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T D Allen

Publications and source records attributed to T D Allen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export.

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

Animals↗
PubMed1997

Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.

The death of small intestinal epithelial cells has been characterized and quantitated after irradiation of mice rendered homozygously null for the p53 gene. In wild-type animals homozygous for p53 a rapid (4.5 h) elevation of p53 protein was observed in the proliferative compartment of the crypts after 8 Gy of irradiation. Cells underwent cell death by apoptosis in this region. We had reported previously a total repression of apoptosis in small intestinal crypt epithelia 4.5 h after the gamma-irradiation (8 Gy) of p53 homozygously null animals. Thus, while 400 apoptotic cells were observed in 200 half crypts taken from wild-type animals at 4.5 h, this fell to background levels (10-30) in the p53 null animals (Merritt et al., 1994) and did not increase by 12 h. However, we have now found a delayed initiation of a p53-independent apoptosis after 8 Gy of gamma-radiation: at 24 h, approximately 100 apoptotic cells were observed in 200 half crypts. This late wave of apoptosis was not observed after 1 Gy of gamma-radiation. The morphological appearance of this p53-independent apoptosis suggested that death may have arisen as the result of aberrant mitosis. Analysis of the regeneration of crypts 3 days after irradiation of mice with between 11 and 17 Gy showed that there was no significant increase (P=0.135) in the potential of clonogenic cells from the p53 null animals to repopulate the crypts. The data support the idea that a p53-independent apoptotic mechanism permits the engagement of apoptosis, probably by a mitotic catastrophe, after 8 Gy of gamma-irradiation in vivo and that a loss of p53 does not make these epithelial cells radioresistant in vivo to doses of 8 Gy and above. In contrast, irradiation with 1 Gy failed to induce a p53-independent apoptosis in vivo, suggesting that the p53 'sensor' of damage was more sensitive than that engaging the p53-independent mechanism of cell death.

Animals↗
PubMed1997

Three-dimensional visualization of the route of protein import: the role of nuclear pore complex substructures.

The three-dimensional localization of nucleoplasmin and wheat germ agglutinin (WGA) at the nuclear envelope of Xenopus oocytes is demonstrated by microinjecting protein coated gold colloids and examining their distribution using both stereo transmission electron microscopy and field emission in-lens scanning electron microscopy. Localization of many WGA gold particles and nucleoplasmin gold particles at the same nuclear pore complex (NPC) following coinjection is demonstrated. Binding of the WGA gold in the central region of the NPCs appears to form a barrier, preventing the import of nucleoplasmin gold, and includes central localization along radial "tracks" which correspond to the internal filaments connecting the cytoplasmic ring and the central region of the NPC. We suggest that these filaments may in some way be involved in opening and closing of the central channel of the NPC for transport. Transport of nucleoplasmin through the central region of the NPCs appears to be in "single file" regardless of the size of the colloidal gold, and distribution into the nucleoplasm appears to be through the basket rings with no association of the nucleoplasmin gold with the basket filaments being observed.

Animals↗
PubMed1997

Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy.

Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM "working resolution." Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM 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.

Animals↗
PubMed1997

Nuclear pore complex structure in birds.

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.

Animals↗
PubMed1997

Results of urinary tract reconstruction in boys with end stage bladders resulting from obstructive uropathy.

PURPOSE: The records of 17 boys who underwent reconstruction of the lower urinary tract because of end stage bladders resulting from obstructive uropathy were reviewed to evaluate the degree to which they void and factors that favorably impacted outcome. MATERIALS AND METHODS: A total of 17 boys with end stage bladders resulting from obstructive uropathy, including posterior urethral valves in 15 and obstructing ureteroceles in 2, underwent reconstruction of the urinary tract. Procedures consisted of augmentation (autoaugmentation in 3, ileocystoplasty in 2, ileocecal cystoplasty in 1 and colocystoplasty in 10), an ileal ureter in 2, bladder neck revision in 15 and appendicovesicostomy in 8. RESULTS: All patients achieved a low pressure reservoir of adequate volume with stable or improved urinary tracts. A total of 13 patients voided sufficiently well to maintain a favorable life-style, including 5 who were completely catheter-free. With respect to the variables involved in reconstruction, bladder neck revision seemed to correlate best with a good outcome. CONCLUSIONS: In most cases end stage bladders in boys with obstructive uropathy can be reconstructed not only to protect the urinary system, but to preserve some degree of voiding potential as well. Bladder neck revision appears to be particularly helpful in achieving the latter goal. When voiding is inadequate, appendicovesicostomy provides easy access to the bladder for intermittent catheterization.

Adolescent↗
PubMed1997

Laparoscopic creation of a catheterizable cutaneous ureterovesicostomy.

