well as cell motility and actin cytoskeleton organization were studied. We found that cells transfected with wild-type E-cadherin showed an epitheloid morphology, while all cell lines expressing mutant E-cadherin exhibited more irregular cell shapes. Cells expressing E-cadherin mutated in exon 8 showed the most scattered appearance, whereas cells with deletion of exon 9 had an intermediate state. Mutant E-cadherins were localized to the lateral regions of cell-to-cell contact sites. Additionally, both exon 8-mutated E-cadherins showed apical and perinuclear localization, and actin filaments were drastically reduced. MDA-MB-435S cells with initial calcium-dependent cell aggregation exhibited decreased aggregation and, remarkably, increased cell motility, when mutant E-cadherin was expressed. Therefore, we conclude that these E-cadherin mutations may not simply affect cell adhesion but may act in a trans-dominant-active manner, i.e. lead to increased cell motility. Our study suggests that E-cadherin mutations affecting exons 8 or 9 are the cause of multiple morphological and functional disorders and could induce the scattered morphology and the invasive behaviour of diffuse type-gastric carcinomas.

Actins↗
PubMed1999

Intratumoral heterogeneity in breast carcinoma revealed by laser-microdissection and comparative genomic hybridization.

To evaluate the potential cytogenetic heterogeneity in breast carcinoma, several small cell groups (each consisting of 20 to 50 cells) were investigated within paraffin sections. By laser-microdissection, three to seven cell groups were taken per case. The DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR), and the samples were analyzed by CGH for chromosomal gains and losses. Two ductal invasive breast carcinomas, one of them with two lymphnode metastases, were investigated. To compare the results from the small samples, CGH was also performed on DNA isolated from the tumorous regions of three to five serial sections (10(7) to 10(6) cells). The aberrations observed in the microdissected tumor samples were multiple and involved up to 14 different chromosomal or subchromosomal regions. The most frequent changes were gains on chromosomes 12q (14/20) and 20q (16/20), and loss on 13q (12/20). Some aberrations have rarely been detected (e.g., loss on 2p, gain on 8q). Comparing chromosomal imbalances in primary tumors and lymph node metastases, more consistent changes were found between the primary tumor and its corresponding metastases than between both primary tumors. The laser-microdissected samples in general showed more chromosomal aberrations than DNA isolated from several tumor sections. Our CGH results were confirmed by fluorescence in situ hybridization (FISH) for the chromosomal regions of centromere 1 and 20, and 20q13. In addition, microsatellite analyses on 31 samples confirmed our CGH findings for selected chromosome regions 2p and 11q. It can be concluded that there is a distinct intratumoral heterogeneity in primary breast tumors as well as in the corresponding lymph node metastases. The combination of microdissection and CGH enabled us to detect cytogenetic aberrations from important clones which are missed when analyzing DNA extracted from large cell numbers.

Breast Neoplasms↗
PubMed1999

20q13.2 amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast.

The 20q13 region harboring recently described putative oncogenes is frequently amplified in invasive ductal carcinoma (IDC). The aim of this study was to examine the 20q13 copy number in intraduct hyperplasia (IH), atypical duct hyperplasia (ADH), and ductal carcinoma in situ (DCIS) adjacent to IDC. In 5 patients, comparative genomic hybridization (CGH) after laser microdissection revealed 20q13 amplification in four of five cases of IH, in all of three cases of IH with atypia, all five of DCIS, and all five of IDC. Fluorescence in situ hybridization (FISH) confirmed the amplification at 20q13.2 in IH in the two specimens analyzed. The amplification rate, however, was higher in DCIS and IDC. In phenotypically normal ductal epithelium normal values were found for 20q13 copy number by FISH (n=2) and CGH (n=5). Although the number of cases presented here is small, our results suggest that mutations in the 20q13.2 region in IH may be associated with accelerated proliferation and hyperplasia of the ductal epithelium. Progression to DCIS and ICD is accompanied by a further increase in the 20q13.2 copy number.

Breast Neoplasms↗
PubMed1999

Anatomic localization of 24- and 96-h particle retention in canine airways.

Long-term retention of particles in airways is controversial. However, precise anatomic localization of the particles is not possible in people. In this study the anatomic location of retained particles after shallow bolus inhalation was determined in anesthetized, ventilated beagle dogs. Fifty 30-cm(3) boluses containing monodisperse 2.5-micron polystyrene particles (PSL) were delivered to a shallow lung depth of 81-129 cm(3). At 96 h before euthanasia, red fluorescent PSL were used; at 24 h, green fluorescent PSL and (99m)Tc-labeled PSL were used. Clearance of (99m)Tc-PSL was measured during the next 24 h. Sites of particle retention were determined in systematic, volume-weighted random samples of microwave-fixed lung tissue. Precise particle localization and distribution was analyzed by using gamma counting, conventional fluorescence microscopy, and confocal microscopy. Within 24 h after shallow bolus inhalation, 50-95% of the deposited (99m)Tc-PSL were cleared, but the remaining fraction was cleared slowly in all dogs, similar to previous human results. The three-dimensional deposition patterns showed particles across the entire cross-sectional plane of the lungs at the level of the carina. In these locations, 33 +/- 9.9% of the retained particles were found in small, nonrespiratory airways (0.3- to 1-mm diameter) and 49 +/- 10% of the particles in alveoli; the remaining fraction was found in larger airways. After 96 h, a similar pattern was found. These findings suggest that long-term retention in airways is at the bronchiolar level.

