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H Spring

Publications and source records attributed to H Spring.

At least 55 records · Page 3Linked to original sources
PubMed1991

Faithful in vivo transcription termination of Xenopus laevis rDNA. Correlation of electron microscopic spread preparations with S1 transcript analysis.

DNA sequencing and subsequent functional in vitro analysis of the Xenopus laevis rDNA transcription termination has led to the identification of three transcription termination sequence elements: T1, located at the 3' end of the 28S rDNA; T2, a putative processing site 235 bp downstream of T1; T3, the principal terminator positioned 215 bp upstream of the gene promoter. As demonstrated for nuclear run-off assays, T3 was found to be the main terminator for Xenopus rDNA transcription. These in vitro data are in obvious contradiction to results obtained by electron microscopic (EM) spread preparations from rapidly isolated amplified oocyte nucleoli, i.e., an rDNA chromatin probe thought to represent the in vivo situation, indicative of transcription termination at sites T1-2. However, most interestingly, T3 had--again by the EM method--been identified as the exclusive terminator for NTS spacer transcription units. In order to answer the question of whether read-through transcription of the complete rDNA spacer sequence is obligatory for 40S pre-rRNA in vivo transcription, we analyzed several hundreds of spread rRNA genes from Xenopus oocyte nucleoli in great detail, applying two different spreading procedures, e.g., dispersal of amplified oocyte nucleoli shortly in detergent-free or detergent containing low-salt media prior to the EM spreading technique. Quantitation of EM spreads resulted in the finding that read-through rDNA spacer transcription beyond T1-2 termination sites (i.e., indicative of T3 transcription termination) can be visualized for the in vivo situation at a frequency of less than 3% of rRNA genes analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Working with the confocal scanning UV-laser microscope: specific DNA localization at high sensitivity and multiple-parameter fluorescence.

The use of fast-staining DNA-specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals. Here we report the characteristics and application of a CSLM, which was adapted for UV-excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple-parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double-stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.

Animals↗
PubMed1990

Mucoepidermoid carcinoma of uterine cervix stage IB. Long-term follow-up, histochemical and immunohistochemical study.

Twelve women with mucoepidermoid carcinoma of the cervix uteri were followed for 2-15 years after diagnosis. Three patients died within 14 months. All had lymph node metastases and/or vascular involvement and exhibited tumor invasion to a depth of 1.2-3.2 cm. Mucoepidermoid carcinoma is defined as a tumor with the appearance of squamous cell carcinoma without any glandular pattern and with demonstrable intracellular mucin. The mucin is best demonstrated by alcian blue and periodic acid-Schiff-diastase. In 265 cases of squamous cell carcinoma, stage IB, lymph node metastases were present in 14%. In the cases of mucoepidermoid carcinoma, the prevalence of nodal metastases was 33%. Because mucoepidermoid carcinomas appear to be more aggressive lesions than squamous cell carcinomas are, it may be advisable to stain all cervical squamous carcinomas for mucin if they demonstrate finely vacuolated cytoplasm and lack peripheral palisading. Immunohistochemical studies for carcinoembryonic antigen (CEA), keratin, and epithelial membrane antigen were positive in all tumors to varying degrees. The detection of CEA may be of additional help in establishing a diagnosis.

Adult↗
PubMed1990

Methodical aspects of 3-D reconstruction of chromatin architecture in mouse trophoblast giant nuclei.

Precise 3-D reconstruction of the spatial organization of murine trophoblast giant-cell chromatin is a prerequisite for detailed investigations on the fine structural changes in chromatin-fibre organization during the trophoblast endomitotic cell cycle. It appears very likely that sequential fine structural changes in the chromatin arrangement are concomitant with the changes in the gene expression pattern of these cells during the early phase of murine gestation. The complex intra-nuclear chromatin organization of mouse trophoblast giant nuclei was investigated in permanent tissue preparations which had been stained with a DNA-specific dye. The spatial chromatin arrangement was examined in fluorescence with a confocal scanning laser microscope. Serial optical sections were recorded and subjected to a computer-based 3-D reconstruction method which is suitable even for very complex biological structures.

Algorithms↗
PubMed1990

Towards light microscopic imaging of hydrated 'native' ribosomal RNA genes. A combined video microscopic and transmission electron microscopic analysis.

Investigations of the higher-order structure of 'native' genes as well as antibody localization to defined gene chromatin areas require elaborate light microscopic imaging of the chromatin structure of interest, such as visualization of unfixed and unstained gene transcription units. Since all our present structural information on the, relatively, small nucleolar rRNA genes has been obtained using transmission electron microscopy of spread genes following fixation, staining and complete dehydration, this type of EM specimen was our first specimen for LM imaging. Using Normarksi differential interference contrast video microscopy, it was found that spread EM chromatin can be sufficiently visualized by light microscopy to allow precise correlation with the conventional TEM image. In addition, it is demonstrated that video-enhanced LM can provide enough contrast enhancement for rapid visualization of spread 'native' rRNA genes in slightly fixed, fully hydrated, unstained gene chromatin.

Animals↗
PubMed1990

Localization of cathepsin B in two human lung cancer cell lines.

