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T D Allen

Publications and source records attributed to T D Allen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract.

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from "lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.

Animals↗
PubMed1996

Nuclear pore clustering is a consistent feature of apoptosis in vitro.

Two cell lines which show different patterns of DNA fragmentation have been examined for variations of their nuclear morphology during apoptosis. FDCP-Mix, a pluripotent murine haemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis either by drugs or withdrawal of growth factor (IL-3) was compared with the human lymphoid leukemia cell line MOLT-4, a cell line which undergoes apoptosis without production of a typical DNA 'ladder'. The nuclear morphology of FDCP-Mix cells was consistent after apoptotic induction by drug or by growth factor withdrawal. Apoptotic nuclear morphology for MOLT-4 and FDCP-Mix showed variations in the distribution, density and texture of the electron dense nuclear marginations. Despite these differences, clustering of nuclear pore complexes (NPCs) after treatment with the topoisomerase II inhibitor etoposide was a common phenomenon for both cell lines. Moreover, pore clustering for FDCP-Mix nuclei occurred independently from the way in which apoptosis was induced, either by growth factor withdrawal or etoposide treatment. In a novel approach, we visualised the clustering of NPCs three-dimensionally by field emission in-lens scanning electron microscopy (FEISEM).

Journal Article↗
PubMed1996

Immunoelectron microscopic localization of growth factors and other markers in human long-term bone marrow cultures.

Ultrastructural immunocytochemical characterization of human long-term bone marrow cultures has shown positive localization for growth factors on cell surface and on extracellular matrix (ECM). In some cases double-labelling indicated co-localization of growth factors and specific cell surface labels. Specific markers for endothelial cells and fibroblasts showed that growth factor (GM-CSF, G-CSF and b-FGF) were present at the surface of these cell types. Both scanning and transmissiorr electron microscopy indicated intense labelling for growth factors on the extracellular matrix. Double-labelling of heparan sulphate proteoglycans and GM-CSF showed a co-localization of the labelling, which indicated the binding of growth factor to the extracellular matrix.

Bone Marrow Cells↗
PubMed1996

Accessing nuclear structure for field emission, in lens, scanning electron microscopy (FEISEM).

Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding, its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces.

Animals↗
PubMed1995

Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy.

FDCP-Mix, a pluripotent murine hemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis by either drugs or withdrawal of growth factor (interleukin-3) was studied after treatment with the topoisomerase II inhibitor etoposide (0.5-4 microM). An increase in autolytic activity was the major early morphological change within the cytoplasm, with mitochondria as the main target for autolytic digestion. Despite this macroautophagy, thin sections showed a high number of mitochondria, suggesting mitochondrial proliferation as a result of drug treatment. This observation of an increase in the number of mitochondria was confirmed by flow cytometric studies of mitochondrial overall mass. Multiparameter flow cytometry of cells double stained with propidium iodide and nonyl-acridine orange gave an accurate assay for mitochondrial mass in relation to cell cycle stages. The increase in mitochondrial mass was found in all cell cycle stages. The results suggest a drug-induced proliferation of mitochondria separate from the processes involved in the doubling of mitochondrial mass during the cell cycle and a decline of mitochondria in the later stages of apoptosis.

Acridine Orange↗
PubMed1995

Structural and functional organization of the nuclear envelope.

The double-membrane nuclear envelope is punctuated by pores where the two membranes are joined. These pores are stabilized by the elaborate nuclear pore complex, which is anchored to the inner membrane by the nuclear lamina, as well as to other nuclear and cytoskeletal structures. Recent experiments have identified proteins involved in the stability of this organization as well as in the function of the nuclear pore complex, which we relate here to newer aspects of nuclear envelope structure.

Animals↗
PubMed1995

Metastasis-associated 5T4 oncofoetal antigen is concentrated at microvillus projections of the plasma membrane.

The 5T4 oncofoetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. A cDNA encoding the 5T4 molecules has been isolated and the extracellular portion contains several leucine-rich repeats which have been implicated in cellular interactions. To study the cell biological role of 5T4 molecules, murine L cells (A9 derivative) were stably transfected with 5T4 cDNA under the control of the CMV immediate-early promoter. The 5T4-expressing cells exhibited a more spindle-shaped morphology compared to the vector alone transfected cells. Confocal immunofluorescence microscopy revealed a 'polkadot' pattern of 5T4 antigen expression, heterogeneous in intensity between cells, but distributed over the entire cell surface. Transmission and scanning electron microscopy showed that the 5T4 antigen is concentrated at microvillus projections of the plasma membrane both in the transfected A9 cells and in various carcinoma cell lines. Such projections express an array of surface molecules which function in cell adhesion and motility. This association of 5T4 antigen with microvillus projections was also observed in various carcinoma cell lines. 5T4 expression in A9 cells was also associated with an altered pattern of cell division, decreased cell-substratum adhesion and increased cellular motility. These results support the hypothesis that 5T4 molecules may have a direct function in trophoblast and tumour cell invasion processes.

3T3 Cells↗
PubMed1995

Pericyte differentiation.

