bluuebunny/crossref_metadata_2025_split huggingface dataset:
Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat
Authors: Sawada Masaaki , Sester Ulrike , Carlson John C. 
Published: May 2005

AbstractPlasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro‐elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca2+ ‐sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme‐mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.
TGF‐β1 inhibits DNA synthesis and phosphorylation of the retinoblastoma gene product in a rat liver epithelial cell line
Authors: Whitson Robert H. , Itakura Keiichi 
Published: May 2005

AbstractIn the rat liver epithelial cell line, WB, the ability of TGF‐β1 to inhibit DNA synthesis was shown to correlate with its ability inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum‐starved, then refed with serum‐containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H‐thymidine at this time. When TGF‐β1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF‐β1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF‐β1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF‐β1. If TGF‐β1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF‐β1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF‐β1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose‐response for inhibition of DNA synthesis by TGF‐β1 was shifted to the right. Inhibition of pRb phosphorylation by TGF‐β1 was also lost in higher passage WB cells. Thus, the passage‐dependent loss of sensitivity to inhibition of DNA synthesis accompained the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF‐β1.
The effect of age on the response of the detrusor to intracellular mechanical stimulus: DNA replication and the cell actin matrix
Authors: Karim Omer M. A. , Seki Narihito , Pienta Kenneth J. , Mostwin Jacek L. 
Published: May 2005

AbstractBenign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myoctyte ultrastructure and consequent effects these might have in modifying the response of the destrusor to mechanical stimulus, we studied differences in dyanmic nuclear‐cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which spcifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2–3 wee) and old (8–12 mon) guinea pgis. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracelluar stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti‐α‐smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor mycocytes showed dose dependent disruption of the actin component of the cytoskeleton.These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the destrusor in response to outflow obstruction in infancy and adulthood. Furthurmore, they suggest that (1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; (2) there appears to be a decrease in myocyte nuclear elasticity with ageing; (3)release of nuclear template restrictions increases activity of DNA polymerase α in young, but not old, detrusor myocytes; and (4) mechanico‐chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that (1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and (2) integrity of nuclear actin may function to maintain the spatial template restriction of DNA polymerase α activity.
Flow cytometric analysis of porcine preadipocytes
Authors: Wright J. T. 
Published: May 2005

AbstractIn this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal‐vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta (TGF‐β). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD‐1 cell surface antigen was greater in cultures supplemented with 2% pig serum and 10nM dexamethasone than in other media. In cultures, GH, TNF‐α and TGF‐β each inhibited lipid deposition, whereas TNF‐α and TGF‐β, but not GH, inhibited AD‐1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF‐α and TGF‐β was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.
Development of a sensitive peptidase assay: In search of cell associated proteases responsible for the cleavage of prepro TGFα
Authors: Brown S. B. , Krause D. , Townsend E. , Ellem K. A. O. 
Published: May 2005

AbstractA radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGFα) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N‐terminal to an octapeptide which is cognate to the N‐terminal cleavage sequence of TGFα. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell‐associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane‐bound prepro TGFα. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo‐ and endo‐peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1–2% of total cell activity of these enzyme classes.
Osteoporosis and vitamin D
Authors: Nordin B. E. Christopher , Morris Howard A. 
Published: May 2005

AbstractBone “density” (bone mass/bone volume) declines with age from the menopause in women and from about age 55 in men. This fall in bone density (osteoporosis) weakens the bones and leads to a progressive rise in fracture rates, particularly in women. Many risk factors contribute to the bone‐losing process, but one which attracts increasing attention is calcium absorption.The main physiological regulator of calcium absorption is vitamin D. This is manufactured in the skin under the influence of UV‐light and then converted to more potent metabolites in the liver and kidney. Although the serum levels of the most potent metabolite 1,25(OH)2D3 (calcitriol) are generally normal in osteoporotic women, treatment with small doses of calcitriol (about 0.25 μg daily) has a remarkable effect on absorptive performance and slows down the rate of bone loss. Improved synthetic metabolites are under development.There is likely also to be greatly increased scope for the use of vitamin D itself in osteoporosis. With advancing age, there is a tendency for men and women to be exposed to less and less sunlight, which is the main natural source of vitamin D. Vitamin D levels, therefore, decline with age, particulaly in those who are housebound, and are found to be low in most reported series of hip fractures. It is likely that this form of vitamin D “insufficiency” has an adverse effect on calcium absorption in the elderly which accelerates bone loss and increases the risk of hip fracture and can be treated with small doses of vitamin D or its 25‐hydroxy derivative. This offers a second important role for vitamin D in the prevention and management of osteoporosis.
Role of vitamin D in bone resorption
Authors: Suda Tatsuo , Takahashi Naoyuki , Abe Etsuko 
Published: May 2005

