Biomedical subjects

C Molenaar

Publications and source records attributed to C Molenaar.

7 recordsLinked to original sources

PubMed2004

Alignment of the cell nucleus from labeled proteins only for 4D in vivo imaging.

Studies of protein dynamics by 4D (3D + time) confocal microscopy in vivo are hampered by global cell motion. The time between the acquisitions of the 3D images is in the order of minutes. Therefore, it is not to be expected that the cell as a whole remains fixed in the water basin on the stage. This superimposes a motion on the protein dynamics that has to be removed. We present a robust registration technique to align the cell images that does not require the a priori establishment of point-to-point correspondences. Instead, it uses the distribution of the labeled proteins. After correction for the translation, the 3D rotation of the cell is estimated. A robust intrinsic body coordinate system is constructed via the inertia tensor from the intensity distribution. By combining basis transformation to this intrinsic coordinate system, we can calculated the rotation matrix in a conceptual and computational straightforward manner. We have evaluated the performance of this approach in three experiments with human osteaosarcoma cells (U-2 OS), where the nuclear proteins Histon H4 and PML were visualized. The PML is concentrated in several dozen nuclear spots. Expression of Histon H4 results in a total nuclear staining. The registration results for both channels computed independently are very similar. Practically, this means that only the labeled material needs to be observed and still registration of the cell as a whole can be achieved.

Algorithms↗

PubMed2001

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Animals↗

PubMed2001

Methods for visualizing RNA processing and transport pathways in living cells.

Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs. Also, the picture emerges that RNA molecules travel through the nucleus at much faster speed, equaling that of free diffusion, than previously anticipated. Still, a major challenge proves to be the development of a microscopic detection technique that allows specific, in vivo, detection of low levels of RNA species by fluorescence in situ hybridization, without interfering fluorescent background signals derived from non-hybridized probe sequences and autofluorescent cell components. By applying photoactivatable caged fluorochrome-, molecular beacon-, or fluorescence resonance energy transfer (FRET)-based detection methods an important step in the future of living cell analysis has already been made.

Animals↗

PubMed2000

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy.

The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.

Antineoplastic Agents↗

PubMed2000

Molecular cloning and characterization of mouse CD97.

The EGF-TM7 family (CD97 and EMR1) is a group of class II seven-span transmembrane receptors predominantly expressed by cells of the immune system. Recently, we have identified CD55, a regulatory molecule of the complement cascade, as a cellular ligand of human CD97 (hCD97). In this study, the molecular properties of mouse CD97 (mCD97) are described. Like hCD97, mCD97 has an extended extracellular region with several epidermal growth factor-like (EGF) domains. Due to alternative RNA splicing, isoforms with three and four EGF domains exist, designated mCD97(EGF1,2,4) and mCD97(EGF1,2, 3,4) respectively. All EGF domains, except for the N-terminal one, possess a calcium-binding site. In a third isoform mCD97(EGF1,2,X,3, 4), a sequence of 45 amino acids was found between the second and third EGF domain that does not correspond to any known protein module. Using newly generated mCD97 mAb, we show that analogous to the blood expression pattern of hCD97, mCD97 can be found on lymphoid and myeloid cells. Adhesion of mouse erythrocytes and splenocytes to COS cells expressing mCD97(EGF1,2,4) or mCD97(EGF1,2, 3,4) could be blocked by mouse CD55 (mCD55) antibody, identifying mCD55 as a cellular ligand for mCD97. Consistent with the necessity of directly linked EGF domains for the integrity of the CD55-binding site on hCD97, no adhesion was detected to the largest mouse isoform mCD97(EGF1,2,X,3,4). Remarkably, we found that the interaction between CD97 and CD55 is phylogenetically restricted, as indicated by the selective adhesion of primate erythrocytes to hCD97 transfectants, and of mouse and rat erythrocytes to mCD97 transfectants respectively.

Amino Acid Sequence↗

PubMed1999

Synthesis, processing, and transport of RNA within the three-dimensional context of the cell nucleus.

Fluorescence in situ hybridization and immunocytochemical techniques have contributed significantly to our current understanding of how transcription, RNA processing, and RNA transport are spatially and temporally organized in the cell nucleus. New technologies enabling the visualization of nuclear components in living cells specifically advanced our knowledge of the dynamic aspects of these nuclear processes. The picture that emerges from the work reviewed here shows that the positioning of genes within the three-dimensional nuclear space is of crucial importance, not only for its expression, but also for the efficient processing of its transcripts. Splicing factors are recruited from speckles to sites of active transcription, which can be present within, at the periphery, or at a relatively large distance from speckles. Furthermore, results are discussed showing that transcripts are exported by means of random diffusion.

Animals↗

PubMed1989

Appearance of IgG and IgA antibodies in human bile after tetanus toxoid immunization.

Humans immunized intramuscularly with one dose of tetanus toxoid exhibited IgG, and in some cases IgA antibody, in their bile as well as serum. Both isotypes appeared in bile transiently with titres declining after about day 10 for both classes. These kinetics resembled those of the serum IgA response but were markedly different to those for IgG antibody in serum. Measured IgG titres in bile were between 0.07 and 4.2% of those in paired sera, and IgA titres were between 6.8 and 124% of sera. Peak responses in bile, while generally of smaller size, exceeded those of paired sera when expressed as antibody/mg of IgG or IgA present. This calculation showed that during the peak response bile was up to nineteen-fold more abundant in IgG antibody than was serum taken at the same time, and up to forty-five-fold more for IgA. Enrichment of antibody in bile is not consistent with the Ig of bile being solely conferred by plasma, and may mean the involvement of local synthesis too. This study indicates that tetanus toxoid immunization of humans results in biliary antibody and raises the possibility of intra-hepatic antibody production for export to the intestinal tract in man.

Adolescent↗