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Biomedical subjects

J K Taylor

Publications and source records attributed to J K Taylor.

At least 19 recordsLinked to original sources
PubMed2001

Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity.

BACKGROUND & AIMS: Cdx2 is critical in intestinal proliferation and differentiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activation domain can modulate its function. METHODS: The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fused to the Gal4-DNA binding domain. In vivo phosphorylation was studied by metabolic labeling with (32)P-orthophosphate. To study a potential phosphorylation site, polyclonal antibodies were generated: CNL was raised against amino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which serine 60 was phosphorylated. RESULTS: A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine reduces incorporation of (32)P-orthophosphate substantially. S60-phosphorylation decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhibited with the mitogen-activated protein kinase inhibitors PD98059 or UO126. P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative compartment of the intestinal epithelial layer. In contrast, CNL recognizes Cdx2 predominantly in the differentiated compartment. CONCLUSIONS: The Cdx2 activation domain is phosphorylated at serine 60 via the mitogen-activated protein kinase pathway. S60-phosphorylated and S60-nonphosphorylated Cdx2 have different transcriptional activity, as well as different spatial expression patterns in the intestinal epithelium.

Amino Acid Sequence↗
PubMed1999

Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli.

The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bcl-xL is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-xL in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-xL. We show that this inhibitor reduces Bcl-xL RNA and protein in a concentration-dependent, sequence-specific manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results offer direct evidence that Bcl-xL is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-xL in keratinocyte and epithelial cell survival.

Apoptosis↗
PubMed1999

The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides.

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.

Apoptosis↗
PubMed1999

CREB-binding [corrected] protein interacts with the homeodomain protein Cdx2 and enhances transcriptional activity.

Cdx2 encodes for a homeodomain protein that is expressed in intestinal epithelial cells. The Cdx2 protein triggers intestinal differentiation in cell lines and is necessary for maintenance of the intestinal phenotype in mice. CBP (cAMP response element-binding protein) is a transcriptional co-activator that interacts with many transcription factors and components of the basal transcriptional machinery. In this study, we demonstrate that CBP is markedly induced upon differentiation of the Caco-2 intestinal cell line and augments Cdx2-dependent transcriptional activity. Cdx2 interacts with the amino-terminal domain of CBP, and the two proteins coexist in vivo within the same nuclear protein complex. Moreover, expression of the CBP domain that interacts with Cdx2 acts as a dominant-negative inhibitor of transcriptional activation by Cdx2. These findings demonstrate a direct interaction between an intestinal homeodomain protein and CBP and suggest that CBP participates in the network of transcriptional proteins that direct intestinal differentiation.

3T3 Cells↗
PubMed1999

Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides.

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.

Alternative Splicing↗
PubMed1997

Activation of enhancer elements by the homeobox gene Cdx2 is cell line specific.

Cdx2 is a caudal-related homeodomain transcription factor that is expressed in complex patterns during mouse development and at high levels in the intestinal epithelium of adult mice. Cdx2 activates transcription of intestinal gene promoters containing specific binding sites. Moreover, Cdx2 has been shown to induce intestinal differentiation in cell lines. In this study, we show that Cdx2 is able to bind to two well defined enhancer elements in the HoxC8 gene. We then demonstrate that Cdx2 is able to activate transcription of heterologous promoters when its DNA binding element is placed in an enhancer context. Furthermore, the ability to activate enhancer elements is cell-line dependent. When the Cdx2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line, but was unable to activate transcription in NIH3T3 cells. These data suggest that there are cell-specific factors that allow the Cdx2 activation domain to function in the activation of enhancer elements. We hypothesize that either a co-activator protein or differential phosphorylation of the activation domain may be the mechanism for intestinal cell line-specific function of Cdx2 and possibly in other tissues in early development.

3T3 Cells↗
PubMed1997

Coronal leakage: effects of smear layer, obturation technique, and sealer.

Coronal microleakage may be a major factor in the etiology of treatment failure. This study examined the effect of obturation technique, sealer, and the presence of smear layer on coronal microleakage. Two hundred extracted human teeth were assigned to 20 treatment groups. Groups were examined with the smear layer present or smear layer removed (17% REDTA). Access cavities were exposed to artificial saliva then Pelikan Ink. Teeth were cleared and linear dye penetration measured. When all groups with the smear layer removed were compared with all groups with the smear layer present, significantly less leakage was seen when the smear layer was removed. Ultrafil displayed significantly more leakage than all other groups. Vertical compaction of lateral condensation and Thermafil obturations significantly reduced leakage. AH-26 displayed significantly less leakage than Roth's 811 sealer. These results indicate that removal of the smear layer, the use of AH-26, and vertical compactin have cumulative effects in reducing coronal leakage.

Analysis of Variance↗
PubMed1997

Radiographic and histologic periapical findings of root canal treated teeth in cadaver.