Pericytes are defined in vivo by their location: They are embedded within the basement membrane of microvessels. They form an integral part of the microvascular wall and are believed to participate in angiogenesis, although their precise role is not clear. Pericytes derived from the retinal microvasculature have been cultured and identified by a series of phenotypic characteristics that clearly distinguishes them from other stromal cells such as smooth muscle cells. Pericytes in vitro form multicellular nodules rich in extracellular matrix. This matrix becomes mineralized in the presence of growth medium containing serum, without exogenous beta-glycerophosphate. These results indicate that pericytes represent primitive mesenchymal cells able to differentiate into an osteogenic phenotype. Pericyte differentiation also is defined by alterations in their response to transforming growth factor beta 1 and changes in the synthesis and/or deposition of various extracellular matrix proteins such as laminin, Type IV collagen, tenascin, Type X collagen osteonectin, and thrombospondin-1. Angiogenesis is associated commonly with mineralization. These data suggest that pericytes may contribute to mineralization in vivo.

Animals↗
PubMed1995

Shortcomings of color Doppler sonography in the diagnosis of testicular torsion.

PURPOSE: We determined the shortcomings of color Doppler sonography for the diagnosis of testicular torsion. MATERIALS AND METHODS: We reviewed 5 cases of testicular torsion in which findings on color Doppler sonography were inconsistent with surgical findings. RESULTS: Color Doppler sonography showed blood flow in 2 patients who had necrotic testes at surgery. In 3 other patients blood flow was also normal by color Doppler sonography of whom 2 were treated for epididymitis with subsequent loss of the testes and 1 underwent salvage by immediate exploration. CONCLUSIONS: Testicular torsion is a clinical diagnosis requiring consideration of many variables. Color Doppler sonography is helpful but should not be regarded as the definitive discriminator.

Adolescent↗
PubMed1995

Evidence for the direct involvement of lamins in the assembly of a replication competent nucleus.

Monoclonal antibodies linked to paramagnetic immunobeads (Dynabeads) have been used to investigate the distribution of lamin B3 in fractions of Xenopus egg extracts. Lamin B3 behaved as if it were completely soluble and did not co-precipitate with membrane fractions. Sperm pronuclei assembled in lamin depleted egg extracts were compared to pronuclei assembled in mock depleted extracts by field emission in-lens electron scanning microscopy (FEISEM). This technique revealed that the surface structures of the nuclear envelopes, including nuclear pores, appeared to be identical, indicating that lamin depletion does not affect nuclear envelope assembly. One-dimensional and two-dimensional gel electrophoresis was used to analyze soluble proteins co-precipitated with lamin B3 on Dynabeads. Our results indicate that two major species (molecular mass: 105 kDa and 57 kDa) specifically co-precipitate with lamin B3 as well as several minor species. At least three proteins which co-precipitate with lamin B3 were identified as nuclear matrix proteins. Lamin B3 was separated from these proteins and re-inoculated into lamin depleted extracts. This resulted in partial rescue of both lamina assembly and DNA replication. These results imply that lamin B3 is directly involved in the assembly of structures required for the initiation of DNA replication.

Animals↗
PubMed1994

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Cell Nucleus↗
PubMed1994

Infundibulopelvic stenosis: a long-term followup.

We followed 21 patients with congenital infundibulopelvic stenosis, a rare obstructive disorder of the intrarenal collecting system, for a median of 11 years (range 2 to 28 years). Of these patients 19 (90%) had evidence of bilateral renal disease. In particular, 10 patients had bilateral infundibular pelvic stenosis, 6 a contralateral cystic dysplastic kidney and 3 a congenitally absent kidney. Extended observations of the patients with this disorder revealed that end stage renal disease or renal insufficiency developed in 8 patients (37%), all with bilateral renal anomalies. Renal biopsies in patients with end stage renal failure revealed widespread areas of renal dysplasia proximal to the stenotic infundibulum with focal or global glomerulosclerosis of the glomeruli, which was not involved in the dysplastic process. These pathological findings are consistent with the presence of a renal hyperfiltration injury. Our data suggest that the prognosis of infundibulopelvic stenosis is dependent upon the extent of total renal mass involved with the disease process and the duration of followup.

Adolescent↗
PubMed1994

Unsuspected ureterocele and ureteral duplication.

Four patients presenting with high grade unilateral vesicoureteral reflux were discovered at surgery to have an unsuspected duplex system with a ureterocele associated with the upper pole of the refluxing kidney. No preoperative radiographic study was diagnostic for duplication or ureterocele but when studies were reviewed retrospectively subtle evidence suggesting this possibility could be identified in a few cases. This rare situation should be considered whenever massive unilateral vesicoureteral reflux is encountered.

Child↗
PubMed1993

Scanning electron microscopy of polytene chromosomes (I).

The structure of polytene chromosomes has been observed by conventional scanning electron microscopy and also with a high resolution 'in lens' field emission instrument. Surface imaging with secondary electron emission has characterized condensed chromatin and regions known for their RNA synthetic potential (nucleoli, Balbiani rings and puffs). In DNA-rich bands pairing of chromatids appears so perfect that individual chromomeres cannot be visualized as discrete units. In the interbands only chromatid bundles appear as elements, but not individual chromatids. High resolution scanning electron microscopy allows resolution at the nucleosome level. Improved localization of chromosomal structures is demonstrated, as in the case of the proposed separation of the prepupal 'Balbiani ring 1'. Surface images of the RNA synthetic centres of the salivary gland cell are presented. Transcripts of the Balbiani ring template can be observed in the condensed and gradually unfolded state, allowing measurement of unit ribonucleoparticles. The surfaces of puffs have been visualized and are characterized by multiple supercoiled loop structures, which are described according to size, conformation and distribution.

Animals↗