Biomedical subjects

M J Atkinson

Publications and source records attributed to M J Atkinson.

At least 19 recordsLinked to original sources

PubMed1999

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.

Adult↗

PubMed1999

Mapping of a cadherin gene cluster to a region of chromosome 5 subject to frequent allelic loss in carcinoma.

Cadherin adhesion molecules define cellular interactions during embryogenesis and morphogenesis, while later in life they are responsible for maintaining tissue integrity. Mutation and loss of expression of cadherins have been implicated in the progression of some malignant tumors, suggesting that cadherins may also act as tumor/metastasis suppressor genes. To determine the extent to which cadherin loci could be affected by allelic losses, we used radiation hybrid mapping to define the chromosomal position of five cadherin genes. A cadherin gene cluster consisting of three genes was identified on the short arm of chromosome 5. This region of the genome is subjected to frequent allelic loss in malignant disease.

Alleles↗

PubMed1999

Cadherin-11 is highly expressed in rhabdomyosarcomas and during differentiation of myoblasts in vitro.

Rhabdomyosarcomas bear a morphological and genetic resemblance to developing skeletal muscle. Apart from myogenic marker genes (bHLH factors, myosin, actin), cell adhesion molecules such as N-cadherin and N-CAM have been reported to be expressed both in rhabdomyosarcomas and during myogenesis. The present study demonstrates the expression of another cadherin, cadherin-11, in rhabdomyosarcomas and during differentiation of myoblasts in vitro: cadherin-11, a predominantly mesenchymal cell adhesion molecule, is highly expressed in embryonal rhabdomyosarcomas and alveolar rhabdomyosarcomas, which do not bear the Pax-3-FKHR fusion previously described. Cadherin-11 is down-regulated in normal skeletal muscle and after myotube formation in vitro. The results of this study suggest that cadherin-11 might be involved in myogenesis and that rhabdomyosarcomas may re-express or fail to down-regulate cadherin-11. Since alveolar rhabdomyosarcomas bearing the t(2;13) translocation do not express cadherin-11, it is postulated that Pax-3 and cadherin-11 might be linked and involved in the same myogenic pathway.

Animals↗

PubMed1999

Functional loss of E-cadherin and cadherin-11 alleles on chromosome 16q22 in colonic cancer.

Proteins of the cadherin family regulate cellular adhesion and motility and are believed to act as tumour suppressors. Previous studies have identified frequent mutation and allelic inactivation of the E-cadherin (cadherin-1) locus in diffuse gastric cancer. At least two other cadherin genes, P-cadherin (cadherin-3) and OB-cadherin (cadherin-11), have been mapped close to the E-cadherin gene on chromosome 16q22. As this region of the genome is frequently deleted in malignancy, multiple cadherin loci may be affected by losses of chromosome 16q22. The expression of mRNA transcripts from polymorphic alleles of the E-cadherin and cadherin-11 genes was examined in 30 cases of colonic, gastric, and renal carcinoma. In gastric cancer, loss of expression of one allele was restricted to the E-cadherin locus, whilst in renal carcinoma neither locus was affected. In colonic cancers, loss of expression of one E-cadherin allele was detected in 5 of 22 cases, whilst loss of a cadherin-11 allele was seen in 5 of 23 cases. This functional loss of cadherin gene expression may be due to gene deletion, inactivation or recombination. As no evidence of cadherin gene mutation was observed in the remaining transcripts, we can conclude that these two genes are only indirectly involved in the pathogenesis of colorectal cancer.

Blotting, Northern↗

PubMed1999

1,25 dihydroxyvitamin-D3 attenuates the confluence-dependent differences in the osteoblast characteristic proteins alkaline phosphatase, procollagen I peptide, and osteocalcin.

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)2D3stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)2D3 (5 x 10(-8) M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2. 3-fold increase of OC secretion, but a decrease of PICP levels to 0. 36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)2D3 did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)2D3 stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)2D3 stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples.