Aerosols↗
PubMed1998

Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester.

The aim of this study was to investigate the possibility of using the benzyl ester of hyaluronic acid (HYAFF 11), a recently developed semisynthetic resorbable material, as a scaffold for the culture of human nasoseptal chondrocytes in tissue-engineering procedures of cartilage reconstruction. Different techniques such as immunohistochemistry, scanning electron microscopy, and confocal laser scanning microscopy were used to study the behavior, morphology, and phenotype expression of the chondrocytes, which were initially expanded and then seeded on the material. The nonwoven cell carrier allowed good viability and adhesivity of the cells without any surface treatment with additional substances. Furthermore, the cultured cells expressed cartilage-specific collagen type II, indicating that they were able to redifferentiate within the scaffold of HYAFF 11 and were able to retain a chondrocyte phenotype even after a long period of in vitro conditions. Nevertheless, the expression of collagen type I, which was produced by dedifferentiated or incompletely redifferentiated chondrocytes, was noticeable. Additional data were obtained by subcutaneous implantation of samples seeded with human cells in the in vivo model of the athymic nude mouse. The results after 1 month revealed the development of tissue similar to hyaline cartilage. This study is promising for the use of this scaffold for tissue engineering of cartilage replacements.

Animals↗
PubMed1998

Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor.

Intravital lectin perfusion was combined with computer-guided scanning digital microscopy to map the perfused elements of the vasculature in tumor-bearing mice. High-precision composite images (spatial precision 1.3 micron and optical resolution 1.5 micron) were generated to permit exact positioning, reconstruction, analysis, and mapping of entire tumor cross-sections (c. 1 cm in diameter). Collation of these mosaics with nuclear magnetic resonance maps in the same tumor plane identified sites of rapid contrast medium uptake as tumor blood vessels. Digitized imaging after intravital double labeling allowed polychromatic visualization of two different types of mismatched staining. First, simultaneous application of two lectins, each bearing a different fluorochrome, revealed organ-specific differential processing in the microvascular wall. Second, sequential application of two boluses of one lectin, bearing different fluorochromes successively, distinguished between double-labeled microvessels, representing efficiently perfused vascular segments, and single-labeled microvessels, with inefficient or intermittent perfusion. Intravital lectin perfusion images of blood vessels in the vital functional state thus highlighted biologically significant differences in vessel function and served as high-resolution adjuncts to MR imaging.

Adenocarcinoma↗
PubMed1997

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-micron and 15-micron paraffin-embedded tissue sections from prostatic carcinoma.

Interphase fluorescence in situ hybridization (FISH) was performed on 15-micron-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific alpha-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-micron sections with signal counts obtained from 5-micron sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (chi 2 test), we transferred the signal frequencies into a cytogenetic classification (-7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-micron thick sections in all 19 tumors, polysomy 7 (> 3 spots) in 18/19 cases, and monosomy 7 (-7) in 13/19 cases. In 5-micron sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-micron or 15-micron sections, the following discrepancies were noted: in 15-micron sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-micron sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-micron) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-micron) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers.

Adenocarcinoma↗
PubMed1997

Numerical aberrations of chromosome 7 detected in 15 microns paraffin-embedded tissue sections of primary cutaneous melanomas by fluorescence in situ hybridization and confocal laser scanning microscopy.

Fluorescence in situ hybridization (FISH) to sections of formalin-fixed paraffin-embedded archival tissues allows the detection of gene or chromosome copy number changes in interphase cell nuclei within the histological context and thus may be of particular interest in tumor pathology. In this report, we describe the application of FISH to thick (15 microns) paraffin sections of 7 primary cutaneous malignant melanomas. A chromosome 7-specific centromeric DNA probe was used to detect numerical aberrations of chromosome 7. By optical sectioning using confocal laser scanning microscopy (CLSM) only complete, uncut interphase cell nuclei were scored. The mean percentage (+/- SEM) of melanoma cell nuclei with three hybridization spots was (20.7 +/- 2.8)%; (6.8 +/- 1.0)% of nuclei showed one spot and (5.0 +/- 1.2)% four or more spots. The frequency distribution of spot numbers among melanoma cell nuclei and normal keratinocyte nuclei was significantly different (chi(2) = 176.8, df = 5, p < 0.001). Trisomy 7 was detected in all 7 cases analyzed, mostly associated with monosomy 7 or polysomy 7. The approach used in our study and the data obtained could be useful for further studies designed to investigate a possible involvement of chromosome 7 in melanocytic tumor progression.

Adult↗