bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of diethylnitrosamine carcinogenesis in rat liver and oesophagus
Authors: Balansky Roumen M. , Blagoeva Penka M. , Mircheva Zwetanka I. , De Flora Silvio
Published: May 2005

A series of 16 experiments, using a total of 2,000 BD6 rats, was designed in order to assess the ability of 8 individual agents or their combinations to modulate the liver and oesophageal carcinogenesis induced by multiple doses of diethylnitrosamine (DEN). Of the antioxidants tested, sodium selenite, ascorbic acid, and butylated hydroxytoluence generally exhibited protective effects on both types of tumors. In contrast, retinoic acid behaved as a promoter of DEN hepatocarcinogenesis, but this effect could be eliminated by its combination with either selenite or butylated hydroxytoluene. Caffeine and theophyline, when individually assayed, were devoid of significant protective effects, and the later methylxanthine stimulated oesophageal tumorigenesis when administered afer exposure to the carcinogen. Caffeine tended to decrease tje multiplicityof tumors and potentiated the inhibitory effect of selenite in the liver. Irrespective of combination with caffeine, treatment with phwnobarbital before each DEN injection tended to reduce the multiplicity of both liver and oesophageal tumors. On the other hand, the metabolic inhibitoe diethyldithiocarbamate, given after each DEN injection, dramatically enhancedd the incidence and multiplicity of oesophageal tumors. Thus, on the whole, modulation of DEN carcinogenesis varied depending on test agents, their conbinations, dosages, treatment schedules, and target organ.
Increased expression of basic fibroblast growth factor in hyperoxic‐injured mouse lung
Authors: Powers Michael R. , Planck Stephen T. , Berger John , Wall Michael A. , Rosenbaum James T.
Published: May 2005

Basic fibroblast growth factor (bFGF) is a mitogenic polypeptide for a wide variety of cell types and has been immunolocalized in the rodent and human lung. We investigated the mRNA and protein expression of bFGF in hyperoxic‐injured adult mouse lungs using northern blot analysis and immunohistochemistry. Mece (6–8weeks) were continuously exposed to 80% osygen up to 4 days. Levels of bFGF mRNA were increased from room air control on days 3 and 4 of hyperoxia. mRNA levels of acidic fibroblast growth factor (aFGF), fibronectin, and transin/stromelysin were also examined in this injury model. Similar to bFGF, the fibronectin and transin/stromelysin mRNA levels were increased after 3 days of hyperoxia. In contrast, the aFGF mRNA levels were gradually reduced on each day of hyperoxia. A rabbit polyclonal anti‐bFGF antibody was used to determine the distribution and levels of expression in the hyperoxic‐injured lungs. The room air control and day 1 hyperoxic‐exposed lungs exhibited staining for bFGF in the basement membranes of the blood vessels, airways, and alveoli. Patchy but intense alveolar staining was prominent on day 4 of hyperoxia. The bFGF immunoreactivity of blood vessels and airways unaffected by the hyperoxia exposure. These results suggest that bFGF may play a role in the alveolar response to hyperoxic‐induced injury by virtye of the altered mRNA levels and protein distribution in this injury model.
Evidence for a regulatory element controlling amino aced transport system L in Chinese hamster ovary cells
Authors: Collarini Ellen J. , Campbell George S. , Oxender Dale L.
Published: May 2005

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO–;tsO25C1 has a temperature‐sinsitive mutationin leucyl‐tRNA synthetase. At the nonpermissive temperature of 39oC, CHO–tsO25C1 cells are unable to charge leucyl‐tRNA and behave as though starved for leucine by increasing their system L transport activity two‐ to fourfold. From the temperature‐sensitive cell line, we have isolated a regulatory mutant cell, CHO–C11B6, that has constitutively elevated system L transport activity. The CHO–C11B6 cell line retains the temperature‐sensitive leucyl‐tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads of increased intracellular leucine levels, which compensate for the defective. Hybrid cells formed by fusion of the temperature‐sensitive CHO–;tsO25C1 cells the temperature‐resistant CHO–C11B6 cells show temperature‐sensitive growth and temperature‐dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant‐acting element that is defective or absent in the regulatory mutant CHO–C11B6 cell line.
Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK‐3α in A431 cells
Authors: Yang Shiaw‐Der , Yu Jau‐Song , Wen Zin‐Der
Published: May 2005