Alkaline Phosphatase↗

PubMed1999

Loss of immunohistochemical E-cadherin expression in colon cancer is not due to structural gene alterations.

E-cadherin, a transmembrane cell adhesion molecule, has been observed to have an altered pattern of immunoreactivity in several types of carcinomas. In lobular breast cancer, loss of immunoreactivity has been shown to be due either to out-of-frame deletions or to nonsense mutations of the E-cadherin gene. We analysed 29 cases of completely resected colon carcinoma with immunohistochemistry using the HEC-D1 antibody. Normal protein expression similar to that in the adjacent nonmalignant mucosa was seen in 6 cases, whereas 23 tumours had reduced or absent E-cadherin expression. In the 8 cases with no expression of E-cadherin revealed by immunohistochemistry, the entire E-cadherin cDNA sequence was analysed. In these cases, sequence analysis failed to reveal any cDNA mutations despite the negative immunohistochemistry. Possible explanations for this discrepancy include regulatory defects in the E-cadherin promoter, abnormalities at the translation or protein processing levels and mutations in other parts of the gene that were not investigated by the cDNA analysis (e.g. intronic sequences), which could play a role in causing abnormal processing of the E-cadherin protein.

Adult↗

PubMed1999

Estrone and estradiol-17 beta concentration in tissue of the scleractinian coral, Montipora verrucosa.

Spawnings of scleractinian corals are affected by light, temperature, and other environmental cues, but no studies elucidate physiological mechanisms that regulate coral gametogenesis. We hypothesized that estrogens may act as bioregulators of coral reproduction. Estrone (E1) and estradiol-17 beta (E2) concentrations were measured in homogenates of tissue and skeleton from M. verrucosa. Tissue samples were collected monthly throughout the year, and more frequently in July and August around spawning. Steroids were extracted with diethyl ether, purified via celite chromatography and assayed with radioimmunoassay. Non-specific binding in coral tissue varied with sample weight and was elevated relative to standards. Monthly mean E1 ranged from 20-70 ng E1 g ash-free dry weight (AFDW)-1, with highest values in April. Smaller asynchronous peaks occurred in early July, prior to spawning. Monthly mean E2 ranged from 8-25 ng E2 g AFDW-1, with highest values in February and March. Peaks in E2 preceded peaks in E1, indicating metabolism of a pool of estrogen. E1 was positively correlated with protein concentration, which is consistent with a bioregulatory role of estrogens. Estrogen peaks in spring and prior to the July spawn corroborate our hypothesis that estrogens regulate coral gametogenesis and spawning.

Animals↗

PubMed1998

Loss of the differentiated phenotype precedes apoptosis of ROS 17/2.8 osteoblast-like cells.

Osteoblast cells, recruited from mesenchymal precursors, initiate the final phase of bone remodeling by secreting the protein components of the bone matrix. Upon completion of remodeling, some of these osteoblasts may further differentiate, giving rise to matrix-embedded osteocytes and bone lining cells. The fate of the remaining osteoblasts is unknown, although by analogy with other cell systems, apoptotic cell death may be involved. We induced and characterized the apoptotic process in ROS 17/2.8 osteosarcoma cells by growing and maintaining confluent cultures in low serum medium. At confluence, but prior to apoptosis, the levels of collagen type I, alkaline phosphatase, and osteocalcin mRNAs declined abruptly. Expression of two housekeeping genes (ribosomal protein RPS6 and GAPDH) remained unchanged. Some 72 hours later cells began to show morphological and biochemical features of apoptosis, namely, chromatin condensation, membrane budding, and internucleosomal degradation of genomic DNA. We conclude that serum starvation-induced apoptosis of ROS 17/2.8 cells can serve as a model for investigating the mechanisms of osteoblastic apoptosis.

Animals↗

PubMed1998

Mapping of a mouse mammary tumor virus integration site by retroviral LTR--arbitrary polymerase chain reaction.

