bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential transport and processing of variant mouse mammary tumor virus glycoproteins
Authors: Platt Emily J. , Kain Steven R. , Goodman Laurie J. , Firestone Gary L.
Published: May 2005

The transport and proteolytic processing of two individual gene isolates of the mouse mammary tumor virus (MMTV) glycoprotein were compared in transfected rat HTC hepatoma cells. Plasmids were constructed such that the MMTV glycoprotein genes were constitutively expressed from the promoter within the Rous Sarcoma Virus 5′ Long Terminal Repeat in the absence of other MMTV proteins. An isolate of the GR strain MMTV glycoprotein was efficiently transported and processed resulting in the localization of MMTV glycoproteins at the cell surface and in the extracellular environment. Moreover, the kinetics of acquisition of endoglycosidase H resistant oligosaccharide side chains and the rate of endoproteolytic cleavage of the glycosylated polyprotein expressed in transfected cells were virtually identical to that observed in viral‐infected rat hepatoma cells. In contrast, a natural variant of the C3H strain MMTV glycoprotein expressed in transfected cells was retained in an intracellular compartment by a heavy chain binding protein (Bip)‐independent pathway in an endoglycosidase H sensitive and uncleaved form. This MMTV glycoprotein isolate was retained early in the exocytic pathway and displayed a half‐life of approximately 45 min in transfected cells. Only a minor fraction of the expressed C3H variant glycoprotein was detected at the cell surface but was not externalized. Our results suggest that the variant C3H MMTV glycoprotein contains one or more mutations that preclude its efficient transport through the exocytic pathway.
Glucocorticoid and retinoid regulation of alpha‐2 type I procollagen promoter activity
Authors: Perez Jose R. , Shull Susan , Cutroneo Kenneth R. , Gendimenico Gerard J. , Capetola Robert J. , Mezick James A.
Published: May 2005

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the proα2(1) collagen gene required for the glucocorticoid‐mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5′ flanking region fragment from −2048 to +54 and the intronic fragment from +418 to +1524 of the mouse α2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid‐mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the ppossible role of the first intron fragment in the dexamethasone‐mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was trasnfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone‐mediated response was then localized within the 5′ flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions −2048 to −981 and −506 to −351 were required for the dexamethasone response of gene activity. However, the DNA stretch from −981 to −506 was not. Analysis of the DNA sequences of these regions revelaed a singel GRE at −1023 to −1018 and a modified doublet at −873 to −856. The doublet GRE contains and A/T strand switch of the third base pair as compared to the single GRE and is not necessary for dexamethasone regulation of gene activity. All‐trans‐retionic acid increased CAT activity of the same pR40 CAT construct transfected in the mouse fibroblasts. DNA sequencing revealed a RARE and a modified RARE in the stretch of DNA from −981 to −506. Deletion of only the latter DNA region eliminated the elevation of CAT activity elicited by all‐trans‐retinoic acid. Our results indicate that the single GRE and the RARE are required for glucocorticoid and retinoic acid regulation of proα2(I) collagen gene activity, respectively.
Replacement of lysine‐181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling
Authors: Wu Lan‐Hsin , Mauzy Camilla , Egloff Ann Marie , Chung Fu‐Zon , Zhu Guochang , Mirzadegan Taraneh
Published: May 2005

A conserved aspartic acid residue in the third transmembrance region of many of the G protein‐coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS‐7 cells transiently expressing the cloned receptor cDNAs. Using 125I‐ET‐I as the radioactive peptide ligend in displacement binding studies, the wild type receptor displayed a typical non‐isopeptide‐selective binding profile with similar IC50 values (0.2–0.6 nM) for all three endothelin peptides (ET‐1, ET‐2, and ET‐3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC 50 values for ET‐1 (5nM), ET‐2 (27nM), and ET‐3 (127nM) but displayed a much larger increase in 1C50 value for SRTX 6c ( > 10uM). The lysine mutant receptor still elicited full incositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10nM of ET‐1, 100nM of ET‐2, or 1uM of ET‐3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These demonstrate that lysine‐181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist‐SRTX5c. © 1992 Wiley‐Liss, Inc.
Phosphorylation of vinculin in human platelets spreading on a solid surface
Authors: Hagmann Jörg , Burger Max M.
Published: May 2005