The signal transducrion mechanism of protein kinase FA/GSK‐3α by tyrosine phosphorylation in A431 cells was investigated. Kinase FA/GSK‐3α was found to exist in a highly tyrosine‐phosphorylated/activated state in resting cells but could be tyrosine‐dephosphorylated and inactivated to ∼60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37oC for 30 min, as demonstrated by metabolic32P‐labeling the cells, followed by immunoprecipitation and two‐dimensional phosphoamino acid analysis and by immunodetection in an antikinase FA/GSK‐3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase FA/GSK‐3α was found to be rephosphorylated on tyrosine residue and reactivated to ∼130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase FA/GSK‐3α can be modulated in a reversible manner by short‐term and long‐term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up‐regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down‐regulation of PKC, the results further suggest that the TPA‐mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase FA/GSK‐3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.
Dual mechanism of protein‐tyrosine phosphorylation in concanavalin A‐stimulated platelets
Authors: Torti Mauro , Ramaschi Giuseppe , Sinigaglia Fabiola , Balduini Cesare
Published: May 2005

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65‐, 85‐, 95‐, and 120‐kDa bands, which were tyrosine phosphorylated also in thrombin‐stimulated platelets; the second group (80‐, 135‐, and 150‐kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl α‐D‐mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A‐induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl‐Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein‐tyrosine phosphorylation in Con A‐stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.
Tyrosine kinase regulates phospholipase D activation at a point downstream from protein kinase C in osteoblast‐like cells
Authors: Kozawa Osamu , Suzuki Atsushi , Oiso Yutaka
Published: May 2005

It has recently been shown that the activation of protein kinase C (PKC) induces protein tyrosine phosphorylation in osteoblast‐like MC3T3‐E1 cells. We previously reported that the activation of PKC stimulates phosphatidylcholine‐hydrolyzing phospholipase D in these cells. In this study, we examined whether protein tyrosine kinase is involved in the PKC‐induced activation of phospholipase D in MC3T3‐E1 cells. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), an activator of PKC, in a dose‐dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also dose‐dependently suppressed the TPA‐induced formation of choline. Sodium orthovandate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the TPA‐induced formation of choline in a dose‐dependent manner. These results strongly suggest that protein tyrosine kinase regulates phospholipase D activity at a point downstream from PKC in osteoblast‐like cells.
Estrogen modulation of osteoclast lysosomal enzyme secretion
Authors: Kremer M. , Judd J. , Rifkin B. , Auszmann J. , Oursler M. J.
Published: May 2005

Osteoclast‐mediated bone resorption is accomplished by secretion of lysosomal proteases into an acidic extracellular compartment. We have previously demonstrated that avian osteoclasts and human osteoclast‐like giant cell tumor cells respond in vitro to treatment with 17β‐estradiol (17β‐E2) by decreased bone resorption activity. To better understand the mechanism by which this is accomplished, we have investigated the effects of 17β‐E2 treatment on lysosomal enzyme production and secretion by isolated avian osteoclasts and multinucleated cells from human giant cell tumors in vitro. Isolated cells were cultured with bone particles in the presence of either vehicle or steroid. The conditioned media and cells were harvested, and the levels of cathepsin B, cathepsin L, β‐glucuronidase, lysozyme, and tartrate‐resistant acid phosphatase (TRAP) activities were determined. There was a steroid dose‐dependent decrease in secreted levels of these enzymes. Cell‐associated levels of cathepsin L, β‐glucuronidase, and lysozyme decreased, whereas cell‐associated levels of cathepsin B and TRAP increased. These changes were measurable at 10−10 M and maximal at 10−8 M 17β‐E2. The changes were detectable at 4–18 h of treatment and increased through 24 h of treatment. The response was steroid specific, since the inactive estrogen isomer, 17β‐E2, failed to alter the activity levels. Moreover, the effects of 17β‐E2 were blocked when the cells were treated simultaneously with the estrogen antagonist ICI182–780 in conjunction with 17β‐E2. Human osteoclast‐like cells obtained from giant cell tumors of bone responded similarly to estrogen with respect to cathepsin B, cathepsin L, and TRAP activities. However, secretion of β‐glucuronidase and lysozyme were not altered by treatment with 10−8 M 17β‐E2. These data indicate that estrogen effects on osteoclast resorption activity may be mediated by decreasing the secretion of cathepsin B, cathepsin L, and TRAP.
MAPKAP kinase 2 is activated by heat shock and TNF‐α: In vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade
Authors: Engel Katrin , Ahlers Annette , Brach Marion A. , Herrmann Friedhelm , Gaestel Matthias
Published: May 2005

