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Message Levels (message + level)
Selected AbstractsTemporal expression of growth factors and matrix molecules in healing tendon lesionsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2005Linda A. Dahlgren Abstract Overuse tendon injuries are common among elite and recreational athletes. Tendon healing may be enhanced at the cellular level through the use of exogenous growth factors; however, little is known about the endogenous expression of growth factors in healing tendon. This study describes the temporal expression of insulin-like growth factor-I (IGF-I), transforming growth factor-,1 (TGF-,1), and collagen types I and III in healing tendon lesions. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8 or 24 weeks following injury. Gene expression was evaluated using Northern blot analysis (collagen types I and III), real time PCR (IGF-I and TGF-,1), and in situ hybridization. Protein content was assayed by dye-binding assay (collagen types I and III), radioimmunoassay (IGF-I), ELISA (TGF-,1), and immunohistochemistry. Samples were also processed for differential collagen typing. DNA and glycosaminoglycan content, and routine H&E staining. Microscopically, lesions progressed from an amorphous, acellular lesion soon after injury to scar tissue filled with collagen fibers and mature fibroblasts organized along lines of tension. Early lesions were characterized by immediate increases in expression of growth factors and collagen. Message levels for TGF-,1 peaked early in the wound healing process (1 week), while IGF-I peaked later (4 weeks), as the regenerative phase of healing was progressing. In the first 2 weeks after lesion induction, tissue levels of IGF-I protein actually decreased approximately 40% compared to normal tendon. By 4 weeks, these levels had exceeded those of normal tendon and remained elevated through 8 weeks. Message expression for collagen types I and III increased by 1 week following injury and remained elevated throughout the course of the study. Collagen type I represented the major type of collagen in healing tendon at all time points of the study. Based on these results, IGF-I, administered exogenously during the first 2 weeks following injury, may provide a therapeutic advantage by bolstering low endogenous tissue levels and enhancing the metabolic response of individual tendon fibroblasts. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Wilms' tumor 1 message and protein expression in bone marrow failure syndrome and acute leukemiaPATHOLOGY INTERNATIONAL, Issue 10 2007Takashi Iwasaki Wilms' tumor 1 (WT1) is a useful marker for the diagnosis of acute leukemia and myelodysplastic syndromes (MDS). In the current study quantitative reverse transcription,polymerase chain reaction and immunostaining were used simultaneously to examine the relationship between WT1 RNA and protein level and also to evaluate WT1 as a tool to differentiate aplastic anemia (AA) and MDS refractory anemia (RA). Three types of WT1 messages (total, exon 5(+) and KTS(+)) and WT1 immunostaining of these diseases were analyzed. An increase of all three WT1 messages in high-grade MDS and acute leukemia was observed as compared with the normal control, whereas there was no significant difference in WT1 message between AA and RA, suggesting that WT1 message is not a good tool to discriminate AA and RA. No significant difference was observed between normal and RA, except for exon 5 message. Three WT1 message levels had a significant correlation, suggesting that the total WT1 message is sufficient for clinical practice. Positive immunostaining of WT1 was observed only in the portion of acute leukemia and overt leukemia (OL) transformed from MDS with a high WT1 message level, suggesting the relatively high detection threshold of WT1 protein with the immunostaining method. [source] Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitorFEBS JOURNAL, Issue 21 2000Laurie A. Graham The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5.5 °C (,T = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11,13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis >,3 °C. Exposure of small larvae (11,13 mg) to 4 weeks of cold (4 °C) caused an ,,20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 °C. Exposure of large larvae (100,120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 °C. Thus, thermal hysteresis increased to similar levels (> 4 °C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased >,20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins. [source] Effect of Osteoblast-Targeted Expression of Bcl-2 in Bone: Differential Response in Male and Female Mice,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2005Alexander G Pantschenko Abstract Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss. Introduction: Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function. Materials and Methods: Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR. Results and Conclusions: Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 ± 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 ± 0.1% and 20.1 ± 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone. [source] The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Peter V.N. Bodine Abstract Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-,1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-,1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival. © 2005 Wiley-Liss, Inc. [source] Nicotinic acetylcholine receptor-subunit mRNAs in the mouse superior cervical ganglion are regulated by development but not by deletion of distinct subunit genesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2008G. Putz Abstract Mice with deletions of nicotinic ACh receptor (nAChR) subunit genes are valuable models for studying nAChR functions. We could previously show in the mouse superior cervical ganglion (SCG) that the absence of distinct subunits affects the functional properties of receptors. Here, we have addressed the question of whether deletions of the subunits ,5, ,7, or ,2 are compensated at the mRNA level, monitored by reverse transcription and quantitative real-time polymerase chain reaction. Relative to our reference gene, ,3, which is expressed in all SCG nAChRs, mRNA levels of ,4 showed little change from birth until adult ages in intact ganglia of wild-type mice. In contrast, ,4 declined sharply after birth and was barely detectable in adult animals. ,5, ,7, and ,2 subunit message levels also declined, though more slowly and less completely than ,4. The subunits ,6 and ,3 were detected by conventional polymerase chain reaction at very low levels, if at all, whereas ,2 was never seen in any of our samples. The developmental profile of nAChR mRNA levels in the three knockout strains did not differ markedly from that of wild-type mice. Likewise, message levels of nAChR subunits were similar in cultures prepared from either wild-type or knockout animals. Our observations indicate a developmental regulation of nAChR subunit mRNAs in the SCG of mice after birth that was not affected by the three knockouts under investigation. © 2007 Wiley-Liss, Inc. [source] Genome-wide analysis of the general stress response in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 4 2001Chester W. Price Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the ,B transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce ,B activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to 33P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which ,B expression was controlled by an inducible promoter. On the basis of their significant ,B -dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the ,B regulon. Of these genes, 30 were known previously or inferred to be ,B dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible ,B recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the ,B -dependent messages for seven representative genes. For all seven, the 5, end of the message lay near typical ,B recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover. [source] Wilms' tumor 1 message and protein expression in bone marrow failure syndrome and acute leukemiaPATHOLOGY INTERNATIONAL, Issue 10 2007Takashi Iwasaki Wilms' tumor 1 (WT1) is a useful marker for the diagnosis of acute leukemia and myelodysplastic syndromes (MDS). In the current study quantitative reverse transcription,polymerase chain reaction and immunostaining were used simultaneously to examine the relationship between WT1 RNA and protein level and also to evaluate WT1 as a tool to differentiate aplastic anemia (AA) and MDS refractory anemia (RA). Three types of WT1 messages (total, exon 5(+) and KTS(+)) and WT1 immunostaining of these diseases were analyzed. An increase of all three WT1 messages in high-grade MDS and acute leukemia was observed as compared with the normal control, whereas there was no significant difference in WT1 message between AA and RA, suggesting that WT1 message is not a good tool to discriminate AA and RA. No significant difference was observed between normal and RA, except for exon 5 message. Three WT1 message levels had a significant correlation, suggesting that the total WT1 message is sufficient for clinical practice. Positive immunostaining of WT1 was observed only in the portion of acute leukemia and overt leukemia (OL) transformed from MDS with a high WT1 message level, suggesting the relatively high detection threshold of WT1 protein with the immunostaining method. [source] Halofuginone inhibition of COL1A2 promoter activity via a c-Jun,dependent mechanismARTHRITIS & RHEUMATISM, Issue 10 2002Tracy L. McGaha Objective The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor , signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription. Methods The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying ,2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on ,2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry. Results Treatment of fibroblasts with 10,8M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the ,3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen ,2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression. Conclusion Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun,dependent manner. [source] | |||||||||||||