AbstractThe idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti‐rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1α,25(OH)2D3 is involved in bone resorption. 1α,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte‐macrophage lineage. 1α,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1α,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1α,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1α,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.
Protein‐DNA interactions at the H4‐Site III upstream transcriptional element of a cell cycle regulated histone H4 gene: Differences in normal versus tumor cells
Authors: van der Houven van Oordt C. Willemien , van Wijnen Andre J. , Carter Ruth , Soprano Kenneth , Lian Jane B. , Stein Gary S. , Stein Janet L. 
Published: May 2005

AbstractUpstream sequences of the H4 histone gene FO108 located between nt –418 to –213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4‐Site III) that binds factor H4UA‐1. Based on methylation interference, copper‐phenanthroline protection, and competition assays, we show that H4UA‐1 interacts with sequences between nt −345 to −332 containing an element displaying sequence‐similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA‐1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase‐treatment of nuclear proteins inhibits formation of the H4UA‐1 protein/DNA complex, although a complex with higher mobility (H4UA‐1b) can be detected; both complexes share identical protein‐DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4‐Site III protein/DNA interactions by directly altering protein/protein associations. H4‐Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA‐1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA‐1/H4‐Site III interaction, concomitant with an increase of the H4UA‐1b/H4‐Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA‐1, whereas H4UA‐1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA‐1 and H4UA‐1b binding activities may reflect deregulation of H4‐Site III interactions in transformed cells. We propose that H4‐Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4‐FO108 histone gene transcription.
Making transgenic livestock: Genetic engineering on a large scale
Authors: Wall R. J. , Hawk H. W. , Nel Neil 
Published: May 2005

AbstractThe feasibility of introducing foreign genes into the genomes of cattle, goats, pigs, and sheep has only recently been demonstrated. Studies have thus far focused on improving growth efficiency or directing expression of pharmaceutical proteins to the mammary glands of these species. The general strategy for producing transgenic livestock and mice is similar. In addition to the obvious difference in scale between mice and livestock experiments, there are noteworthy obstacles that significantly reduce the efficiency of producing transgenic livestock. Low embryo viability, low transgene integration rates, and high animal costs contribute to project costs that can easily exceed hundreds of thousands of dollars. A better understanding of the mechanisms that govern transgene integration should lead to improved efficiencies. But, the full potential of the transgenic livestock system will not be fully realized until: (1) gene constructs can be designed that function in a reproducible, predictable manner; and (2) the genetic control of physiological processes are more clearly elucidated. Newly emerging approaches may resolve at least some of these issues within the next decade.
Downregulation of histone H4 gene transcription during postnatal development in transgenic mice and at the onset of differentiation in transgenically derived calvarial osteoblast cultures
Authors: Gerbaulet Stephan P. , van Wijnen Andre J. , Aronin Neil , Tassinari Melissa S. , Lian Jane B. , Stein Janet L. , Stein Gary S. 
Published: May 2005

