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Biomedical subjects

E J Murray

Publications and source records attributed to E J Murray.

At least 37 records · Page 2Linked to original sources
PubMed1994

Heterogeneity of growth and turnover in the femurs and humeri of calcium-replete and -deficient C57BL/6 and SENCAR mice at sexual maturity.

The effects of dietary calcium intake on the composition and turnover of the femur and humerus were compared in two mouse strains that differ in growth kinetics and phorbol ester-induced signal transduction. C57BL/6 (control) and SENCAR (large) mice were fed calcium-deficient (0.02% Ca) or -sufficient (0.6% Ca) diets from 10 to 14 weeks of age. Bone mass was determined gravimetrically. Calcium and magnesium were determined by atomic absorption spectrophotometry, while phosphorus was determined colorimetrically. Turnover was estimated pharmacokinetically in [3H]tetracycline-labeled bone. Femur and humerus lengths, breadths, masses, and ash and mineral contents were higher in SENCAR mice than in C57BL/6 mice at 10 weeks of age and after being fed 0.02% or 0.6% calcium diets for four weeks. Relative formation was higher in C57BL/6 mice than in SENCAR mice from 10 to 14 weeks of age, resulting in greater net increases (0.6% Ca diet) or lower net decreases (0.02% Ca diet) in the calcium contents of the femurs and humeri of C57BL/6 mice, compared to SENCAR mice. Calcium-deficient feeding stimulated higher relative rates of bone resorption in both strains and affected the net changes in bone calcium contents. Thus, hereditary factors in SENCAR and C57BL/6 mice that regulate formation rates, not resorption rates or the response to dietary calcium intake, appear to modulate net changes in humerus and femur calcium contents at maturity.

Age Factors↗
PubMed1993

Bone morphogenetic protein inhibits differentiation and affects expression of helix-loop-helix regulatory molecules in myoblastic cells.

Bone morphogenetic protein (BMP) reproducibly induces chondrogenesis and osteogenesis when implanted into skeletal muscle. The exact identity of the cell that responds to BMP is not known. Furthermore, controversy exists regarding the possibility that myoblastic cells may transdifferentiate to chondrocytes and osteoblasts under the influence of BMP. We have therefore, undertaken studies on the effects of BMP on differentiation in L6 and C2C12 cells, two rodent myoblastic cell lines. To gain insights into the mechanisms of action of BMP, we have studied the effects of BMP on the levels of expression of the four known myogenic determination genes: myogenin, Myo D, herculin, and myf-5. BMP inhibited myogenesis in myoblastic cells. Convincing evidence of transdifferentiation of myoblasts to chondrocytes or osteoblasts was not seen. BMP inhibited the expression of all four myogenic determination genes.

Alkaline Phosphatase↗
PubMed1993

PTH-induced osteoblast contraction is mediated by cysteine proteases.

E-64d, a membrane-permeable cysteine protease inhibitor, was tested for its ability to inhibit PTH-induced contraction in intact mouse MC-3T3-E1 osteoblastic cells. Incubation of MC-3T3-E1 cells with vehicle (DMSO) or E-64c, a nonpermeant cysteine protease inhibitor, in the presence or in the absence of PTH had no effects on cAMP production or on morphology from 0 to 90 minutes after addition. In contrast, treatment with E-64d markedly attenuated PTH-induced contraction in these cells. These findings suggest that cysteine proteases, such as the calcium-activated neutral proteases (calpains), are involved in PTH-induced osteoblastic contraction. The observation that cysteine protease activity mediates PTH-induced osteoblastic contraction also suggests that endogenous inhibitors, such as calpastatin, may also be present in the osteoblast and play a role in the regulation of stimulus-response coupling in bone. This mechanism may provide another regulatory point at which bone cells may be pharmacologically manipulated in clinical situations characterized by excessive bone resorption.

Animals↗
PubMed1993

Strain-dependent differences in vertebral bone mass, serum osteocalcin, and calcitonin in calcium-replete and -deficient mice.