We demonstrated the cysteine proteinase cathepsin B in two human lung tumor cell lines by cytochemical and immunocytochemical methods. The cell lines were derived from a squamous cell carcinoma of the lung (HS-24) and a metastasis to the adrenal gland from an adenocarcinoma of the lung (SB-3). For comparison and control, normal human lung fibroblasts cells (Wi-38) were also investigated. Intracellular cathepsin B activity was detected in all three cell lines. SB-3 and the normal fibroblast cells showed almost equal cathepsin B activity, which was considerably stronger than that in the HS-24 cells. Specific inhibitors for cathepsin B (E64, leupeptin, antipain) suppressed its activity completely. Stefin A, the physiological cathepsin B inhibitor, was less effective; this might depend on its limited penetrability into living cells. Localization of the cathepsin B was performed by conventional immunofluorescence microscopy and laser scanning microscopy. With specific anti-cathepsin B antibodies, the enzyme was localized in HS-24, SB-3, and Wi-38 fibroblast cells within perinuclear granules representing the lysosomal compartment. In the SB-3 cells, we additionally localized a minor fraction of the enzyme bound to the plasma membrane in a speckled distribution, accessible to the antibodies from the outside. This direct demonstration of cathepsin B distribution supports biochemical data about the dual localization of the enzyme in tumor cells. It also supports the possibility of a direct involvement of cathepsin B in the degradation of the extracellular matrix, and thus a contribution of the enzyme in invasion and metastasis.

Adenocarcinoma↗
PubMed1988

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos.

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 micron) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.

Animals↗
PubMed1988

DNA-fluorescence of mammalian intra-nucleolar chromatin detected by confocal laser scanning microscopy (CLSM).

The complex spatial DNA distribution in the mammalian interphase nucleus was investigated in Feulgen stained thick sections through mouse trophoblast giant nuclei after Lowicryl embedding. DNA-fluorescence was visualized using confocal laser scanning microscopy. Our results show that the spatial arrangement of major interphase chromatin areas can be precisely documented, including the distribution of small intra-nucleolar chromatin zones.

Animals↗
PubMed1988

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification.

Light microscopic autoradiographs of 3H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.

Animals↗
PubMed1986

Synthesis, antitumor activity, distribution and toxicity of 4-[4-[bis(2-chloroethyl)amino]phenyl]-1-hydroxybutane-1 1-bisphosphonic acid (BAD), a new lost derivative with increased accumulation in rat osteosarcoma.

The aim of this study was to investigate whether the newly synthesized bisphosphonic acid-linked N-Lost derivative BAD retains bone-seeking and cytostatic properties. The paper describes experiments on mutagenicity in vitro and on toxicity in vivo. BAD is characterized by very low mutagenic activity toward histidine auxotrophic Salmonella typhimurium strains. Cytotoxic effects were tested in rat osteosarcoma and in Walker carcinosarcoma 256B. The LD50 of i.v. injected BAD was 146 mg/kg. Acute toxicity is probably caused by calcium complexing of the bisphosphonate part of the molecule. Labeling experiments showed moderate accumulation in bone and osteosarcoma, as well as in lung metastases. BAD effected high tumor growth inhibition in osteosarcoma and Walker carcinosarcoma-bearing rats and marked prolongation of survival; histologic and radiographic examination revealed rapid calcification of osteosarcoma and lung metastases. BAD-pretreatment produced protective effects against osteolysis induced by intratibially implanted Walker carcinosarcoma ascites cells. The cytostatic efficacy of equitoxic doses of BAD in rat osteosarcoma is comparable to that of dacarbazine and in Walker carcinosarcoma to that of melphalan.

Animals↗
PubMed1985

Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy.

The three-dimensional arrangement of the chromatin components within the nucleolar organizer regions (NORs) from living oocyte nuclei was investigated. As a suitable cell system we chose vitellogenic oocytes of the orthopteran insect Acheta. This cell type is particularly attractive for analysis of nucleolar chromatin, since structural and functional aspects of NORs during early oogenic stages (including the association of NORs with amplified rDNA copies) are particularly well known (Lima-de-Faria 1974). In the course of the present study we first identified putative chromosomal NORs in isolated nuclei of mid-diplotene oocytes according to morphological characteristics using differential interference contrast (DIC) or phase contrast light microscopy. The presence of actively transcribing chromosomal NORs during this late stage of Acheta oogenesis obviously had been overlooked by previous investigators, probably due to difficulties of chromosome visualization. For a more detailed ultrastructural analysis, NORs were gently sedimented from opened nuclei and processed for sectioning using a modified "end-embedding" procedure (Mott and Callan 1975; Spring and Franke 1981). A small number of thick and thin sections could be made from individual NORs. Sections were analyzed by light microscopy, conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Whereas the structural connection of NORs to the chromosome axis and also the general arrangement of active nucleolar genes within the NOR complex could be seen with TEM, the visualization of individual nucleolar genes and the organization of transcription complexes was only possible using bright field STEM of thick sections at low temperature.

Animals↗
PubMed1984

Intraosseously transplantable osteosarcoma with regularly disseminating pulmonary metastases in rats.

An intratibially transplantable osteogenic sarcoma in rats, that regularly disseminates osteogenic metastases in the lung, is described. The tumor take rates exceeded 90% 30 days after transplantation. Pulmonary metastases were diagnosed in about 99% of the animals, while metastases in the kidneys (14%), lymph nodes (13%) and liver (3%) occurred less frequently. The mean survival time of untreated animals varied between 36 and 48 days, depending on the age at transplantation. Histologic and scintigraphic findings are reported.

Animals↗