Nephrectomy and creation of a cutaneous ureterovesicostomy for intermittent catheterization of the bladder traditionally requires two surgical procedures performed through separate incisions. Herein we report completion of these procedures using a transperitoneal laparoscopic approach, with the ureterovesicostomy stoma created at one of the laparoscopic working ports. The clinical course was remarkable for a shortened postoperative hospitalization (48 hours) with minimal incisional pain, and an excellent long-term result with complete bladder emptying and resolution of urinary infections. Laparoscopic application of the Mitrofanoff principle for creation of a catheterizable cutaneous ureterovesicostomy combines the advantages of both, allowing optimal preservation of ureteral vascularity, minimal morbidity, and efficient bladder evacuation.

Adolescent↗
PubMed1997

The processing, transport and heterologous expression of Epstein-Barr virus gp110.

Epstein-Barr virus (EBV) glycoprotein gp110 has substantial structural and sequence homology with herpes simplex virus (HSV) gB and gBs of other alpha- and betaherpesviruses but unlike HSV gB localizes differently in infected cells and is absent from virions. To facilitate the analysis of EBV gp110, antisera were raised to fragments of gp110 expressed in a bacterial system. They recognized a protein of the predicted size in recombinant bacterial lysates, in lymphoblastoid cells and in recombinant vaccinia virus-gp110 infected cells. gp110 from all sources possessed a high-mannose type of N-glycosylation implying that gp110 has not passed through the Golgi. Immunofluorescence and immuno-electron microscopy confirmed this conclusion and demonstrated that, in contrast to HSV gB, the majority of immunoreactive gp110 was present at the nuclear membrane or endoplasmic reticulum (ER) but not at the cell membrane. Unexpectedly, a truncated version of gp110 lacking the hydrophobic C-terminal region, despite forming dimers analogous to HSV dimers, was transported in a similar manner to full-length gp110. Two chimeric proteins constructed by replacing the N- and C-terminal domains of gp110 with corresponding regions of gp340/220 were also transported to the nuclear membrane/ER. These data suggest that unlike HSV gB both the N- and C-terminal portions of EBV gp110 contain independent signals sufficient to direct the molecule to the ER/nuclear membrane. Specific transport of gammaherpesvirus gB homologues to the nuclear membrane, from where herpesviruses bud, suggests that they may be involved in the egress of virus from the nucleus.

Biological Transport↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗
PubMed1997

Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly.

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare 'dimples' (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular 'star-ring' subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5'-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N',N'-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Animals↗
PubMed1997

[Study on expression of adherent proteins related to regulation of hematopoiesis in long-term cultured human bone marrow stromal cells].

OBJECTIVE: To study the molecular basis of bone marrow stromal cell supporting hematopoiesis. METHODS: Immunoelectron microscopic localization of adherent protein related to regulation of hematopoiesis was performed by immunocolloidal gold labelling technique in the adherent layer of long-term cultured human bone marrow stromal cells (LTCHBMSC). RESULTS: Adherent proteins were found to be expressed on LTCHBMSC. Double labelling revealed co-expression of these proteins and some specific markers for endothelial cells (vWF) and fibroblasts (Fb-sp). It was suggested that endothelial cells and fibroblasts in LTCHBMSC could synthesize and secrete adherent proteins, which bound to the surface of stromal cells and extracellular matrix. CONCLUSION: Adherent proteins and all kinds of hematopoietic growth factors secreted by stromal cells constitute a complex network supporting hematopoiesis.

Bone Marrow Cells↗
PubMed1997

Endoscopic management of infected enlarged prostatic utricles and remnants of rectourethral fistula tracts of high imperforate anus.

PURPOSE: Infected enlarged prostatic utricles and infected remnant fistula tracts of high imperforate anus are usually managed by a suprapubic, transtrigonal or posterior sagittal approach. We describe a minimally invasive endoscopic approach to these entities. MATERIALS AND METHODS: We treated 12 patients with infected enlarged prostatic utricles and 4 with infected remnant fistula tracts using endoscopic techniques. Specifically a resectoscope with a bulb electrode or a cystoscope with a Bugby electrode was used to fulgurate circumferentially the dilated utricle or remnant fistula. After fulguration a Councill catheter was placed in the lesion for 3 to 5 days and urine was diverted via a suprapubic tube for 2 to 3 weeks. Obliteration of the abnormality was verified by a voiding cystourethrogram. RESULTS: Using this technique median postoperative hospital stay was 2 days (range 0 to 7). The enlarged prostatic utricle or remnant fistula tract was completely obliterated in 87% of the cases (62% after 1 and 25% after 2 treatments). Of our patients 13% had a significant (greater than 50%) decrease in utricular cyst size although a urethral abnormality persisted. Postoperative morbidity was minimal. One patient (6%) had a fever for 3 days postoperatively and none has had a urethral stricture during a median followup of 2 years (range 3 months to 4 years). CONCLUSIONS: Although it is not a panacea, electrofulguration of an enlarged prostatic utricle and/or remnant fistula of imperforate anus is a simple procedure that has a high rate of success, does not require prolonged hospitalization and is associated with minimal morbidity.

