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Susan Smith

Publications and source records attributed to Susan Smith.

At least 19 recordsLinked to original sources
PubMed2004

A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2.

BACKGROUND: Human telomeres are coated by the telomere repeat binding proteins TRF1 and TRF2, which are believed to function independently to regulate telomere length and protect chromosome ends, respectively. RESULTS: Here, we show that TRF1 and TRF2 are linked via TIN2, a previously identified TRF1-interacting protein, and its novel binding partner TINT1. TINT1 localized to telomeres via TIN2, where it functioned as a negative regulator of telomerase-mediated telomere elongation. TIN2 associated with TINT1, and TRF1 or TRF2 throughout the cell cycle, revealing a partially redundant unit in telomeric chromatin that may provide flexibility in telomere length control. Indeed, when TRF1 was removed from telomeres by overexpression of the positive telomere length regulator tankyrase 1, the TIN2/TINT1 complex remained on telomeres via an increased association with TRF2. CONCLUSIONS: Our findings suggest a dynamic cross talk between TRF1 and TRF2 and provide a molecular mechanism for telomere length homeostasis by TRF2 in the absence of TRF1.

Base Sequence↗
PubMed2004

Resolution of sister telomere association is required for progression through mitosis.

Cohesins keep sister chromatids associated from the time of their replication in S phase until the onset of anaphase. In vertebrate cells, two distinct pathways dissociate cohesins, one acts on chromosome arms and the other on centromeres. Here, we describe a third pathway that acts on telomeres. Knockdown of tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest. Chromosomes aligned normally on the metaphase plate but were unable to segregate. Sister chromatids separated at centromeres and arms but remained associated at telomeres, apparently through proteinaceous bridges. Thus, telomeres may require a unique tankyrase 1-dependent mechanism for sister chromatid resolution before anaphase.

Anaphase↗
PubMed2004

Treatment of AIDS related non-Hodgkin's lymphoma with combination mitoguazone dihydrochloride and low dose CHOP chemotherapy: results of a phase II study.

PURPOSE: To evaluate the response and side effects of combination therapy with low dose CHOP chemotherapy and mitoguazone dihydrochloride in patients with non-Hodgkin's lymphoma associated with the acquired immunodeficiency syndrome (AIDS-NHL). METHODS: Eighteen patients newly diagnosed with intermediate or high-grade AIDS-NHL were treated with low dose CHOP as follows: day 1, cyclophosphamide 350 mg/m(2), intravenously (IV); doxorubicin 25mg/m(2) IV; vincristine 2mg IV; and prednisone 100mg given orally on days 1 through 5. In addition, mitoguazone dihydrochloride was given at a dose of 600 mg/m(2) IV on days 1 and 15 of each 28-day treatment cycle. RESULTS: Seventeen males and one female patient were accrued. Twelve patients had high-grade pathologies while the remainder had an intermediate grade pathology (diffuse large cell). The median CD4+ lymphocyte count was 98/dl (range 1-924). Three patients (17%) reported an AIDS-defining illness prior to lymphoma diagnosis. Of 14 evaluable patients, 6 (43%) achieved a complete remission and 5 (35%) a partial remission. The median failure free and overall survival times were 6.5 and 8.4 months, respectively. Major toxicity was hematologic with grade 3 or 4 neutropenia in 72%; two patients died of neutropenic sepsis. CONCLUSIONS: Mitoguazone in combination with low dose CHOP is a safe regimen, associated with a response rate of 79% (CR 43%, PR 36%, 95% CI=49-95%). These preliminary results suggest no major improvement in terms of response over use of CHOP without mitoguazone.

Adult↗
PubMed2004

Functional subdomain in the ankyrin domain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation.

