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K M Wan

Publications and source records attributed to K M Wan.

14 recordsLinked to original sources
PubMed1999

The nuclear matrix prepared by amine modification.

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.

Amines↗
PubMed1997

The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus.

The nucleus is an intricately structured integration of many functional domains whose complex spatial organization is maintained by a nonchromatin scaffolding, the nuclear matrix. We report here a method for preparing the nuclear matrix with improved preservation of ultrastructure. After the removal of soluble proteins, the structures of the nucleus were extensively cross-linked with formaldehyde. Surprisingly, the chromatin could be efficiently removed by DNase I digestion leaving a well preserved nuclear matrix. The nuclear matrix uncovered by this procedure consisted of highly structured fibers, connected to the nuclear lamina and built on an underlying network of branched 10-nm core filaments. The relative ease with which chromatin and the nuclear matrix could be separated despite extensive prior cross-linking suggests that there are few attachment points between the two structures other than the connections at the bases of chromatin loops. This is an important clue for understanding chromatin organization in the nucleus.

Cells, Cultured↗
PubMed1994

The B1C8 protein is in the dense assemblies of the nuclear matrix and relocates to the spindle and pericentriolar filaments at mitosis.

The B1C8 monoclonal antibody detects a 180-kDa nuclear matrix-specific protein. The protein is a component of the dense, metabolically active bodies or assemblies revealed by resinless section electron microscopy of the nuclear matrix. These assemblies are scattered through the nuclear interior, enmeshed in a complex network of 11-nm filaments. Resinless section electron microscopy of immunogold-stained nuclear matrix preparations shows B1C8 located in many but apparently not all the assemblies. In this regard, the B1C8 antigen resembles previously studied nuclear matrix proteins such as the H1B2 protein. The speckled pattern of nuclear immunofluorescence by B1C8 reflects this labeling of the dense assemblies in the nuclear matrix. Somewhat unusual is the faint staining of cytoplasmic microtubules by B1C8, which appears to be due to a weakly cross-reacting protein. During cell division, the B1C8 antigen redistributed drastically, showing the dispersion of nuclear matrix assemblies at mitosis. Speckles of B1C8 fluorescence first coalesced at prophase within the nuclear interior and then scattered into numerous cytoplasmic speckles by prometaphase. At metaphase, the B1C8 speckled cytoplasmic staining had become even more widely distributed and finely grained. Also, intense labeling appeared at the mitotic pole and on the spindle fibers themselves. The reassembly of B1C8 antigens into larger cytoplasmic speckles began at anaphase and finally, at telophase, most B1C8 labeling redistributed into speckles in the re-forming nuclei.

Antibodies, Monoclonal↗
PubMed1992

A normally masked nuclear matrix antigen that appears at mitosis on cytoskeleton filaments adjoining chromosomes, centrioles, and midbodies.

mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed H1B2 antibody detected a protein of 240 kD in the nuclear matrix fractions. The H1B2 antigen was completely masked in immunofluorescently stained interphase cells. However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope. The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments. Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves. The H1B2 antigen showed remarkable behavior at mitosis. As cells approached prophase the antigen became unmasked to immunofluorescent staining without the removal of chromatin. First appearing as a bright spot, the antibody staining spread through the nucleus finally concentrating in the region around the condensed chromosomes. The antibody also brightly stained the spindle poles and, more weakly, in a punctate pattern in the cytoskeleton around the spindle. As the chromosomes separated at anaphase, H1B2 remained with the separating daughter sets of chromosomes. The H1B2 antigen returned to the reforming nucleus at telophase, but left a bright staining region in the midbody. Immunoelectron microscopy of resinless sections showed that, in the mitotic cell, the H1B2 antibody did not stain chromosomes and centrioles themselves, but decorated a fibrogranular network surrounding and connected to the chromosomes and a fibrogranular structure surrounding the centriole.

Antigens, Nuclear↗
PubMed1989

Chromatin architecture and nuclear RNA.

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large clumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.

Cell Nucleus↗
PubMed1984

Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well-preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.

Animals↗
PubMed1983

Many cytoskeletal proteins associate with the hela cytoskeleton during translation in vitro.

Observations that cytoskeletal proteins assemble in vivo close to the time and site of synthesis have been confirmed and extended by an in vitro translation system. HeLa cytoskeletons prepared with Triton in a translation-extraction buffer without reticulocyte or wheat germ lysate efficiently incorporate 35S-methionine into polypeptides, and are stable during this translation. Cytoskeletal proteins translated in this way associate with the HeLa cytoskeleton independent of the concentration of soluble proteins. These associations are puromycin-resistant before the proteins are complete; the protein associations made in vitro show only minor differences from those made in vivo. The protein associations are not simply a consequence of protein solubility in the buffers used, as the associations require initiation in vivo. These results indicate that many cytoskeletal proteins associate with the cytoskeleton during translation.

Cell Fractionation↗
PubMed1983

Regulation of actin mRNA levels and translation responds to changes in cell configuration.

