PubMed Health⌕ Search

Biomedical subjects

Mark E Bolander

Publications and source records attributed to Mark E Bolander.

At least 19 recordsLinked to original sources
PubMed2006

Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma.

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.

Adaptor Proteins, Signal Transducing↗
PubMed2006

High WT1 expression is associated with very poor survival of patients with osteogenic sarcoma metastasis.

PURPOSE: Although metastasis is the primary determinant of poor survival of patients with osteogenic sarcoma, some patients live much longer than others, indicating metastatic heterogeneity underlying survival outcome. The purpose of the investigation was to identify genes underlying survival outcome of patients with osteogenic sarcoma metastasis. EXPERIMENTAL DESIGN: We have used microarray to first compare mRNA expression between normal bone and osteogenic sarcoma specimens, identified genes overexpressed in osteogenic sarcoma, and compared expression of the selected gene between a poorly metastatic (SAOS) and two highly metastatic cell lines (LM8 and 143B). Finally, expression of the selected gene was assessed by immunostaining of osteogenic sarcoma samples with known survival outcome. RESULTS: Microarray analysis revealed 5.3-fold more expression of WT1 mRNA in osteogenic sarcoma compared with normal bone and >2-fold overexpression in 143B and LM8 cells compared with SAOS. Furthermore, WT1 mRNA was absent in normal bone (10 of 10) by reverse transcription-PCR but present in osteogenic sarcoma-derived cell lines (5 of 8). One hundred percent (42 of 42) of low-grade osteogenic sarcoma specimens expressed no WT1 as determined by immunostaining; however, 24% (12 of 49) of the high-grade specimens showed intense staining. Mean survival of patients with high-grade metastatic osteogenic sarcoma but low WT1 staining (27 of 37) was 96.5 +/- 129.3 months, whereas mean survival of patients with high-grade metastatic osteogenic sarcoma having intense staining (10 of 37) was 18.3 +/- 12.3 months (P > 0.0143). All splice variants of WT1 mRNA, including a hitherto unknown variant (lacking exons 4 and 5), were found to be expressed in osteogenic sarcoma. CONCLUSION: WT1 seems to be associated with very poor survival of patients with osteogenic sarcoma metastasis.

Base Sequence↗
PubMed2006

Follow-up study of long-term survivors of osteosarcoma in the prechemotherapy era.

Osteosarcoma is the most common primary bone sarcoma. Several studies published in the 1960s established that approximately one fifth of patients survive when treated with surgery alone. There is no information, however, about the long-term consequences of osteosarcoma. It is especially relevant to know if these patients are at risk for a second malignancy. We reviewed all clinical records from long-term (defined as more than 10 years) osteosarcoma survivors treated at Mayo Clinic in the prechemotherapeutic era from 1900 to 1960. We re-reviewed histological sections for most cases. Patients or next of kin provided follow-up information during telephone interviews. Rates of second malignancy were compared with expected rates in the population at large. We identified 465 patients treated for osteosarcoma. Of these patients, 83 (17.8%) were long-term survivors, including 19 who were alive up to 65 years after treatment. Of the 7 patients with pulmonary metastases, 3 died. A second malignancy developed in 26 patients, 15 of whom died of the malignancy. Although long-term survivors of osteosarcoma have a higher incidence of a second malignant tumor than a normal population, this increase was not statistically significant. No demographic or histological variables predicted long-term survival.

Age Distribution↗
PubMed2006

Expression of a novel alternatively spliced UCP-2 transcript in osteogenic sarcoma.