In human cells, telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 binds TRF1 via its ankyrin domain and poly(ADP-ribosyl)ates it. Overexpression of tankyrase 1 in telomerase-positive cells releases TRF1 from telomeres, resulting in telomere elongation. The tankyrase 1 ankyrin domain is classified into five conserved subdomains, ARCs (ankyrin repeat clusters) I to V. Here, we investigated the biological significance of the ARCs. First, each ARC worked as an independent binding site for TRF1. Second, ARCs II to V recognized the N-terminal acidic domain of TRF1 whereas ARC I bound a discrete site between the homodimerization and the Myb-like domains of TRF1. Inactivation of TRF1 binding in the C-terminal ARC, ARC V, either by deletion or point mutation, significantly reduced the ability of tankyrase 1 to poly(ADP-ribosyl)ate TRF1, release TRF1 from telomeres, and elongate telomeres. In contrast, other ARCs, ARC II and/or IV, inactivated by point mutations still retained the biological function of tankyrase 1. On the other hand, ARC V per se was not sufficient for telomere elongation, suggesting a structural role for multiple ARCs. This work provides evidence that specific ARC-TRF1 interactions play roles in the essential catalytic function of tankyrase 1.

Amino Acid Sequence↗
PubMed2004

Histological and immunohistological studies on cartilage.

The material properties of dense avascular connective tissues such as cartilage present unique challenges to the development of any prospective histological procedures. This chapter documents some of the protocols we have developed in our laboratories for the histological and immunohistochemical evaluation of extracellular-matrix components in normal and pathological articular cartilage. Illustrative examples are provided of specimens from an ovine meniscectomy model of osteoarthritis. A major strength of this experimental animal model is that spatial and temporal changes in joint matrix components can be followed longitudinally in an animal population of well-defined pedigree that minimizes biological variation. Furthermore, a systematic experimental approach can be adopted with this model using cartilage specimens from early predegenerative stages to later advanced stages of joint degeneration. Significantly, the histological changes observed in this model closely parallel changes evident in joint tissues during osteoarthritis in humans and have provided significant insights into these degenerative processes.

Animals↗
PubMed2004

Histochemical visualization of the cartilage hyaladherins using a biotinylated hyaluronan oligosaccharide bioaffinity probe.

Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.

Aggrecans↗
PubMed2003

TRF1 is degraded by ubiquitin-mediated proteolysis after release from telomeres.

Mammalian telomeres are coated by the sequence-specific, DNA-binding protein, TRF1, a negative regulator of telomere length. Previous results showed that ADP-ribosylation of TRF1 by tankyrase 1 released TRF1 from telomeres and promoted telomere elongation. We now show that loss of TRF1 from telomeres results in ubiquitination and degradation of TRF1 by the proteasome and that degradation is required to keep TRF1 off telomeres. Ubiquitination of TRF1 is regulated by its telomere-binding status; only the telomere-unbound form of TRF1 is ubiquitinated. Our findings suggest a novel mechanism of sequential post translational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres.

Adenosine Diphosphate Ribose↗
PubMed2003

Perlecan, the multidomain heparan sulfate proteoglycan of basement membranes, is also a prominent component of the cartilaginous primordia in the developing human fetal spine.

The aim of this study was to localize perlecan in human fetal spine tissues. Human fetal spines (12-20 weeks; n=6) were fixed in either Histochoice or 10% neutral buffered formalin, routinely processed, paraffin-embedded, and 4-microm sagittal sections were cut and stained with toluidine blue, H&E, and von Kossa. Perlecan, types I, II, IV, and X collagen, CD-31, aggrecan core protein, and native and delta-HS 4, 5 hexuronate stub epitopes were immunolocalized. Toluidine blue staining visualized the cartilaginous vertebral body (VB) rudiments and annular lamellae encompassing the nucleus pulposus (NP). Von Kossa staining identified the VB primary center of ossification. Immunolocalization of type IV collagen, CD-31, and perlecan delineated small blood vessels in the outer annulus fibrosus (AF) and large canals deep within the VBs. Perlecan and type X collagen were also prominently expressed by the hypertrophic vertebral growth plate chondrocytes. Aggrecan was extracellularly distributed in the intervertebral disk (IVD) with intense staining in the posterior AF. Notochordal tissue stained strongly for aggrecan but negatively for perlecan and types I and II collagen. Type I collagen was prominent in the outer AF and less abundant in the NP, while type II collagen was localized throughout the IVD and VB. The immunolocalization patterns observed indicated key roles for perlecan in vasculogenic, chondrogenic, and endochondral ossification processes associated with spinal development.