The activation of MAPKAP kinase 2 was investigated under heat‐shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor‐α (TNF‐α). MAPKAP kinase 2 activity was determined using the small heat‐shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10–15 min after stimulation either by heat shock or TNF‐α. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF‐α treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.
Cortisol downregulates osteoblast α1(I) procollagen mRNA by transcriptional and posttranscriptional mechanisms
Authors: Delany Anne M. , Gabbitas Bari Y. , Canalis Ernesto
Published: May 2005

Glucocorticoids decrease osteoblast proliferation and type I collagen production, and this may play a role in the development of glucocorticoid‐induced osteoporosis. Osteoblast‐enriched cultures derived from fetal rat calvaria were used to determine the mechanisms by which cortisol decreases α1(I) procollagen expression in bone cells. A 24 h treatment with cortisol decreased collagen synthesis in these cultures in a dose‐dependent manner. Cortisol decreased α1(I) procollagen transcripts in a dose‐ and time‐dependent manner as well. Repression of α1(I) procollagen transcripts was evident as early as 2 h of treatment and was maximal after 48 h of treatment. Nuclear run‐off assays showed that cortisol downregulated transcription of the α1(I) procollagen gene. In addition, pretreatment with cortisol decreased the stability of α1(I) procollagen mRNA in transcription‐arrested osteoblast cultures. The ability of cortisol to downregulate α1(I) procollagen transcripts was sensitive to cycloheximide treatment, suggesting that the gene is under “secondary control” by glucocorticoids. Since cortisol decreases α1(I) procollagen gene transcription in osteoblasts but does not affect α1(I) procollagen gene transcription in fibroblasts, we suggest that the mechanisms controlling glucocorticoid repression of collagen expression are cell‐type specific.
Long‐term exposure to chromium(VI) oxide leads to defects in sulfate transport system in chinese hamster ovary cells
Authors: Lu Yi‐Yi , Yang Jia‐Ling
Published: May 2005

Chromium(VI) resistant Chinese hamster ovary (CHO) cell lines were established in this study by exposing parental CHO‐K1 cells to sequential increases in CrO3 concentration. The final concentration of CrO3 used for selection was 7 μM for Cr7 and 16 μM for Cr16 cells. Cr16‐1 was a subclone derived from Cr16 cells. Next, these resistant cells were cultured in media without CrO3 for more than 6 months. The resistance of these cells to CrO3 was determined by colony‐forming ability following a 24‐h treatment. The LD50 of CrO3 for chromium(VI) resistant cells was at least 25‐fold higher than that of the parental cells. The cellular growth rate, chromosome number, and the hprt mutation frequency of these chromium(VI) resistant cells were quite similar to their parental cells. The glutathione level, glutathione S‐transferase, catalase activity, and metallothionine mRNA level in Cr7 and Cr16‐1 cells were not significantly different from their parental cells. Furthermore, Cr16‐1 cells were as sensitive as CHO‐K1 cells to free‐radical generating agents, including hydrogen peroxide, nickel chloride, and methanesulfonate methyl ester, and emetine, i.e., a protein synthesis inhibitor. The uptake of chromium(VI) and the remaining amount of this metal in these resistant and the parental cell lines were assayed by atomic absorption spectrophotometry. Experimental results indicated that a vastly smaller amount of CrO3 entered the resistant cell lines than their parental cells did. A comparison was made of the sulfate uptake abilities of CHO‐K1 and chromium(VI) resistant cell lines. These results revealed that the uptake of sulfate anion was substantially reduced in Cr7 and Cr16‐1 cells. Extracellular chloride reduced sulfate uptake in CHO‐K1 but not in Cr16‐1 cells. Therefore, the major causative for chromium(VI) resistance in these resistant cells could possibly be due to the defects in SO42‐/C1− transport system for uptake chromium(VI).
Unidirectional potentiation of binding between two anti‐FBP MAbs: Evaluation of involved mechanisms
Authors: Casaline Patrizia , Mezzanzanica Delia , Valota Olga , Adobati Elena , Tomassetti Antonella , Colnaghi Maria I. , Canevari Silvana
Published: May 2005