The effects of dietary calcium intake on vertebral bone mass, composition, and turnover (calcium deposition and resorption) were determined in 10- and 14-week-old C57BL/6 (small) and SENCAR (large) mice. Total vertebral mass, percent ash, calcium, magnesium, and phosphorus were higher in SENCAR mice than in C57BL/6 mice at 10 weeks of age and after being fed 0.02% or 0.6% dietary calcium for 4 additional weeks. Relative calcium deposition was higher in C57BL/6 than in SENCAR mice, while relative calcium resorption was similar in both strains. The rate of resorption was higher in mice fed 0.02% dietary calcium than in those fed 0.6% dietary calcium. Thus, C57BL/6 mice gained vertebral calcium, while it remained unchanged or declined in SENCAR mice under conditions of both calcium depletion and calcium repletion. Serum osteocalcin (an index of bone formation) was higher in C57BL/6 mice than in SENCAR mice. Mathematically significant correlations between osteocalcin levels and vertebral calcium resorption and the net vertebral calcium loss were observed only in SENCAR mice. The serum calcitonin concentration was correlated with the amount of vertebral calcium resorbed in SENCAR mice, but not in C57BL/6 mice. Thus, vertebral resorption and formation are more tightly coupled in 10- to 14-week-old SENCAR mice than in C57BL/6 mice. In addition, remodeling appears to dominate vertebral calcium dynamics in SENCAR mice, while growth dominates in C57BL/6 mice during this period. Rodents have frequently been dismissed as potential models of bone aging based on the expectation that continued growth, rather than remodeling, dominates skeletal dynamics. These data clearly demonstrate that increases in body mass ("growth") are not invariably associated with continued vertebral growth. In this murine model, both heredity and dietary calcium intake modulate vertebral bone mass, turnover dynamics, and composition at sexual maturity. These differences in the development and regulation of vertebral bone mass in small C57BL/6 and large SENCAR mice suggest that animal, as well as clinical, models provide useful insights into the cellular and hormonal mechanisms of somatotype-dependent vertebral growth.

Analysis of Variance↗
PubMed1993

Identification of calcium-activated neutral protease activity and regulation by parathyroid hormone in mouse osteoblastic cells.

Calcium-activated neutral protease activity was detected in mouse MC3T3-E1 cell extracts. Inclusion of the cysteine protease inhibitor, E64c, reduced the activity, while pretreatment of intact cells with 10 nM parathyroid hormone for 90 minutes increased it. The presence of calpains in solubilized cells was confirmed by Western blotting using an antibody specific for the 80 K catalytic subunit. These results, combined with the observation that preincubation with a membrane-permeable cysteine protease inhibitor ablates 50% of the PTH-induced osteoblastic retraction, suggest that calpain-catalyzed hydrolysis of regulatory enzymes or structural proteins plays a role in mediating its short-term effects in bone.

Animals↗
PubMed1992

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist.

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothioglucose, which has disease-modifying activity in treated patients, acts as an IL-1 functional antagonist in this system.

3T3 Cells↗
PubMed1992

Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells.

Although much is known about the hormonal regulation of osteoblastic cell differentiation, much less is known about the nuclear regulatory molecules that affect this process. We analyzed the expression of several regulatory molecules of the helix-loop-helix (H-L-H) group in primary mouse calvarial cells and in MC3T3-E1 mouse osteoblastic cells in situations representing different degrees of cellular differentiation. H-L-H class regulators are known to participate directly in directing cell fate and differentiation decisions in other mesodermal lineages. Two of the molecules that we studied, Id and E12, have well-established roles in this process. The other, mTwi, the murine homolog of the Drosophila twist gene, is a newly cloned mammalian H-L-H gene. Levels of E12 RNA remained unchanged during differentiation. On the other hand, in both primary osteoblastic cells and MC3T3-E1 cells, the abundance of Id and mTwi declined with cell maturation; mTwi less dramatically than Id. That Id expression is causally related to differentiation is suggested by the finding that MC3T3-E1 cells transfected with an Id-expression plasmid fail to undergo differentiation. We conclude that helix-loop-helix regulatory genes are expressed in mouse osteoblastic cells, where they are likely to participate in differentiation. The E12 gene product is likely to function as a positive modulating factor. In contrast, Id inhibits differentiation, probably by sequestering other H-L-H gene regulators, including E12, in inactive complexes. The precise role of mTwi is more speculative at this time, but the observed pattern of expression is consistent with a role in early and midmesodermal specification that is terminated as cells differentiate.

3T3 Cells↗
PubMed1992

Doxorubicin inhibits differentiation and enhances expression of the helix-loop-helix genes Id and mTwi in mouse osteoblastic cells.