Vinculin is a cytoskeletal protein believed to be involved in linking microfilaments to the cell membrane. it is a substrate for the Ca2+ ‐ and phospholipid‐dependent protein kinase C. We show here that when human platelets attach and spread on a solid surface, the α isoforms of vinculin become phosphorylated at serine and/or threonine residues. Phosphorylation is dependent on adhesion to a surface, since suspended, unattached platelets can produce filopodia but no phosphorylation of vinculin. Phosphorylation is also dependent on actin polymerization, as it does not occur when platelets had been pretreated with cytochalasin B. Most likely, protien kinase C is responsible for the phosphorylation of vinculin, since phosphorylation also occurs when platelets are treated with a phorbol ester, which activates protein kinase C, and is blocked by treatment with a staurosporine derivative which inhibits this enzyme. These results suggest that phosphorylation plays a role in anchoring vinculin at sites of microfilament‐membrane interaction. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐beta1 induces expression of statin during differentiation of human promonocytic leukemia cells
Authors: Lgnotz Ronald A.
Published: May 2005

Transforming growth factor‐Beta (TGF‐β) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP‐1, we have shown that TGF‐β inhibits their proliferation and promotes differentiation into cells exhibiting macrophage‐like properties. Therefore, a key question is whether TGF‐β influences the expression of genes associated with proliferation and/or growth inhibition. TGF‐β treatment of THP‐1 cells results in downregulation of expression of c‐myc. We also observe that TGF‐β1‐treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP‐1 cells in response to TGF‐β1 ttreatment. These results suggest that TGF‐β1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells. © 1992 Wiley‐Liss, Inc.
Induction of an immune response through the idiotypic network with monoclonal anti‐idiotype antibodies in the carcinoembryonic antigen system
Authors: de Moraes Jane Z. , Lopes J. Daniel , Carneiro Célia R. W. , Buchegger Franz , Mach Jean‐Pierre
Published: May 2005

Anti‐idiotype antibodies can mimic the conformational epitopes of the original antigent and act as antigent substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigent (CEA), BALB/c mice were immunized with the prevously described anti‐CEA monoclonal antibody (MAB) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti‐lds (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I‐labeled CEA to MAb 5.D11. Abesence of reactivity of anti‐ld MAbs with normal mouse lgG was first demontrated by the fact that anti‐ld MAbs were not absorbed by passage through a mouse lgG column, and second because they bound specifically to non‐reduced MAb 5.D11 on Western blots. Anti‐5.D11 MAbs did not inhibit binding to CEA of MAb10.B9, another anti‐CEA antibody obtained in the same fusion as 5.D11, or that of several anti‐CEA MAbs reported in an international workshop, with the exception of two other anti‐CEA MAbs, bothe directed against the GOLD IV epitope. When applied to an ld‐anti‐ld competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti‐ld MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recongnizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti‐idiotype antibodies as antigen substitutes. © 1992 Wiley‐Liss, Inc.
Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes
Authors: Shalhoub Victoria , Conlon Donna , Stein Gary S. , Lian Jane B. , Tassinari Melissa , Quinn Cheryl , Partridge Nicola
Published: May 2005

To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone‐like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid‐treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10 ‐7 M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c‐fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000‐fold elevated in glucocorticoid‐differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGFβ was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run‐on assay to mRNA levels indicates that glucocorticoids exert both transcriptional and post‐transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoid promote changes in gene expression involved in cell‐cell and cell‐extracellular matrix signaling mechanism that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue‐like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures. © 1992 Wiley‐Liss, Inc.
Expression of histone and alkaline phosphatase genes in UMR 106‐01 rat osteoblast‐like cells exposed to the hoechst dye H33342
Authors: Zheng M. H. , Wysocki S. , Papadimitriou J. M. , Wood D.
Published: May 2005