The monoclonal antibody MOv19 directed to a folate binding protein shows temperature‐dependent potentiation of binding of the noncompeting monoclonal antibody MOv18 to the relevant antigen, but the mechanism involved in this phinomenon had remained unclear. Use of chimeric versions of both monoclonal antibodies and the F(ab′)2 and fan fragments of MOv19 revealed an increment in MOv18 binding in all combinations irrespective of the orgin of the Fc portin of the monoclonal antibody. The potentiating effect of bivalent MOv19 fragments on 125l‐MOv18 binding was similar to that of the entire monoclonal antibody and occurred at saturating concentrations of both reagents at which monovalent binding prevails. Similarly, the monovalent fragment also induced a significant increase in MOv18 bunding. Howener, the potentiation sccurred only at very high concentrations of antibody fragment. Homologous inhibition was drastically reduced using MOv19 Fab fragment, suggesting a low binding stability of the monovalent reagent. Immunoblotting analysis and binding in the presence of exogenous purified folate binding protein indicated a cross‐linking between soluble and cell surface molecules mediated by the bivalent monoclonal antibodies. The extentof the increase in MOv18 binging at O°C with high amounts of exogenous folate binding protein was lower than that obtained at 370C in the absence of added molecule. Release of 125l‐MOv18 from the cell surface was significantly higher in the absence of MOv19 than in its presence. Affinity constant values of 125l‐MOv18 binding evaluated in the presence of MOv19 or control monoclonal antibody MINT5 were comparable, whereas the number of binding sites per cell detected by 125l‐MOv18 was significantly higher in the presence of MOv19 than MINT5. Together, the data suggest that monoclonal antibody MOv19 induces a conformational change of the molecule it binds that increases the number of antigenic sites anvailable for MOv18 binding and, in turn, the binding stability of the latter, MOv19 bivalency also contributes to the MOv18 binding increment by cross‐linking released and cell surface–anchored folate binding protein molecules. © Wiley‐Liss, Inc.
Expression of gamma‐glutamyl transpeptidase by renal epithelial cells occurs on a cell‐by‐cell basis and is inhibited by the chronic TPA treatment
Authors: Chen Jen‐Li , Amsler Kurt
Published: May 2005

Abstract Upon attaining a confluent density, populations of the renal epithelial cell line, LLC‐PK1, express progressively many properties characteristic of the renal proximal tubule cell, including gamma‐glutamyl transpeptidase activity. Expression of transpeptidase activity was inhibited reversibly by chronic treatment with the phorbol ester tumor promoter, 12‐o‐tetradecanoylphorbol‐13‐acetate(TPA). TPA treatment inhibited expression of transpeptidase activity regardless of whether added prior to or following appearance of the activity. Increased transpeptidase activity in postconfluent cell populations was due to an increased enzyme Vmax with no change in substrate Km. TPA‐treated cell populations. exhibited a low Vmax similar to subconfluent populations. Detection of transpeptidase activity at the individual cell level by enzyme histochemistry demonstrated that near‐confluent cell populations possesed few transpeptidase activity–positive cells. Progressive expression of transpeptidase activity in the cell population was due to an increasing proportion of cells in the population possessing transpeptidase activity. There was a parallel increase in the proportion of cells expressing transpeptidase protien, detected by immunofluorescence. TPA treatment inhibited apperance of both transpeptidase activity and transpeptidase protein in virtually all cells of the population. These results demonstrate that expression of transpeptidase activity in populations of LLC‐PK1 cells occurs on a cell‐by‐cell basis and reflects expression of transpeptidase protein. Chronic treatment with TPA inhibits reversibly expression of transpeptidase activity and protein, suggesting a role for protein kinase C in regulating expression of this proximal tubule–specific property. © Wiley‐Liss, Inc.
Influence of portein tyrosine phosphorylation on the experssion of the c‐myc oncogene in cancer of the large bowel
Authors: Heruth Daniel P. , Wetmore Lori A. , Leyva Albert , Rothberg Paul G.
Published: May 2005