UNLABELLED: The success of root canal treatment can be subjectively evaluated both clinically and radiographically. Normally, the recall radiograph is the main factor in evaluating success or failure. OBJECTIVES: This study evaluated periapical areas of root canal treated teeth by correlating radiographic and histologic findings. STUDY DESIGN: Jaws were resected from cadavers and radiographed. Those teeth that had received root canal treatment were evaluated for success or failure based on radiographic criteria. Teeth and surrounding bone were then removed en bloc and prepared for light microscopy. Untreated teeth without periapical pathosis were examined as controls. RESULTS: Root canal treated teeth classified as failures were found to consistently have inflammatory resorptive lesions at the periapices. In contrast, those treated teeth classified as radiographically successful showed varying reactions ranging from normal uninflamed to mildly inflamed. CONCLUSIONS: Those classified as failure showed consistent inflammation. However, the majority of our examined treated teeth were radiographically normal and exhibited no periapical inflammation.

Cadaver↗
PubMed1997

CDX1 protein expression in normal, metaplastic, and neoplastic human alimentary tract epithelium.

BACKGROUND & AIMS: CDX1 is an intestine-specific transcription factor expressed early in intestinal development that may be involved in regulation of proliferation and differentiation of intestinal epithelial cells. We examined the pattern of CDX1 protein expression in metaplastic and neoplastic tissue to provide insight into its possible role in abnormal differentiation. METHODS: Tissue samples were stained by immunohistochemistry using an affinity-purified, polyclonal antibody against a peptide epitope of CDX1. RESULTS: Specific nuclear staining was found in epithelial cells of the small intestine and colon. Esophagus and stomach did not express CDX1 protein; however, adjacent areas of intestinal metaplastic tissue intensely stained for CDX1. Adenocarcinomas of the stomach and esophagus had both positive and negative nuclear staining for CDX1. Colonic epithelial cells in adenomatous polyps and adenocarcinomas had a decreased intensity of staining compared with normal colonic crypts in the same specimen. CONCLUSIONS: CDX1 may be important in the transition from normal gastric and esophageal epithelium to intestinal-type metaplasia. The variability in expression of CDX1 in gastric and esophageal adenocarcinomas suggests more than one pathway in the development of these carcinomas. The decrease of CDX1 in colonic adenocarcinomas may indicate a role for CDX1 in growth regulation and in the maintenance of the differentiated phenotype.

3T3 Cells↗
PubMed1997

Comparison of intestinal phospholipase A/lysophospholipase and sucrase-isomaltase genes suggest a common structure for enterocyte-specific promoters.

Intestinal phospholipase A/lysophospholipase (IPAL) is an intestine-specific brush-border enzyme expressed during development and along the intestinal crypt-villus axis in a pattern similar to another well characterized brush-border enzyme, sucrase-isomaltase (SI). A tissue-specific DNase I hypersensitive site was identified in chromatin from intestinal nuclei immediately upstream from the transcriptional start site of the IPAL gene. Footprinting analysis showed that two DNA elements within the IPAL promoter were protected by intestinal nuclear proteins. The IPAL-FP1 element was shown to be a monomer binding site for Cdx1 and Cdx2, intestine-specific homeobox proteins. Moreover, this site was important for transcriptional activation of the promoter in intestinal cell lines via interaction with Cdx proteins. Nuclear proteins from both liver and intestine interacted with the IPAL-FP2 element, forming a complex consistent with binding to HNF1. Cdx and HNF1 binding sites have also been shown to be the two major regulatory elements responsible for transcriptional activation of the SI gene promoter, which directs intestine-specific transcription in transgenic mice. These findings suggest that enterocyte genes that are expressed in similar developmental patterns may be regulated by the interaction of common DNA elements and their associated transcription factors.

Animals↗
PubMed1996

Corrosion at the interface. A possible solution to cobalt-chrome heads on titanium alloy stems.

The low-wear characteristics of cobalt-chrome femoral heads matched with the excellent biocompatibility and low modulus of titanium alloy femoral stems constitute the preferred combination used by many orthopaedic surgeons performing total hip arthroplasty. The combination of these materials in a synovial fluid environment, however, has proven to result in extensive crevice corrosion and metallosis of the surrounding tissues. This study investigates an alternative to the conventional mating of dissimilar metals at the head-neck junction. Five cobalt-chrome heads premated with titanium alloy sleeves were investigated by gross examination, dissecting microscopy, and scanning electron microscopy. Examination by both gross examination and dissecting microscope revealed no signs of corrosion. Scanning electron microscope examination revealed slight crevice corrosion in the only head with a +15-mm neck length.

Alloys↗
PubMed1995

Use of acetabular models in planning complex acetabular reconstructions.

The number of patients requiring revision total hip arthroplasty continues to increase each year. Accurate preoperative planning is a key factor in obtaining a good result. Radiographs provide little information concerning the actual extent of the acetabular defects. Computed tomography-generated models of the acetabulum can provide the surgeon with accurate information concerning the size and location of the defects. Evaluation of radiographs and models in 24 cases showed that radiographs alone failed to detect all 13 anterior wall defects (P < .001), 8 of 18 posterior wall defects (44.4%, P < .001), and 8 of 19 segmental central defects (42%, P < .001), all of which were easily identified with the models. This study showed that preoperative planning based on the foam models accurately predicted the actual implant used in 22 of 24 cases (92%).