BACKGROUND: Development of chemoresistance is common in patients with osteogenic sarcoma (OGS); however, the underlying mechanism is largely unknown. Many anticancer drugs exert their therapeutic action by generating reactive oxygen radicals, which might be countered by the cancer cell through induction of uncoupling protein 2 (UCP-2). UCP-2 has been shown to be able to protect tumor cells from the cytotoxic actions of chemotherapeutic drugs. Because OGS is seldom completely cured by current chemotherapy regimens, we hypothesized that increased expression of UCP-2 underlies this phenomenon. The primary initial interest of our research was to evaluate the level of UCP-2 mRNA in OGS. METHODS: The level of UCP-2 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR) comparing expression in normal-bone-derived specimens and OGS-derived specimens. Semiquantification of mRNA expression was achieved by radioactive RT-PCR. Nucleotide sequencing was performed using automated instruments. RESULTS: Interestingly, we failed to observe induction of UCP-2 mRNA in OGS tumor specimens and OGS-derived primary cell lines compared to the expression level in normal bone. However, we found expression of a hitherto unknown UCP-2 transcript in eight of eight OGS-derived and one EWS-derived cell lines and in nine of ten OGS biopsy specimens but in only one of six normal bone-derived specimens. Thus, tumor samples express both types (normal and the novel one) of UCP-2 mRNAs, whereas normal bone expresses only the wild-type form. Further experiments identified the novel mRNA species as an alternatively spliced UCP-2 transcript (termed UCP-2as). UCP-2as has a 22-nucleotide insertion from the 3' end of intron 3 that introduces an early stop codon in exon 4, which theoretically can produce a protein 79 amino acids long. CONCLUSIONS: We have identified a hitherto unknown UCP-2 transcript. Expression of the novel transcript appears to be OGS-specific, implying a function advantageous to the tumor.

Bone Neoplasms↗
PubMed2006

Bicuspid aortic valve and ascending aortic aneurysm are not associated with germline or somatic homeobox NKX2-5 gene polymorphism in 19 patients.

BACKGROUND: Bicuspid aortic valve is the most common congenital anomaly, occurring in 1% to 2% of the population. It is the most common reason for aortic valve replacement, and such individuals are at significantly increased risk of aortic complications. Despite the clinical significance of bicuspid aortic valve, its genetic basis remains unclear. The homeobox gene NKX2-5 occupies a central position in the hierarchy of cardiac determinants, and mutations in this gene are associated with bicuspid aortic valve in mice. We therefore investigated the presence of mutations in NKX2-5 among patients with bicuspid aortic valve and associated aneurysm. METHODS: Germline DNA was extracted from peripheral blood leukocytes and somatic DNA from diseased aortic tissues of 19 patients with bicuspid aortic valve and associated aortic aneurysm. Three patients with trileaflet aortic valve and aneurysm served as control subjects. The entire NKX2-5 coding sequence, including intron-exon boundaries, was screened for mutation by means of polymerase chain reaction, followed by DNA sequencing. RESULTS: Direct sequencing revealed a change in somatic (aortic) DNA 239A-->G, leading to synonymous amino acid alteration of Glu21Glu in one patient with bicuspid aortic valve and 1 control subject. There were no other alterations detected in the coding regions of germline or somatic genes. A known polymorphic change in the 3' untranslated region adjacent to exon 2 was detected in both bicuspid aortic valve and control samples. Discrepancies between germline and somatic DNA sequences were observed. CONCLUSION: Our study fails to demonstrate an association between bicuspid aortic valve and NKX2-5 mutation, as has been seen in mice. Our findings support the importance of sequencing somatic, as well as germline, DNA.

Aortic Aneurysm, Thoracic↗
PubMed2005

The use of microorganisms for the formation of metal nanoparticles and their application.

Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The development of reliable experimental protocols for the synthesis of nanomaterials over a range of chemical compositions, sizes, and high monodispersity is one of the challenging issues in current nanotechnology. In the context of the current drive to develop green technologies in material synthesis, this aspect of nanotechnology is of considerable importance. Biological systems, masters of ambient condition chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macroscale. Recent studies on the use of microorganisms in the synthesis of nanoparticles are a relatively new and exciting area of research with considerable potential for development. This review describes a brief overview of the current research worldwide on the use of microorganisms in the biosynthesis of metal nanoparticles and their applications.

Bacteria↗
PubMed2005

Comparative evaluation of two two-dimensional gel electrophoresis image analysis software applications using synovial fluids from patients with joint disease.