The de novo integration of retroviral genomes within the mammalian genome is believed to contribute to the tumorigenic process. Integration may result in the disruption or inappropriate transcription of key regulatory genes. We describe the application of an arbitrarily primed PCR method for the mapping and cloning of genomic integration sites of the mouse mammary tumor virus (MMTV). We have amplified DNA sequences between a selected retroviral MMTV-LTR and random sites in the 3' flanking DNA. Using this technique we were able to visualize several proviral integration sites present in a MMTV-induced mammary tumor derived cell line that were absent from the germ line. Cloning and sequencing of the PCR product corresponding to one site established its identification as an unique 3' flanking sequence.

Animals↗

PubMed1998

In vitro differentiation potential of a new human osteosarcoma cell line (HOS 58).

Cultured rodent osteoblastic cells reiterate the phenotypic maturation of osteoblasts seen in vivo. Under appropriate culture conditions this maturation is a stepwise sequence of phenotypic changes culminating in the production of a mineralised matrix. Although individual components of the osteoblast phenotype are apparent in transformed osteosarcoma cell lines, the co-ordination of the maturation sequence appears to be compromised. Because to date no comparable human cell differentiation system has been developed we investigated the recently introduced HOS 58 osteosarcoma cell line up to 3 months in culture. Proliferation, the secretion of osteoblast specific proteins, gene expression and mineralisation were analysed at different time points. Low-density HOS 58 cultures exhibit rapid proliferation and high levels of c-myc, collagen type I and osteopontin mRNAs. This phenotypic stage was maximum between the 4th and 5th days of culture. As cell density increased expression of these genes declined and by day 14 the predominant mRNAs was alkaline phosphatase. Osteocalcin secretion was detected after confluence at an increasing level. In the presence of ascorbate and beta-glycerophosphate the production of alkaline phosphatase and collagen type I increased coincident with the elaboration of a Von Kossa staining matrix. Nevertheless no proper mineralisation of the collagenous matrix was detectable by electron microscopy. Hence, the human osteosarcoma cell line HOS 58 expressed a rather differentiated phenotype with further maturation during a culture period of 21 days. We conclude that the developmental sequence exhibited by the HOS 58 human osteosarcoma cell line is comparable to that described for primary rat osteoblasts. However, in detailed analysis considerable differences to other species are evident. Further evaluation of the HOS 58 system and comparison to other human osteoblast cell lines will be necessary to establish the most appropriate differentiation model for human bone cell cultures.

Adult↗

PubMed1998

The use of confluence stages does not decrease the overall variability in primary human osteoblasts but can give additional information on differentiation in vitro.

Primary cultures of human osteoblast-like cells are frequently used to study osteoblast function. Due to inhomogeneous growth of primary osteoblasts in culture, it is of interest if the use of confluence stages for analysis would reduce the overall variability. Consequently, we have tested the influence of degree of confluence and passage number on the growth and differentiation of human primary osteoblast-like cells. Phenotypic features of primary human osteoblast-like cells were compared at four successive cell densities defined as stage of confluence I (50%), stage II (75%), stage III (100%) and stage IV (5 days post confluence). The stability of the system was also tested by comparing these observations obtained using cells from the 2nd and 4th passages. As a sign for further differentiation, the number of AP-positive cells increased with a decrease in proliferation. The secretion of procollagen-I decreased to 50% during culture while procollagen I mRNA doubled from proliferation to confluence. A constant activity of alkaline phosphatase, procollagen-I secretion and procollagen I gene expression over passages was seen together with a decrease in growth. The paper introduces a potential model of osteoblastic differentiation in vitro for human primary osteoblast-like cells. We were able to show an increasing differentiation with a decrease in proliferation and the stability of this differentiation behaviour over cell passages in this model, but we were not able to reduce the overall variability.

Adult↗

PubMed1997

The differential effects of mood on patients' ratings of life quality and satisfaction with their care.