Acetabulum↗
PubMed1993

Management of bone loss in revision total hip arthroplasty using custom cementless femoral components.

Forty-seven consecutive revision total hip arthroplasties were performed using porous ingrowth custom-made prostheses designed from plain radiographs and computed tomography scans. At an average follow-up period of 30 months (range, 2-4 years), one revision has been required for loosening. The average Harris pain score was 39 and the average total score was 84 at the last follow-up evaluation. Complications included nine cases (19%) with intraoperatively detected fractures of the proximal femur treated with cerclage wires and seven cases (15%) with subsidence greater than 3 mm. There were no postoperative infections and only two dislocations. At the time of surgery local particulate bone graft was used in all cases. Thirty-four percent required structural graft to restore bone stock; no case required grafting for prosthesis stability. Management of bone loss in total hip arthroplasty using a custom cementless prosthesis appears to provide an attractive alternative to massive bone grafting.

Adult↗
PubMed1993

Comparison of glucocorticoid-induced effects in prolactin-dependent and autonomous rat Nb2 lymphoma cells.

Cultured Nb2 node rat lymphoma cells require lactogenic hormone for their proliferation. We reported previously that dexamethasone (Dex) inhibits prolactin (PRL)-induced mitogenesis and, in the absence of mitogen, induces apoptosis of Nb2 cells. Both antiproliferative and cytolytic effects of Dex on Nb2 cells appear to involve glucocorticoid (Type II) receptor mediation. In this study, we compared Dex effects in PRL-dependent Nb2 cells (Nb2) with SFJCD1 (SF), a clone of Nb2 cells that proliferates independently of exogenous PRL. Proliferative assays involved a 72-hr incubation in a chemically defined, serum-free medium where ovine PRL (1 ng/ml) was added to Nb2 cells but not to SF cells. Both cell lines were responsive to the antiproliferative effects of Dex in a dose (6.25-200 nM)-dependent fashion of comparable sensitivity and magnitude. Co-incubation with the glucocorticoid receptor antagonist, RU 486, prevented the antiproliferative effect of Dex in both cell lines. In the same medium devoid of PRL, Dex was cytolytic to Nb2 cells and fragmented DNA in a fashion reflective of apoptosis, but was ineffective in SF cells. A dual chamber incubation system revealed no evidence that SF cells produced cytokines that were mitogenic or anticytolytic to Nb2 cells. Both Nb2 and SF cells fragmented DNA in a fashion indicative of apoptosis in the presence of the Ca2+ ionophore, A23187 (1 microM). These studies reveal a basic difference in glucocorticoid responsiveness between the PRL-dependent Nb2 cell line and its PRL-independent subclone, SF. While both cell lines exhibit functional glucocorticoid receptors and the necessary intranuclear machinery for apoptosis, the pathway mediating the latter is inhibited or dysfunctional in SF cells.

Animals↗
PubMed1990

Kinetics of germinal center development in lymph nodes of young and aging immune mice.

Recent findings imply that germinal center paucity in old mice, at least in part, results from a defect in the mechanisms responsible for the transport of antigens to lymphoid nodules (follicles) and the consequent impairment of the antigen retaining reticulum (ARR) of follicular dendritic cells (FDCs). The present objective was to observe the kinetics of lymph node germinal center development in old mice having antigen transport and ARR deficits. Germinal center development was monitored in popliteal (PLN) and axillary (AXLN) lymph nodes of 6-8 wk and 23-mo-old horseradish peroxidase (HRP) immune C57BL/6 mice. Using the selective binding of germinal center B cells for peanut agglutinin (PNA), germinal centers were identified in serial vibratome sections following histochemical labeling with PNA-peroxidase conjugates at times 0, 15 min, 1, 3, 5, and 10 days after footpad challenge with 8 micrograms HRP. To follow the fate of preexisting (environmental antigen-induced) germinal centers and the development of de novo (HRP-induced) germinal centers, it was essential to distinguish between these germinal centers. Accordingly, PNA positive germinal centers associated with HRP-retaining (peroxidase positive) ARR were identified as de novo germinal centers and germinal centers not associated with a peroxidase positive ARR were classified as preexisting germinal centers. Kinetic analysis of PNA positive germinal centers showed the following: 1) Preexisting, environmentally-induced germinal centers dissociated and disappeared by day 3 as indicated by a decline in their numbers after antigen injection: the process of germinal center dissociation remained unaffected by aging. 2) The latency of de novo germinal center appearance was approximately equal in duration (approximately 3 days) to the disappearance of pre-existing germinal centers. 3) The number and size of de novo HRP-induced germinal centers increased through the experimental period in young lymph nodes, but in old mice these parameters were depressed, resulting in a significant germinal center deficit. 4) The ratio of HRP-retaining ARR to de novo induced germinal centers was 1:1 in young and responder old mice. This ratio was not affected by aging. This finding favored the concept that antigen retention in ARR is a requirement of germinal center development. The observations supported our hypothesis that germinal center development, at least in part, depends on a normal antigen transport by showing that in aged mice with defective antigen transport-related ARR and iccosome deficits there is an impaired development of germinal centers.

Aging↗