We tested the potential impact of tyrosine phosphorylation on the expression of the c‐myc gene in tow colon cancer cell lines, HCT8 and SW837. We found that the protein tyrosine kinase inhibitor genistein causes a decrease in the aboundace of c‐myc RNA and an inhibition of proliferation with a similar dose response. Geldanamycin, a mechanistically different tyrosine kinase inhibitor, also causes a decrease in both the expression of cmyc RNA and proliferation. Genistein has also been found to inhibit topoisomerase II, but the topoisomerase II inhibitor novobiocin did not lower the expression of c‐myc. The most likely interpretation is that inhibition of protein tyrosine kinase activity caused a decrease in c‐myc expression in these cells. The impact of tyrosine phosphorylation on the experssion of the c‐myc gene is further supported by the finding that inhibition of phosphotyrosine phosphatase using orthovanadate causes an increase in the level of c‐myc RNA. The effect of genistein on HCT8 cells is not dependent on the synthesis of new protein and does not involve an allteration in the stability of the massage. Analysis of transcription in the cmyc gene reveals a more complicated picture with a decrease in initiation and an increase in elongation but no net change in transcription. We speculate that the genistein induced reduction in myc experssion is the result of a posttranscriptional intranuclear event(s). © Wiley‐Liss, Inc.
Bipartite structure of the proximal promoter of a human H4 histone gene
Authors: Wright Kenneth L. , Birnbaum Mark J. , van Wijnen Andre J. , Stein Gary S. , Stein Janet L.
Published: May 2005

The proximal promoter of the human H4 histone gene FO108 contains two regions of in vivo protein‐DNA interaction, Sites I and II. Electrophoretic mobility shift assays using a radiolabeled DNA probe revealed that several proteins present in HeLa cell nuclear extracts bound specifically to Site I (nt‐125 to nt‐86). The most prominent complex, designated HiNF‐C, and a complex of greater mobility, HiNF‐C′, were specifically compatable by an Sp1 consensus oligonucleotide. Fractionation of HiNF‐C using wheat germ agglutinin affinity chromatography suggested that, like Sp1, HiNF‐C contains N‐acetylglucosamine moieties. Two minor complexes of even greater mobility, designated HiNF‐E and F, were compatable by ATF consensus oligonucleotides. A DNA probe carrying a site‐specific mutation in the distal portion of Site I failed to bind HiNF‐E, indicating that this protein associated specifically to this region. UV cross‐linking analysis showed that several proteins of different molecular weights interact specifically with Site I. These data indicate that Site I possesses a bipartite structure and that multiple proteins present in HeLa cell nuclear extracts specifically with Site I sequences.
Transcriptional repression of the α1(I) collagen gene by ras is mediated in part by an intronic AP1 site
Authors: Slack James L. , Parker M. Iqbal , Bornstein Paul
Published: May 2005

We have previously shown that transformation of fibroblasts by ras results in transcriptional inhibition of the α1(I) gene. An α1(I)‐hGH chimeric plasmid containing 3.7 kb of 5′ flanking and 4.4 kb of α1(I) transcribed sequence was regulated appropriately by ras in a transient transfection assay. In contrast, a similar plasmid containing α1(I) DNA from −220 to + 500 was virtually unresponsive to ras. The regions from −3700 to −220 and + 500 to + 4400 contributed equally to the ras‐mediated inhibition of the parental plasmid. Deletion analysis indicated that a short fragment, between +500 and +890 in the first intron of the α1(I) gene, was recognized differently in ras‐transformed and wild‐type cells. A previously described AP1 site in this fragment stimulated α1(I) transcription in Rat1 fibroblasts but was inactive in ras‐ transformed cells. Mobility shift assays using nuclear extracts from the two cell types demonstrated differences in binding to the α1(I) AP1 site. We conclude that ras transformation suppresses the function of a cell‐specific enhancer in the first intron of the α1(I) collagen gene.
Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells
Authors: Gimble J. M. , Morgan C. , Kelly K. , Wu X. , Dandapani V. , Wang C.‐S. , Rosen V.
Published: May 2005