The recent directives to improve both the quality and the efficiency of mental health service delivery systems have emphasized the need for evidence based treatment efficacy data, yet recent evidence suggests that quality of life data may be confounded with psychiatric symptomatology. The objective of the current inquiry was to determine whether responses to patient satisfaction measures are equally effected by mood-congruent response bias. Thirty-seven patients from a mood disorders clinic in an urban acute care hospital were asked to rate their current mood, satisfaction with their care, and quality of life. While patient rating of mood were highly correlated with specific quality of life scales and predicted 21% of the variance in global quality ratings, the more objective satisfaction indicators were not. For the clinician, these data suggest that clinically depressed patients may view their support system and care givers in negative or biased perspective.

Female↗

PubMed1996

A defective Vkappa A2 allele in Navajos which may play a role in increased susceptibility to haemophilus influenzae type b disease.

The antibody response to H. influenzae type b (Hib) is pauciclonal, and is dominated by antibodies using the VkappaA2 gene. Navajos have a 5-10-fold increased incidence of Hib disease compared with control populations. We hypothesized that a polymorphism in one of the genes in this oligoclonal response may lead to increased disease susceptibility. Since the predominant A2+ anti-Hib antibodies have high avidity for Hib and can be unmutated, the A2 Vkappa gene was analyzed. Over half of the Navajos studied, but only one control individual, had a new allele of A2, termed A2b, with three changes from the published A2 germline sequence. One of the changes was in the recombination signal sequence, suggesting that the A2b allele might not undergo V-J rearrangement very frequently. This possibility was confirmed by analyzing the relative frequency of non-productive A2 rearrangements in A2a/b heterozygous Navajos. Many fewer A2b rearrangements were observed, showing that the A2b allele is defective in its ability to undergo rearrangement. The prevalence of this allele in Navajos may play a role in their increased susceptibility to invasive Hib disease. If so, it would underscore the importance of the germline Ig repertoire for protective antibody responses to pathogenic bacteria in unimmunized children.

Alleles↗

PubMed1996

Type-1 plasminogen activator inhibitor in human renal cell carcinoma.

In experimental models, plasminogen activator-mediated degradation of the extracellular matrix is inhibited by type-1 plasminogen activator inhibitor (PAI-1). PAI-1 has also been shown to protect tumour stromal tissue from autoproteolytic activities and may thus substantially promote tumour growth and metastasis formation. Human renal cell carcinoma (RCC) cells express significant amounts of plasminogen activator activity. In the present study, the expression of its specific inhibitor PAI-1 has been investigated in 32 cases of RCC and compared with adjacent non-tumour renal tissues. RCC tissue exhibited higher levels of PAI-1, determined at both the antigen and the mRNA level by ELISA and Northern blot analysis respectively. Immunohistochemical analysis showed that PAI-1 antigen was primarily confined to tumour cells and vascular endothelium, a distribution similar to that previously reported for plasminogen activator activity in RCC. The close co-localization with endogenous plasminogen activator activity may be important in the regulation of RCC-associated proteolysis. The increased expression of PAI-1 and its predominant localization within the tumour may help to conserve tumour tissue integrity and may thus promote RCC progression and metastasis formation.

Blotting, Northern↗

PubMed1996

The diagnostic potential of the chromosome translocation t(2;13) in rhabdomyosarcoma: a Pcr study of fresh-frozen and paraffin-embedded tumour samples.