The proteomic composition of synovial fluid (SF) may hold clues to understanding the molecular basis of arthritis. However, the highly viscous nature and proteomic complexity of SF present a challenge when analyzing results obtained by two-dimensional gel electrophoresis (2D-GE). Several software applications are available for analyzing 2D-GE images. Despite inherent strengths and weaknesses, no comparison between these applications has been reported using SF or any human fluid specimens. We evaluated two common software packages--PDQuest and Progenesis Workstation--for spot detection, matching, and quantitation of 2D-GE images of SF from four patients with arthritic disease. Initially, whole 2D-gel images were analyzed for spot detection, which suggested that PDQuest is more consistent than Progenesis; however, PDQuest appeared to require more user intervention than Progenesis. Subsequently, two small areas (spots well resolved and spots not well resolved) were selected from each gel image, which were analyzed by the software for spot detection, matching, volume, and resolution. These analyses suggest that both tools can quantify well-resolved spots relatively consistently when compared with manual spot detection (the "gold standard"). The "3D viewer" option offered by both tools enables correct spot identification and matching. The strengths and weaknesses of these computer tools can provide guidance in the choice of a particular workstation for identifying biomarkers of arthritis.

Adult↗
PubMed2004

Continuous parathyroid hormone induces cortical porosity in the rat: effects on bone turnover and mechanical properties.

UNLABELLED: We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. INTRODUCTION: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. MATERIALS AND METHODS: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 microg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. microCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. RESULTS AND CONCLUSIONS: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.

Animals↗
PubMed2004

Identification of estrogen-regulated genes during fracture healing, using DNA microarray.

Estrogen deficiency impairs fracture healing, while estrogen treatment of ovariectomized rats accelerates fracture healing. To identify genes regulated by estrogen during fracture healing, we evaluated gene expression in calluses from three groups of rats: sham-operated, ovariectomized, and ovariectomized and treated with estrogen. RNA from calluses harvested 10 days after fracture was subjected to DNA microarray-based analysis of 5147 genes. In total, 52 genes were identified whose mRNA expressions were found to be downregulated by ovariectomy but restored with estrogen. Differential mRNA expression of 4 genes (collagen type 2, extracellular superoxide dismutase, urokinase-type plasminogen activator [ u-PA], and ptk-3) was confirmed by reverse transcription polymerase chain reaction. Further, chondrocytes and chondroclasts were positive for u-PA in the junction between cartilage and bone, implying its importance in resorption and remodeling of callus. Identification of these genes is a prerequisite to understanding the mechanism by which estrogen influences the complex metabolic process of fracture repair.

Animals↗
PubMed2004

Evaluation of different conditions for ligating dumbbell-shaped oligonucleotides.

We tested three different standard ligation conditions (37 degrees C for 30 min, 16 degrees C for 24 h, and 4 degrees C for 48 h) to generate dumbbell-shaped oligonucleotides (ODNs) as transcription factor decoys for SOX9 and alphaA-crystallin binding protein 1 (CRYBP1), which are positive and negative transcriptional regulators for type II collagen expression in chondrocytes. Decoy ODN for CRYBP1 was successfully produced as a "dumbbell" by all three conditions. A small amount of decoy ODN for SOX9, however, remained unligated under all three ligation conditions. Ligation at 4 degrees C for 48 h appeared to be the least desirable for SOX9 ODN. Transfection experiments with the SOX9 ODN ligated in different conditions and a luciferase-based reporter system also supports this conclusion. In general, shorter incubation time produced more acceptable results for this ODN than incubation for a longer time. These data suggest that different ligation conditions should be tested prior to creating dumbbell-shaped ODNs for transfection experiments.

Animals↗
PubMed2004

Oligonucleotide decoy mimicking alphaA-crystallin-binding protein 1 binding site on mouse Col2a1 enhancer stimulates transcription from the adjacent Col2a1 promoter in chondrogenic ATDC5 cell.