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor‐β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein‐2, the pre‐adipocyte BMS2 cells exhibited the expected induction of the osteogenic‐related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre‐adipocytes constitutively expressed mRNA encoding bone morphogenetic protein‐4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
β3‐Adrenergic activation of adenylyl cyclase in mouse white adipocytes: modulation by GTP and effect of obesity
Authors: Bégin‐Heick Nicole
Published: May 2005

Lipolysis and adenylyl cyclase (AC) activation in response to β‐adrenergic agents are abnormally low in white epididymal adipose tissue (WAT) of the ob/ob mouse. The abundance of G‐proteins (Gsα and Giα) linked to AC is also abnormally low. By contrast, β‐adrenergic receptor (β‐AR) levels were previously found to be normal in WAT and elevated in liver. The relative importance of various forms of the β‐AR in mouse WAT was reassessed in view of the discovery of the β3‐AR. The results show that (1) the β3‐AR is mainly responsible for AC activation in lean‐mouse WAT; (2) the β3‐AR is only partly responsible for AC activation in obese mouse WAT; and (3) GTP modulates β3—‐but not β1—‐or β2‐AR activation of AC in a biphasic manner. Therefore, the β3‐AR appears responsible for the well‐known bimodal effect of GTP on β‐adrenergic receptor‐mediated AC activity in WAT.
Overexpression of cellular activity and protein level of protein kinase FA/GSK‐3α correlates with human thyroid tumor cell dedifferentiation
Authors: Lee Tsong‐Tze , Ho Yat‐Sen , Yu Jau‐Song , Yang Shiaw‐Der
Published: May 2005

Abstract Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline‐directed protein phosphorylation, suggesting an association of the proline‐directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase‐3α (kinase FA/GSK‐3α) (a member of the PDPK family) has been optimized for human thyroid tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in thyroid carcinoma (24.2 ± 2.8 units/mg of protein) (n = 7), thyroid adenoma (14.5 ± 2.2 units/mg of protein) (n = 6), and thyroid hyperplasia (8.0 ± 2.4 units/mg of protein) (n = 5) when compared to five normal controls (4.1 ± 1.8 units/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK‐3α in thyroid tumor cells is due to overexpression of protein level and cellular activity of kinase FA/GSK‐3α is involved in human thyroid tumor cell dedifferentiation, supporting an association of PDPK with neoplastic transformation and tumorigenesis. Since kinase FA/GSK‐3α may function as a possible regulator of transcription factors/protooncogenes, kinase FA/GSK‐3α may therefore play an important role in thyroid cell carcinogenesis, especially in its differentiation.
Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb
Authors: Wen G. Y. , Mato A. , Wisniewski H. M. , Malik M. N. , Jenkins E. C. , Sheikh A. M. , Kim K. S.
Published: May 2005

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur‐containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA‐stimulated human short‐term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti‐40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.
Molecular mechanisms of bone resorption
Authors: Teitelbaum Steven L. , Abu‐Amer Yousef , Ross F. Patrick
Published: May 2005

This review focuses on osteoclast ontogeny and function, emphasizing three aspects. We describe how a combination of laboratory models available to study the cell plus examination of the osteopetroses, a family of sclerotic diseases of the skeleton, have yielded major insights into osteoclast ontogeny and function. We proceed to describe the cell and molecular machinery enabling osteoclasts to resorb bone. The final, and most speculative, aspect of the review addresses possible mechanisms by which the osteoclast assumes its characteristic morphology, that of a polarized cell on bone. Since little direct information has been forthcoming as to how the osteoclast polarizes, we draw on other polarized cells. In particular, we examine the role of microtubules and members of the small GTPase family, the latter mediating polarized targeting of intracellular vesicles. In the case of the osteoclast, such vesicles probably represent the origin of the highly convoluted ruffled membrane, the cell's characteristic bone resorptive organ. © 1995 Wiley‐Liss, Inc.
Possible role for protein kinase C in the pathogenesis of inborn errors of metabolism
Authors: Boneh Avihu
Published: May 2005

Protein kinase C (PKC) is a ubiquitous enzyme family implicated in the regulation of a large number of short‐ and long‐term intracellular processes. It is hypothesized that modulation of PKC activity may represent, at least in part, a functional link between mutations (genotype) that lead to the pathological accumulation of naturally occurring compounds that affect PKC activity and perturbation of PKC‐mediated substrate phosphorylation and cellular function in the corresponding diseases (phenotype). This model provides a unifying putative mechanism by which the phenotypic expression of some inborn errors of metabolism may be explained.