The chromosomal translocation t(2;13)(q35;q14) has been reported in alveolar paediatric rhabdomyosarcoma. The rearrangement leads to the juxtaposition of the PAX-3 and FORKHEAD genes and the production of a fusion protein with putative transcriptional regulatory activity. The diagnostic potential of this translocation has been examined using a reverse transcription polymerase chain reaction (RT-PCR) assay to detect translocations in both fresh-frozen and archival formalin-fixed, paraffin-embedded rhabdomyosarcoma. A total of 25 tumours and one cell line were examined. PAX-3-FORKHEAD chimeric mRNAs were amplified by PCR in 8 of 15 cases of alveolar rhabdomyosarcoma. Translocations were detectable in both fresh-frozen tissues (4 of 7) and paraffin-embedded tumours (3 of 7) and in the alveolar rhabdomyosarcoma cell line. Our study confirms that the t(2;13) translocation is not present in embryonal rhabdomyosarcomas, but can be detected in nearly half of alveolar rhabdomyosarcomas, whether fresh-frozen or paraffin-embedded. The PCR-based t(2;13) translocation assay can aid in the diagnosis of rhabdomyosarcoma, but cannot replace a careful histopathological evaluation. It may contribute in further characterizing an otherwise undifferentiated small cell tumour, where it may be indicative of clinical behaviour.

Blotting, Southern↗

PubMed1996

Sites of urokinase-type plasminogen activator expression and distribution of its receptor in the normal human kidney.

The urokinase-type plasminogen activator (uPA) is secreted into the urine at high concentrations and both the uPA protein and mRNA are present in human renal tissue. Normal kidney tissue also expresses the receptor for uPA. Neither the precise sites of uPA mRNA expression, nor the distribution of the uPA-receptor antigen, have been elucidated in the human kidney. In the present study, the sites of uPA mRNA expression were identified by in situ hybridization, and the cellular localization of both uPA and uPA-receptor was determined by immunohistochemical analysis. High-level uPA mRNA expression was restricted to epithelial cells of the convoluted proximal tubules and the thick ascending limb of Henle's loop (the straight part of the distal tubule). However, uPA immunoreactivity was not confined to sites of uPA mRNA expression, but was present in all segments of the tubular epithelium. Tubular epithelial cells also exhibited a consistent immunoreactivity with uPA-receptor antibody, indicative of a co-localization of the uPA antigen and its receptor in the uriniferous epithelium. We propose that the uPA antigen expression in nephron segments lacking demonstrable endogenous uPA synthesis may be the result of a uPA-receptor-mediated uptake of uPA.

Humans↗

PubMed1996

New alleles of IGKV genes A2 and A18 suggest significant human IGKV locus polymorphism.

The human kappa light chain consists of approximately 35 potentially functional IGKV genes. However, an estimation of the diversity in the IGKV repertoire of an individual will be affected by the extent of polymorphisms for the different IGKV genes and their patterns of inheritance. To date, little information is available to indicate the extent of allelic variation of the IGKV genes. We examined the extent of allelism for one IGKV gene pair, the distal region A2 gene and its closely related proximal region duplicate A18. We found two new alleles for A2 and one new allele for A18, and sequenced approximately 1 kilobase flanking each gene. The new A18 allele, unlike the originally described allele, appears to be functional. All these alleles were found at relatively high frequencies in the four ethnic populations studied, with the exception of the defective A2b allele which was highly represented only in Navajos. The originally described A2a allele encodes for the predominant protective antibody against Haemophilus influenzae. Therefore, the patterns of allelic inheritance described for this IGKV gene pair indicate that allelism in the IGKV locus is likely to have a significant impact on immune responses.

Alleles↗

PubMed1995

Inactivation of the bovine spongiform encephalopathy agent by rendering procedures.

Bovine brain infected with the bovine spongiform encephalopathy (BSE) agent was used to spike material processed in pilot scale facsimiles of 12 rendering processes which are used within the European Union, and three which are not. The raw materials for experimental rendering represented those used in practice, and consisted of appropriate proportions of BSE-infected brain tissue, bovine or porcine intestine, and bovine bone. Meat and bone meal, and tallow were produced from the rendered tissues. Suspensions of all the meat and bone meal samples were assayed in inbred mice for BSE infectivity, and two of the tallow fractions were tested similarly. Four of the 15 processes produced meat and bone meal with detectable BSE infectivity. Neither of the tallow samples had detectable infectivity.

Animal Feed↗