Aggrecans↗
PubMed2003

Oral L-arginine and vitamins E and C improve endothelial function in women with type 2 diabetes.

Endothelial dilator function is impaired in people with type 2 diabetes mellitus (T2DM). Prior research indicates that this can be improved with intravenous administration of ascorbate or L-arginine, but whether these agents have this effect when administered by the clinically practical oral route is unknown. To investigate this question, 10 premenopausal women with T2DM and 10 healthy, premenopausal, non-diabetic women received, in random sequence, a 1-week administration of oral L-arginine (9 g daily) or vitamins E (1800 mg) and C (1000 mg) with an intervening 1-week washout period. Flow-mediated brachial artery dilation (FMD) was measured by ultrasonography and forearm blood flow was measured by plethysmography before and following blood pressure cuff-induced forearm ischemia before and after each week of treatment. At baseline, the women with T2DM had lesser FMD responses (0.028 +/- 0.006 cm vs 0.056 +/- 0.008 cm, p < 0.05). Post-ischemic forearm hyperemia was reduced at baseline in T2DM compared with controls (16.4 +/- 1.8 vs 26.0 +/- 1.4 ml 100 ml(-1) min(-1), p < 0.05). Administration of L-arginine caused a 50 +/- 12% increase in FMD in T2DM (p < 0.05) and raised post-ischemic forearm blood flow by 29 +/- 8% (p < 0.05). No significant changes were seen in controls. Administration of vitamins E and C in women with T2DM produced an increase in the brachial artery diameter response of 79 +/- 15% (p < 0.05), but did not significantly increase the hyperemic blood flow response (p = NS). No significant changes in the responses of controls from pre to post vitamin administration were observed. We concluded that administration of two types of oral agents improved measures of endothelial function in people with T2DM.

Administration, Oral↗
PubMed2003

Integrating medical management with diabetes self-management training: a randomized control trial of the Diabetes Outpatient Intensive Treatment program.

OBJECTIVE: This study evaluated the Diabetes Outpatient Intensive Treatment (DOIT) program, a multiday group education and skills training experience combined with daily medical management, followed by case management over 6 months. Using a randomized control design, the study explored how DOIT affected glycemic control and self-care behaviors over a short term. The impact of two additional factors on clinical outcomes were also examined (frequency of case management contacts and whether or not insulin was started during the program). RESEARCH DESIGN AND METHODS: Patients with type 1 and type 2 diabetes in poor glycemic control (A1c >8.5%) were randomly assigned to DOIT or a second condition, entitled EDUPOST, which was standard diabetes care with the addition of quarterly educational mailings. A total of 167 patients (78 EDUPOST, 89 DOIT) completed all baseline measures, including A1c and a questionnaire assessing diabetes-related self-care behaviors. At 6 months, 117 patients (52 EDUPOST, 65 DOIT) returned to complete a follow-up A1c and the identical self-care questionnaire. RESULTS: At follow-up, DOIT evidenced a significantly greater drop in A1c than EDUPOST. DOIT patients also reported significantly more frequent blood glucose monitoring and greater attention to carbohydrate and fat contents (ACFC) of food compared with EDUPOST patients. An increase in ACFC over the 6-month period was associated with improved glycemic control among DOIT patients. Also, the frequency of nurse case manager follow-up contacts was positively linked to better A1c outcomes. The addition of insulin did not appear to be a significant contributor to glycemic change. CONCLUSIONS: DOIT appears to be effective in promoting better diabetes care and positively influencing glycemia and diabetes-related self-care behaviors. However, it demands significant time, commitment, and careful coordination with many health care professionals. The role of the nurse case manager in providing ongoing follow-up contact seems important.