Recent studies in a cell‐free system of human skin fibroblasts support the hypothesis that alteration of PKC activity may represent the functional link between accumulation of sphingolipids and fatty acyl‐CoA esters, and perturbation of cell function in sphingolipidoses and fatty acid oxidation defects, respectively. Further studies will elucidate the effects of these alterations on PKC‐mediated short‐ and long‐term cellular functions in these diseases, as well as the possible role of PKC in the pathogensis of other diseases. © 1995 Wiley‐Liss, Inc.
Synthesis of stromal glycosaminoglycans in response to injury
Authors: Brown C. T. , Applebaum E. , Banwatt R. , Trinkaus‐Randall V.
Published: May 2005

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with 35S‐sulfate for 18 h; GAGs were extracted with 4 M guanidine–HCl. Extracts were chromatographed on Q‐Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS–PAGE. The nonbound fractions from the chromatography were assayed for TGF‐β using Western blot analysis and for hyaluronic acid using an 125I‐radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan‐chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF‐β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF‐β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley‐Liss, Inc.
Purification and stability characterization of a cell regulatory sialoglgycopeptide inhibitor
Authors: Moos Philip J. , Fattaey Heideh K. , Johnson Terry C.
Published: May 2005

Previous attempts to physically separate the cell cycle inhibitory and protease in activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non‐overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two‐fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine‐SDS‐PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed. © 1995 Wiley‐Liss, Inc.
Sea urchin zygote chromatin exhibit an unfolded nucleosomal array during the first S phase
Authors: Imschenetzky MaríA , Puchi Marcia , Gutiérrez Soraya , Montecino Martín
Published: May 2005

To investigate changes in chromatin organization associated with DNA replication during the first stages of development of the sea urchin Tetrapygus niger, we compared micrococcal nuclease (MNase) digestion patterns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragments derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nuclei were larger (1,500 to 410 bp). This result indicates that in zygotes, where active DNA replication is occurring, the major chromatin fraction is represented as unfolded nucleosomes. In contrast, in unfertilized eggs chromatin appears to be organized into polynucleosomes. To determine if the unfolded structure of nucleosomes observed during S phase is related to the level of poly (ADP‐ribosylation) of cleavage stage (CS) histone variants, zygotes were treated with 20 mM 3‐Amino Benzamide (3 ABA) during the interval between 3 and 30 min post‐insemination (p.i.). This treatment with 3 ABA decreases the poly (ADP‐ribosylation) of CS histone variants and inhibits the first S phase in zygotes [Imschenetzky et al. (1991): J Cell Biochem 46:234–241; Imschenetzky et al. (1993): J Cell Biochem 51:198–205]. When the MNase digested patterns of chromatin from these 3 ABA treated and control zygotes were compared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly (ADP‐ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, particular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly (ADP‐ribosylation) of CS histone variants. © 1995 Wiley‐Liss, Inc.
Human parathyroid hormone as a secretory peptide in milk of transgenic mice
Authors: Rokkones Erik , Fromm Sigurd H. , Kareem B. Najma , Klungland Helge , Olstad Ole K. , Høgset Anders , Iversen Jan , Bjøro Kristian , Gautvik Kaare M.
Published: May 2005

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t‐antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS‐PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley‐Liss, Inc.
Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene
Authors: Harris Steven A. , Tau Kimberly R. , Enger Robert J. , Toft David O. , Riggs B. Lawrence , Spelsberg Thomas C.
Published: May 2005

The gene coding for the human wild‐type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β‐estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE‐TK‐luciferase construct. Expression of luciferase from this construct increased ∼25‐fold in hFOB/ER9 cells following 10−9M E2 treatment. This effect on ERE‐TK‐luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10−9M E2 resulted in a 2.5–4.0‐fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley‐Liss, Inc.
Cpn60 is exclusively localized into mitochondria of rat liver and embryonic Drosophila cells
Authors: Martín Carmen San , Flores Ana I. , Cuezva Josí M.
Published: May 2005