Treatment with doxorubicin is associated with decreased levels of expression of muscle-specific genes in myocytes. This may be related to an effect on expression of helix-loop-helix (h-l-h) regulatory molecules since in myoblastic cells, doxorubicin inhibits Myo D expression and enhances Id expression. We have reported that expression of Id and mouse Twist (mTwi), another h-l-h molecule, decline in association with differentiation in osteoblastic cells. We have sought, therefore, to determine the effect of doxorubicin on MC-3T3-E1 osteoblastic cells. Treatment with doxorubicin decreased total cellular protein content, reduced [3H]-leucine incorporation into protein, inhibited proliferation and diminished alkaline phosphatase activity. Glucose utilization and lactate production were not adversely affected. Id expression was increased by doxorubicin treatment under growth conditions but not differentiation conditions. Expression of mTwi was markedly increased under both growth and differentiation conditions. These data support the contention that Id and mTwi may regulate differentiation in osteoblastic cells.

Alkaline Phosphatase↗
PubMed1991

Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

Adenovirus Early Proteins↗
PubMed1990

Two-dimensional gel autoradiographic analyses of the effects of 1,25-dihydroxycholecalciferol on protein synthesis in clonal rat osteosarcoma cells.

The steady state synthesis of L-[35S]methionine-radiolabeled cellular proteins by two rat osteogenic sarcoma cell lines (G2 and C12) was examined by two-dimensional polyacrylamide gel electrophoresis under basal conditions and after 72-h treatments with 10 nM 1,25-dihydroxycholecalciferol or triamcinolone acetonide. Computer analysis resolved 681 spots, with mol wt ranging from 10-105K and isoelectric points ranging from 4.0-8.0. Fourteen spots were abundant (greater than or equal to 2000 parts/million), with the remainder occurring in limited abundance (150-2000 parts/million) in both clones. Only 28 proteins were radiolabeled at significantly different rates by G2 and C12 cells under basal conditions. The high degree of similarity in the identity and relative abundance of proteins synthesized by these distinct subclones suggests that minor changes in the levels of specific intracellular proteins may have major effects on the osteoblastic phenotype. 1,25-Dihydroxycholecalciferol [1,25-(OH)2D3] or triamcinolone acetonide treatment induced qualitative and quantitative changes in the synthesis of specific subsets of proteins, including induction of novel proteins, complete repression of proteins synthesized under basal conditions, and significant increases or decreases in the levels of others. 1,25-(OH)2D3 significantly altered the levels of 13 proteins in G2 cells and 28 proteins in C12 cells. 1,25-(OH)2D3 enhanced the synthesis of two proteins (no. 304 and 2506) in both subclones. The remainder of the proteins affected by 1,25-(OH)2D3 were unique to the subclone. With the exception of protein 304, the changes induced by 1,25-(OH)2D3 differed from those induced by triamcinolone acetonide, suggesting that unique proteins modulate the osteoblastic phenotype in response to these steroids.

Animals↗
PubMed1989

Fetal skull fracture from an automobile accident.

This is a case report of a fetal skull fracture that resulted in fetal death caused by a high-velocity automobile accident. As a 25-year old gravida 3, para 2, was pulling out of a driveway onto a highway, her car was struck in the left front end by a car traveling perpendicular to her vehicle. She died almost instantly from shock caused by massive injuries and internal hemorrhage. She had a fractured rib and multiple fractures of the pelvis as well as bilateral hemothorax and ruptured splenic and renal vessels. The fetus had depressed multiple skull fractures. The probable mechanism of fetal injury, as well as the influence of seat restraints on injury to the mother and fetus, are discussed.

Accidents, Traffic↗
PubMed1987

Site specific demethylation in the promoter of human gamma-globin gene does not alleviate methylation mediated suppression.

Human gamma A-globin genes with different patterns of methylation were synthesized in vitro. These methylated constructs were transfected into mouse L cells and their expression analyzed by S1-nuclease protection assays. We show that methylation downstream of +92 does not affect transcription while methylation downstream of -210 abolishes transcription. To test which of the six CpG dinucleotides between -210 and +92 plays a role in DNA methylation mediated suppression of transcription, we generated mutant gamma-globin genes which lack the three CpGs 5' to the cap site, the three CpGs 3' to the cap site, or all six CpGs. After in vitro methylation, we show that removal of methylated residues 5' or 3' to the cap site did not alleviate suppression, whereas removal of both 5' and 3' methylated residues restored partial expression. These results indicate that CpG methylation at different positions can cause suppression of expression and they are inconsistent with the hypothesis that site-specific demethylations in the promoter region can alleviate suppression. We propose that a minimum length of methylation-free DNA in the promoter area is the only requirement for expression of this promoter by ubiquitous transcription factors, and discuss these results with reference to methylation-free islands.