Adult↗
PubMed2002

Perlecan, the multidomain HS-proteoglycan of basement membranes, is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes.

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.

Animals↗
PubMed2002

Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair.

STUDY DESIGN: The spatial and temporal localization of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in the injured anulus fibrosus was investigated. OBJECTIVE: To assess the involvement of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in anulus fibrosus repair. SUMMARY OF BACKGROUND DATA: Fibroblast growth factor-2 and transforming growth factor-beta have been localized to disc herniation tissue, and alpha-smooth muscle cell actin has been identified in a number of mesenchymal cell types, but their roles have not been evaluated in repair processes in the experimentally injured anulus fibrosus. METHODS: For this study, 32 two adult merinos received a 4-mm deep standard annular incision in their L1L2 and L3L4 discs (lesion group). A similar number of sham-surgery animals served as control subjects. Osteonectin, fibroblast growth factor-2, transforming growth factor-beta, and alpha-smooth muscle cell actin were immunolocalized in sagittal disc sections 3, 6, 12, and 26 months after the operation. Selected specimens also were stained with hematoxylin and eosin, Masson-trichrome, toluidine blue, and picrosirius red. RESULTS: Early focal depletion of proteoglycan was evident in the anulus fibrosus and reorganization of outer annular lamellas 3 to 6 months after the operation. Blood vessel ingrowth and fibroblast infiltration from the outer anulus fibrosus along the plane of the annular defect were maximal 12 months after the operation. Focal upregulation in alpha-smooth muscle cell actin expression was evident with maximal staining in the 12-month lesion samples near infiltrating blood vessels at the lesion site, and also in cells well away from these vessels. Some of the anulus fibrosus cells of the sham sections also stained positively for alpha-smooth muscle cell actin, but this staining was significantly less than in the lesion samples. Staining for fibroblast growth factor-2, transforming growth factor-beta, and osteonectin was strongly localized to blood vessels and cells in the vicinity of the annular lesion. It was maximal 12 months after the operation and diminished by 26 months after the operation. Osteonectin expression also was significantly elevated in outer anulus fibrosus cells distant from the lesion site and its associated blood vessels. In the sham discs, immunoreactivity to fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin was confined to sparsely distributed cells in the anulus fibrosus. No matrix staining was observed. CONCLUSIONS: Immunoreactivity for the noted agents was strongly associated with regions of the annular lesions undergoing matrix reorganization consistent with an active repair response. This response extended as far as the middle third of the anulus fibrosus, which also demarcated the extent of blood vessel ingrowth and cellular infiltration in this model. The alpha-smooth muscle cell actin expression suggested an active involvement of myofibroblasts in the anulus fibrosus repair processes.

Actins↗
PubMed2002

Assessment of the cellular heterogeneity of the ovine intervertebral disc: comparison with synovial fibroblasts and articular chondrocytes.

The objective of this study was to compare and contrast the cellular heterogeneity of ovine intervertebral disc (IVD) annulus fibrosus (AF) and nucleus pulposus (NP) cells, synovial fibroblasts (SFs) and articular chondrocytes (ACs) grown in monolayer and alginate bead culture. Phase contrast microscopy of the monolayers indicated that the SF and AF cells, and the AC and NP cells had similar morphologies. Immunolocalisation of type I, II and VI collagen epitopes in the monolayers, however, demonstrated distinct quantitative and relative differences between the SF and AF, and the AC and NP cells. Immunolocalisation of bead-incorporated proteoglycans (PGs) also demonstrated quantitative and qualitative differences between the SF and AF cells. Quantitation of (35)S-bead PGs indicated that the AF cells synthesised significantly higher PG levels than the SFs, and AC cells synthesised significantly higher PG levels than the NP cells. These data were also consistent with the relative metabolic activities and cellular viabilities displayed by each cell type in bead culture. AF cells shared morphological traits with SFs in monolayer, but displayed distinctly different characteristics in bead culture. Similarly, NP cells shared similarities with ACs in monolayer and bead culture, but overall were less metabolically active. Data presented here support the proposal that the AF and NP cells of the ovine IVD should be considered as specialised fibrochondrocytic cells.