AbstractIn vivo regulation of cell cycle dependent human histone gene expression was examined in transgenic mice using a fusion construct containing 6.5 kB of a human H4 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional control of histone gene expression, as a function of proliferative activity, was determined. We established the relationship between DNA replication dependent H4 mRNA levels (Northern blot analysis) and H4 promoter activity (CAT assay) during postnatal development in a broad spectrum of tissues. In most tissues sampled in adult animals, the cellular representation of H4 gene transcripts declined in parallel with promoter activity. This result is consistent with transcriptional control of H4 gene expression at the cessation of proliferation. Interestingly, while H4 mRNA was detectable at very low levels post‐proliferatively in brain, promoter activity persisted in adult brain, where most of the cells are terminally differentiated. This dissociation between histone gene promoter activity and histone mRNA accumulation points to the possibility of post‐transcriptional regulation of histone gene expression in brain. Cultures of osteoblasts were prepared from calvaria of transgenic mice carrying the H4 promoter/CAT reporter construct. In contrast to the brain, in these bone‐derived cells, we established by immunohistochemistry that the transition to the quiescent, differentiated state is associated with a transcriptionally mediated downregulation of histone gene expression at the single cell level.
A new monoclonal antibody for detection of EGF‐receptors in western blots and paraffin‐embedded tissue sections
Authors: Fernandez A. , Spitzer E. , Perez R. , Boehmer F.‐D. , Eckert K. , Zschiesche W. , Grosse R. 
Published: May 2005

AbstractThe prognostic significance of the epidermal growth factor receptor status (EGF‐R‐status) for certain human tumors requires the development of antibodies useful for clinical application. We used purified receptor preparations to generate monoclonal antibodies immunoreactive with the EGF‐R purified from placenta membranes and A431 tumors. Four of the hybridomas contained antibodies (R2, R3, R5, and R9) which recognized both antigens. Antibody R3 was shown to display the following properties: it binds with a KD value of about 10−9–10−10 M to the receptor, a half maximal inhibition of EGF‐binding is achieved at 5 × 10−8 M, and in Western blots of cell membranes R3 specifically detects the EGF‐R at 0.1 μ/ml. R3 inhibits EGF‐dependent clonogenic growth of NRK cells and completely blocks EGF stimulated autophosphorylation of the receptor. Moreover, R3 also detects EGF‐R in paraffin‐embedded tissue sections taken from human salivary gland, term placenta, and adult skin and mammary carcinomas. Thus, R3 can be used in retrospective diagnostic clinical studies and might help to develop new immunotherapeutic intervention.
Extremely divergent histone H4 sequence from trypanosoma cruzi: Evolutionary implications
Authors: Toro G. Cecilia , Wernstedt Christer , Medina Carlos , Jaramillo Nicolás , Hellman Ulf , Galanti Norbel 
Published: May 2005

AbstractTrypanosoma cruzi presents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, name, corresponds to H4 in other species. We present evidence that histones is H4 by sequencing its amino terminal end. The amino terminal of T. cruzi histone H4, unlike that of other H4s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence of T. cruzi histone H4 differs greatly from homologous sequences of human, yeast, or Tetrahymena.Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T. cruzi histone H4 is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H4 amino terminal sequence varies among different lineages. We postulate a slow‐down in the evolutionary rate of histone H4 amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.
The phosphatidylinositol transfer protein in 3T3 mouse fibroblast cells is associated with the Golgi system
Authors: Snoek G. T. , de Wit I. S. C. , van Mourik J. H. G. , Wirtz K. W. A. 
Published: May 2005

AbstractBy use of indirect immunofluorescence it was shown that the phosphatidylinositol transfer protein (PI‐TP) in 3T3 mouse fibroblast cells is associated with the Golgi system. This was concluded from double‐labeling experiments with TRITC‐labeled Ricin which binds to sugar residues that are specifically processed in the Golgi system. Independent evidence for this association was provided by the fact that dissociation of the Golgi system by brefeldin A was reflected in an extensive redistribution of PI‐TP labeling. In addition, PI‐TP is localized in the cytoplasm and in the nucleus. In exponentially growing cells an enhanced labeling of PI‐TP was observed in the cytosol and in the Golgi system in comparison with quiescent cells. By Western blot analysis and by transfer activity assays, it was confirmed that the concentration of PI‐TP was increased in exponentially growing cells. These results strongly suggest that PI‐TP fulfills a role in the functioning of the Golgi complex.
Interaction of multiple factors with a GC‐rich rlement within the mitogen responsive region of the human transferrin receptor gene
Authors: Miskimins W. Keith 
Published: May 2005

AbstractNuclear factors from HeLa cells were isolated by elution of DNA‐cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from −34 to −79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double‐stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC‐rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but, still contacts the GC‐rich element.