Several reports have claimed that the mitochondrial chaperonin cpn60, or a close homolog, is also present in some other subcellular compartments of the eukaryotic cell. Immunoelectron microscopy studies, using a polyclonal serum against cpn60, revealed that the protein is exclusively localized within the mitochondria of rat liver and embryonic Drosophila cells (SL2). Furthermore, no cpn60 immunoreactive material could be found within the nucleus of SL2 cells subjected to a 1 h 37°C heat‐shock treatment. In contrast to these findings, immunoelectron microscopy studies, using a cpn60 monoclonal antibody, revealed mitochondrial and extramitochondrial (plasma membrane, nucleus) immunoreactive material in rat liver cells. Surprisingly, the monoclonal antibody also reacted with fixed proteins of the mature red blood cell. The monoclonal antibody, as well as cpn60 polyclonal sera, only recognize mitochondrial cpn60 in Western blots of liver proteins. Furthermore, none of the cpn60 antibodies used in this study recognized blotted proteins from rat red blood cells. Therefore, we suggest that the reported extramitochondrial localization of cpn60 in metazoan cells may be due to cross‐reactivity of some of cpn60 antibodies with conformational epitopes also present in distantly related cpn60 protein homologs that are preserved during fixation procedures of the cells. © 1995 Wiley‐Liss, Inc.
Expression of lysyl oxidase from cDNA constructs in mammalian cells: The propeptide region is not essential to the folding and secretion of the functional enzyme
Authors: Kagan Herbert M. , Reddy Vemuri B. , Panchenko Maria V. , Nagan Narasimhan , Boak Andra M. , Gacheru Stephen N. , Thomas Kathleen M.
Published: May 2005

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate‐resistant cell line, LOD‐06, generated by transfecting with full‐length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32–2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including β‐aminopropionitrile, phenylhydrazine, ethylenediamine, α,α′‐dipyridyl, and diethyl‐dithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase purified from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase. © 1995 Wiley‐Liss, Inc.
Molecular cloning of a mouse epithelial protein‐tyrosine phosphatase with similarities to submembranous proteins
Authors: Hendriks Wiljan , Schepens Jan , Bächner Dietmar , Rijss Johan , Zeeuwen Patrick , Zechner Ulrich , Hameister Horst , Wieringa Bé
Published: May 2005

Protein‐tyrosine phosphatases (PTPases) form an important class of cell regulatory proteins. We have isolated overlapping cDNA clones that together comprise an 8 kb transcript encoding a novel murine PTPase which is expressed in various organs. Sequence analysis revealed an open reading frame of 2,460 amino acid residues. The predicted protein, PTP‐BL, is a large non‐transmembrane PTPase that exhibits 80% homology with PTP‐BAS, a recently described human PTPase. PTP‐BL shares some intriguing sequence homologies with submembranous proteins. It contains a band 4.1‐like motif also present in the tumor suppressors neurofibromatosis 2 and expanded, five 80 amino acid repeats also present in the disc‐large tumor suppressor, and a single catalytic phosphatase domain. No obvious homologies to other proteins were found for the N‐terminal region of the protein other than human PTP‐BAS. RNA in situ hybridization experiments show that the PTP‐BL gene is expressed in epithelial cells, predominantly in kidney, lung, and skin. These data suggest a cell cortical localization for PTP‐BL in epithelial cells and a possible role in the morphology and motility of epithelial tissues. © 1995 Wiley‐Liss, Inc.
Facile optical resolution of amino acid esters via hydrolysis by an industrial enzyme in organic solvents
Authors: Kijima Tatsuro , Ohshima Kouji , Kise Hideo
Published: May 2005

Racemic amino acid esters were optically resolved via hydrolysis in organic solvents by the catalysis of an industrial alkaline protease, “Alcalase”. The products which were composed mainly of L‐amino acids were insoluble and easily separated by filtration. The activity of the enzyme and enantiomeric excess of the products were significantly dependent on the nature of solvent and the water content in the reaction media. Generally, high values of enantiomeric excess were obtained at low water contents. Many natural and unnatural amino acids were resolved by this method.