Adolescent↗
PubMed1996

RNP export is mediated by structural reorganization of the nuclear pore basket.

Messenger RNA leaves the cell nucleus as ribonucleoprotein (RNP) particles. The nucleocytoplasmic translocation of the particles takes place through the nuclear pore complex (NPC) and includes two steps: binding to the NPC and transit through its central channel. The NPC basket is a fishtrap-like component of NPC facing the nucleoplasm. Its position in the NPC strongly suggests that it has an important role in the initial steps of macromolecular export from the nucleus. Here we report a cyclic rearrangement of the basket structure in relation to the translocation of a specific messenger RNP (mRNP) of exceptional size, the Balbiani ring RNP particles in the salivary gland cells in Chironomus. We used field emission in-lens scanning electron microscopy (FEISEM), transmission electron microscopy (TEM), and immunocytochemistry to analyse the structural organization of the basket during the mRNP export. Our observations reveal five configurations of the basket which are presented in a model of basket reorganization related to the state of mRNP penetration into the NPC. We suggest that the functional role of the basket is to anchor the mRNP particle to the NPC and position it in correct orientation at the entrance to the central channel of the NPC.

Animals↗
PubMed1996

The nuclear pore complex and lamina: three-dimensional structures and interactions determined by field emission in-lens scanning electron microscopy.

Three dimensional surface imaging was used to examine structural details of the nuclear pore complex. Subsurface structures were uncovered by detergent extraction, proteolysis, mechanical fracturing and combinations of these. Features observed in this way were mostly consistent with previous three-dimensional structures with some novel observations. In addition to cytoplasmic and basket filaments attached to each coaxial ring, we have observed radiating filaments within the central channel. New details of basket organization are presented, showing that basket filaments branch and are woven together to form the basket ring. The "central transporter" is shown to be a regular, consistent structure revealed after removal of overlying internal filaments. The lumenal ring is visualised and we present evidence that the lamina may be attached to the spoke ring complex. Finally we show that there is evidence for a novel structure, the "star ring", sandwiched between the cytoplasmic ring and the membrane.

Animals↗
PubMed1996

Urinary tract reconstruction in children undergoing renal transplantation.

In children, congenital urinary tract anomalies contribute to end-stage renal disease in 20% to 30% of cases. As more and more children with myelomeningocele, posterior urethral valves, prune belly syndrome, and other serious congenital anomalies of the urinary tract survive early infancy, more of these patients will be in need of renal transplantation. Of these, a significant proportion will have persisting abnormal anatomic and physiological characteristics of the urinary tract requiring reconstructive surgery before transplantation. Before undertaking these procedures, urologic evaluation should be performed in all such children. Comprehensive evaluation includes a careful history and physical examination with radiological imaging of the urinary tract (renal ultrasonography and voiding cystourethrography). In selected instances, further evaluation of bladder function and urethral anatomy may require urodynamic evaluation or cystourethroscopy. The goal of these investigations is to ensure that the bladder will hold urine at a low intravesical pressure during the storage phase and that it can be evacuated with certainty. This presentation focuses on the pretransplantation evaluation and the various possible urinary tract reconstructions that may be performed in children destined for renal transplantation.

Child↗
PubMed1996

DNA inclusions within autolytic cytoplasmic vacuoles of hemopoietic stem cell line FDCP-Mix.

FDCP-Mix, a pluripotent routine hemopoietic stem cell line undergoes internucleosomal cleavage of DNA when induced to apoptosis either by drugs or by withdrawal of growth factor (IL-3), and also displays a pattern of nuclear morphology that is typical for apoptosis. However, increased autolytic activity in the cytoplasm precedes the nuclear changes. For etoposide-treated FDCP-Mix cells, mitochondria were identified as a target for autolytic digestion in large autolytic vacuoles, but during this period an increase in the number of mitochondria was observed. The autolytic vacuoles displayed variations in their content. Large, electron-dense inclusions resembling "condensed chromatin" could regularly be found in FDCP-Mix cells treated with low concentrations of etoposide (<4 microM). Confocal fluorescence microscopy and DNAse-gold labeling were employed to demonstrate the presence of DNA in the formation of the electron-dense inclusions within autolytic vacuoles. The identification of mitochondrial macroautophagy, the evidence for an etoposide-induced proliferation of mitochondria, and the fact that electron-dense inclusions are formed at a stage when the morphology of the nucleus is still not effected, suggests that the DNA within the autolytic vacuoles may be of mitochondrial origin.

Animals↗