Animals↗
PubMed1987

Association between the expression of the c-myc oncogene mRNA and the expression of the receptor protein for 1,25-dihydroxyvitamin D3.

Studies in lymphocytes have indicated similarities in the state of activation, the time kinetics, and the pathologic states associated with the expression of the c-myc oncogene, and the expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor protein. Here, we have sought evidence for an association between c-myc and the 1,25-(OH)2D3 receptor protein in mammalian cells other than lymphocytes. Comparing two rat osteogenic sarcoma cell lines, one that produces constitutively relatively high levels of the 1,25-(OH)2D3 receptor protein (ROS 17/2.8) and one in which the 1,25-(OH)2D3 receptor protein is practically undetectable (ROS 2/3), we found that the 1,25-(OH)2D3 receptor-expressing cell line also expressed c-myc mRNA. In contrast, the cell line in which the 1,25-(OH)2D3 receptor was undetectable did not express c-myc mRNA. Furthermore, we transfected mouse skin fibroblasts (NIH 3T3) with a recombinant plasmid carrying the human c-myc oncogene. We found a dramatic increase in the 1,25-(OH)2D3 receptor concentration in five separate clonal lines of NIH 3T3 cells transfected with the c-myc-carrying plasmid compared to their nontransfected counterparts or to NIH 3T3 fibroblasts transfected with the vector plasmid alone. The receptor protein of the transfected cells exhibited biochemical characteristics indistinguishable from those of classical receptors for 1,25-(OH)2D3. The increased expression in the transfected cells appeared specific for the receptor for 1,25-(OH)2D3; receptors for sex steroids were not detected in the nontransfected NIH 3T3 cells and remained undetectable after transfection with c-myc. Moreover, the level of the glucocorticoid receptor protein, which was expressed in the nontransfected cells, did not change upon transfection with c-myc.

Animals↗
PubMed1986

The antiproliferative effect of calcitriol on human peripheral blood mononuclear cells.

Activation of lymphocytes leads to the expression of receptors for the calcitropic hormone calcitriol [1,25(OH)2D3], and calcitriol is a potent inhibitor of interleukin-2 (IL-2) and of lymphocyte proliferation. We used peripheral blood mononuclear cells (PBM) activated in vitro with phytohemagglutinin to study 1) the relationship between 1,25(OH)2D3 receptor expression, IL-2 production, and 1,25(OH)2D3-induced inhibition of PBM proliferation in connection with the cell cycle; 2) the effect of 1,25(OH)2D3 on PBM activation and on the expression of activation-related molecules including the IL-2 receptor, and 3) the role of calcium in the antiproliferative effect of the hormone. 1,25(OH)2D3 receptor expression occurred when PBM entered the G1a phase of the cell cycle. The concentration of the receptor protein reached a peak at G1b and declined during the S phase. 1,25(OH)2D3 inhibited cell proliferation by blocking PBM at the G1a-G1b border. The antiproliferative effect of calcitriol was not caused by hormonal interference with the calcium-dependent activation process nor with the expression of activation-related molecules including the IL-2 receptor. Moreover, this effect was not influenced by extracellular calcium, suggesting that the hormonal action cannot be due to calcium translocation. These findings support the contention that 1,25(OH)2D3-induced inhibition of PBM proliferation is mediated through selective inhibition of IL-2 production.

Antigens, Surface↗
PubMed1983

Beyond behavioural and dynamic therapy.

While historically major philosophical differences between the behavioural and dynamic approaches to therapy have existed, there is no necessary connection between metabeliefs and specific intervention techniques. Behavioural techniques can be used within a tragic vision of life and some psychoanalysts have a comic vision. Both camps want to achieve both objective and subjective changes but differ on how to achieve this. What is needed is a new, higher order theory that goes beyond both camps to help us understand the connections between cognitive, affective, and behavioural systems.

Behavior Therapy↗