Animals↗
PubMed2002

Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration.

STUDY DESIGN: Nerves and blood vessel distribution in discs were localized immunohistochemically and correlated with the proteoglycan contents of normal and degenerate disc tissues. OBJECTIVE: The aim of the present study was to systematically evaluate whether nerve and blood vessel ingrowth was associated with depletion of disc proteoglycans and degenerative changes in an established experimental model of disc degeneration. SUMMARY OF BACKGROUND DATA: Animal models of disc degeneration, allowing longitudinal study of pathogenic mechanisms, are limited. The ovine model enables systematic monitoring of blood vessel and nerve ingrowth during the development of disc degeneration after injury to the anulus fibrosus. METHODS: Merino sheep received a controlled left anterolateral surgical defect in the outer anulus fibrosus of the L1-L2 and L3-L4 discs (lesion group); sham-operated controls received the retroperitoneal anterolateral approach only. Animals were killed 3, 6, 12, and 26 months postoperation, and the discs were collected for histology and compositional and morphologic analyses. Sagittal tissue sections were stained with toluidine blue and hematoxylin and eosin; Type IV collagen immunolocalization visualized blood vessel ingrowth, and nerves were immunolocalized using monoclonal antibodies to growth-associated protein (GAP-43), protein gene product 9.5, and glial fibrillary acidic protein. RESULTS: Compositional and histologic results demonstrated early focal depletion 3-12 months postoperation of glycosaminoglycan associated with lesion development, increased blood vessel and nerve ingrowth, and infiltration of cells from the outer anulus fibrosus along the plane of the original defect. Blood vessel numbers in the outer to mid third of the anulus fibrosus were elevated in the lesion discs 3-6 months postoperation reaching a maximum at 12 months postoperation; nerves immunoreactive with protein gene product 9.5 (also maximal at 12 months postoperation) were often found associated (but not exclusively) with blood vessels, and some nerves were also reactive with GAP-43 and glial fibrillary acidic protein, but only at 12 months postoperation. CONCLUSIONS: Nerve and blood vessel ingrowth into the anulus fibrosis were strongly associated with proteoglycan depletion. The ovine anular lesion model of disc degeneration is a useful experimental model for the systematic evaluation of nerve and blood vessel development after anular injury.

Animals↗
PubMed2002

The telomeric poly(ADP-ribose) polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor, 182-kDa tankyrase-binding protein (TAB182).

Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase, was originally identified through its interaction with TRF1, a negative regulator of telomere length. Tankyrase 1 ADP-ribosylates TRF1 in vitro, and its overexpression induces telomere elongation in human cancer cells. In addition to its telomeric localization, tankyrase 1 resides at multiple subcellular sites, suggesting additional functions for this protein. Here we identify TAB182, a novel tankyrase 1-binding protein of 182 kDa. TAB182 displays a complex pattern of subcellular localization. TAB182 localizes to the nucleus in a heterochromatic staining pattern and to the cytoplasm, where it co-stains with the cortical actin network. TAB182 coimmunoprecipitates with tankyrase 1 from human cells and serves as an acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in vitro. Like TRF1, TAB182 binds to the ankyrin domain (comprising 24 ankyrin repeats) of tankyrase 1. Surprisingly, dissection of this domain reveals multiple discrete and overlapping binding sites for TRF1 and TAB182. Thus, we demonstrate five well conserved ankyrin repeat clusters in tankyrase 1. Although each of the five ankyrin repeat clusters independently binds to TRF1, only three of the five bind toTAB182. These findings suggest that tankyrase 1 may act as a scaffold for large molecular mass complexes made up of multiple binding proteins. We discuss potential roles for tankyrase 1-mediated higher order complexes at telomeres and at other subcellular sites.

Amino Acid Motifs↗