A 48-bp sequence element in intron 1 of the alpha1(II) collagen gene (Col2a1) acts as an enhancer of Col2a1 transcription and contains binding sites for the transcription activator SOX9 and repressor alphaA-crystallin-binding protein 1 (CRYBP1). We hypothesized that abrogating CRYBP1 binding should increase transcription from a promoter associated with the Col2a1 enhancer. We tested this hypothesis by cotransfecting an oligonucleotide (ODN) decoy for CRYBP1 and a luciferase-based reporter vector under the transcriptional control of the Col2a1 promoter linked to the 100-bp enhancer in chondrogenic ATDC5 cells. As a control, we used decoy ODN corresponding to the SOX9 binding site. Transfection with CRYBP1 decoy increased luciferase activity by >2.5-fold in the absence or presence of insulin, whereas SOX9 decoy ODN decreased luciferase activity to about 50% under similar conditions. In addition, the repressive effect of interleukin-1 on Col2a1 transcription through decreasing SOX9 messenger ribonucleic acid (mRNA) expression and increasing CRYBP1 mRNA expression, was counteracted by CRYBP1 decoy ODN. These results provide a rationale for gene therapy in degenerative joint diseases by elevating Col2a1 expression in chondrocytes through oligomimetics of repressor binding sites.

Animals↗
PubMed2003

Two-dimensional gel electrophoresis of synovial fluid: method for detecting candidate protein markers for osteoarthritis.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and Gamma-globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89-0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85-0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

Biomarkers↗
PubMed2003

Determination of the complete cDNA sequence of rat type II collagen and evaluation of distinct expression patterns of types IIA and IIB procollagen mRNAs during fracture repair in rats.

Elucidating the molecular mechanisms that underlie fracture healing is crucial to understanding and devising strategies for the management of fractures, especially those associated with a pathological condition such as diabetes or old age. Cartilage formation, and therefore the expression of type II collagen by chondrocytes, is a critical step in frac-ture healing. Two forms of type II collagen, IIA and IIB, are known to be produced by alternative splicing of the Alpha(1) (II) procollagen gene. We have followed the patterns of expression of these two forms of type II collagen to determine the nature of chondrocyte recruitment during fracture healing. First, we sequenced the rat collagen type II cDNA to design the primers. Second, using a competitive quantitative reverse transcription-mediated polymerase chain reaction, we provide evidence that (1) there is a basal level of type IIA collagen expression during the early stages of fracture healing; (2) transient but sharp up-regulation of IIA expression occurs concomitant with chondrogenesis and endochondral ossification; and (3) type IIB collagen is the predominant mRNA variant expressed at virtually all times during fracture repair.

Animals↗
PubMed2003

Statin stimulates bone morphogenetic protein-2, aggrecan, and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes.

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and (35)S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2 microM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.

Aggrecans↗
PubMed2003

Total joint arthroplasty in human immunodeficiency virus-positive patients: an alarming rate of early failure.

The results of 21 total joint arthroplasties (13 knees, 8 hips) in 15 patients were reviewed. There were 13 men and 2 women with an average age of 38 years (range, 28 to 69 years). The mean follow-up period was 10.2 years (range, 2 to 23 years). Six patients died within an average of 10.6 years (range, 3 to 18 years) of joint arthroplasty. All patients died of AIDS. Deep infection developed in 6 joints. Knee Society scores improved significantly, but 13 repeat surgeries were required. An alarmingly high rate of complications was found after total knee and total hip arthroplasty in these patients with human immunodeficiency virus (HIV). Physicians and surgeons should be made aware of this high rate of complications after joint arthroplasty in patients with HIV and include a frank discussion of this information with their patients who are contemplating total joint arthroplasty.

Adult↗
PubMed2003

Cis-acting intronic elements that regulate cartilage-specific alternative splicing of the type II collagen (Col2) pre-mRNA lie at or near splice site junction sequences flanking exon 2 of the gene.

UNLABELLED: Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. INTRODUCTION: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. MATERIALS AND METHODS: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. RESULTS: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14-21 days) with insulin. CONCLUSION: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Alternative Splicing↗