The role of cell configuration in regulating cell metabolism has been studied, using a system in which cell shape and surface contact can easily be manipulated. The suspension of anchorage-dependent mouse fibroblasts in Methocel results in a coordinate decrease of DNA, RNA, and protein synthesis. These processes are restored upon reattachment of cells to a solid surface. This recovery process has two or more components: a rapid recovery of protein synthesis requiring only surface contact, and a slower restoration of nuclear events which is dependent upon extensive cell spreading (A. Ben-Ze'ev, S.R. Farmer, and S. Penman, Cell 21:365-372, 1980). In the present study, we examined 3T3 cells while in suspension culture and after attachment to a tissue culture dish surface to study cell configuration-dependent expression of specific cytoskeleton protein genes. The 3T3 line of fibroblasts used here shows these responses much more dramatically compared with 3T6 cells previously studied. We demonstrate that whereas total protein synthesis was strongly inhibited upon suspension, actin synthesis was preferentially inhibited, decreasing from 12% of total protein synthesis in control cells to 6% in suspended cells. This occurred apparently at the level of translation of actin mRNA, since the amount of actin mRNA sequences in the cytoplasm was unchanged. Reattachment initiated the rapid recovery of overall protein synthesis which was accompanied by a dramatic, preferential increase in actin synthesis reaching peak values of 20 to 25% of total protein synthesis 4 to 6 h later, but then declining to control values by 24 h. Translation in vitro and hybridization of mRNA to a cloned actin cDNA probe revealed that the induction of actin synthesis was due to increased levels of translatable mRNA sequences in the cytoplasm. These results imply a close relationship among cell cytoarchitecture, expression of a specific cytoskeletal protein gene, and growth control. The expression of the actin gene appears to be regulated at both the level of translation (during suspension) and mRNA production (during recovery).

Actins↗
PubMed1982

The nuclear matrix: three-dimensional architecture and protein composition.

The structural filament network of the nucleus is prepared while still connected to the cytoskeleton. The relatively gentle procedure removes about 98% of the DNA and at least 86% of the histones. The matrix is bounded by an outer nuclear lamina connected to the cytoskeletal framework, as well as the inner filaments. The filaments range in diameter from 3 to 22 nm, and are organized in a three-dimensional anastomosing network in which nucleoli are enmeshed. The nuclear matrix is separated from the cytoskeletal framework by a double detergent and then partitioned into a chromatin fraction and a matrix fraction by nuclease and high salt. Two-dimensional gel electrophoresis shows that the proteins of the cytoskeleton, chromatin and nuclear matrix are very different. A major protein found in all fractions cofocuses with actin. Vimentin is largely associated with the nuclear matrix, probably as a corona external of filaments.

Animals↗
PubMed1980

The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework.

Acridine fluorescence reveals polyribosomes in fibroblasts and Triton-extracted skeletal frameworks; simultaneous phase-contrast images show cellular structure. Polyribosomes appear near nuclei of both intact fibroblasts and skeletal frameworks. Simultaneous autoradiography of cells so examined locates radioactive proteins. After synthesis, most proteins diffuse rapidly through the cytoplasm; intact cells autoradiographed afer a 35S pulse show grains throughout. In sharp contrast, extraction with Triton leaves only radioactive skeletal proteins, which, although released from ribosomes, are near polyribosomes after a pulse. After a chase, skeletal-associated radioactivity is found throughout the framework structure. However, skeletal proteins migrate only if protein synthesis continues. Emetine administered following a pulse block protein migration; skeletal framework radioactivity remains near polyribosomes. This also indicates limited exchange between skeletal framework and soluble cytoplasmic proteins. The fact that proteins insert themselves into the skeletal framework at or near their synthesis site, with limited subsequent exchange, appears to contradict current view of protein self-assembly.

Actins↗
PubMedDate not supplied

Pseudomonas exit-site infections in CAPD patients: evolution and outcome of treatment.

OBJECTIVE: To examine the natural history of Pseudomonas aeruginosa (PSA) exit-site infections (ESI) in patients treated with antibiotics with or without surgical interventions. DESIGN: Retrospective record review from May 1994 to April 1997. SETTING: A single dialysis unit in a district hospital. PATIENTS: The review included 353 patients who had undergone continuous ambulatory peritoneal dialysis (CAPD). OUTCOME MEASURES: The prevalence and etiology of ESI, the treatment regimen for PSA ESI, and the outcome of treatment. RESULTS: The prevalence of ESI was 55%. A total of 131 episodes (range 1-5) of PSA ESI occurred in 78 (40.2%) of the 194 patients who experienced ESI. Among these 78 patients, 4 groups with different outcomes were identified. In group I, 35 patients (44.9%) were treated successfully with antibiotic therapy alone. Among these 35 patients, 4 developed PSA peritonitis at a mean of 5 months (range 2-10 mth) after apparent clinical resolution of PSA ESI. Two of the 4 patients switched to long-term hemodialysis (HD) because of peritoneal failure. In group II, 8 patients (10.3%) responded to a combination of antibiotics and shaving of the external cuff. In group III, 21 patients (26.9%) with recurrent ESI underwent elective Tenckhoff catheter removal and reinsertion. One of the 21 patients had relapse of PSA ESI 14 months after the operation. In group IV, 14 patients (17.9%) had recurrent PSA ESI that failed to respond to multiple courses of antibiotics and shaving of the external cuff. Consent for Tenckhoff catheter removal was not obtained and 4 of these 14 patients subsequently developed PSA peritonitis. One of the 4 patients changed to permanent HD due to peritoneal failure. CONCLUSIONS: Considering the increased risk and the poor outcome of PSA peritonitis in patients with persistent PSA ESI, early Tenckhoff catheter removal is recommended if the patient fails to respond to antibiotics with or without externalization of the external cuff.

Adult↗