The fluorescent Dye H33342 (H342) is a bis‐benzimidazole used for intravital fluorescent staining. In this report, we found that H342 completely abolished histone 2a mRNA but had no effect on alkaline phosphatase gene expression and protein synthesis in UMR 106‐01 rat osteoblast‐like cells. The complete loss of histone 2a mRNA occurred after only 20 min of treatment with H342. This effect is unlikely to be a result of inhibition of DNA synthesis, which was only partly suppressed. The mechanism of the action of H342 on histone 2a mRNA is presently unknown. © 1993 Wiley‐Liss, Inc.
Effect of retinyl acetate on the assembly of the fibronectin extracellular matrix and the processing of the fibronectin receptor β subunit of confluent C3H/10T1/2 mouse embryo fibroblasts
Authors: Suzuki Satoshi S. , Piette Lawrence H.
Published: May 2005

The mouse embryo fibroblast cell line, C3H/10T1/2, synthesized and deposited a large amount of fibronectin especially in the pericellular matrix. Confluent cultures of these cells cultured in the presence of 0.3 μg/ml of retinyl acetate released cell surface fibronectin and the extracellular matrix fibronectin fibrils were disorganized. The immunoblot analysis demonstrated that the number of the fibronectin receptor was decreased in the prolonged culturing of retinyl acetate‐treated cells. Immunoprecipitation of 35S‐methionine pulse‐chase labeled cell extracts by antifibronectin receptor antibody indicated that about one‐half of the pre‐β subunit was processed and converted to the mature form in control cells, and only about one‐fourth of the pre‐β subunit was processed in the retinyl acetate‐treated confluent cells. 1‐deoxymannojirimycin (MNJ), which is an inhibitor of oligosaccharide processing, induced disorganization of the extracellular matrix fibronectin assembly similar to that observed with retinyl acetate. The results of this study suggest that a mechanism of action of retinyl acetate is inhibition of the glycosylation during processing of the fibronectin receptor, a step necessary for fibronectin binding and for assembly of the extracellular matrix. © 1993 Wiley‐Liss, Inc.
Temporally different poly(adenosine diphosphate‐ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins
Authors: Imschenetzky María , Montecino Martín , Puchi Marcia
Published: May 2005

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]‐ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP‐ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of 3H‐thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP‐ribose) neosynthesized on CS histone variants was measured by labeling with 3H‐adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP‐ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP‐ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP‐ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased 3H‐thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP‐ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation. These results also indicate that a poly(ADP‐ribosylation) signal is required for cell division; such signal is temporally different from that related to S phase initiation and occurs at the exit of S phase. © 1993 Wiley‐Liss, Inc.
Detection and characterization of low abundance cellular proteins that specifically increase upon loss of the metastatic phenotype
Authors: Nielsen‐Preiss Sheila M. , Quigley James P.
Published: May 2005

Human epidermoid carcinoma (HEp‐3) cells are highly tumorigenic and metastatic in vivo, but their metastatic phenotype is progressively and uniquely lost upon serial passage in vitro. The nonmetastatic phenotype is fully reversible to the highly metastatic state when HEp‐3 cells are passaged back in vivo.

To study the complex process of metastasis and its possible negative regulation by specific gene products, the expression of specific proteins between the highly metastatic and nonmetastatic HEp‐3 cells was investigated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and subsequent computer assisted analyses. Concomitant with the in vitro loss of metastatic potential of HEp‐3 cells is the elevated expression of a subset of low abundance proteins detectable in 2D‐PAGE but not apparent in high resolution one dimensional PAGE. When the HEp‐3 cells revert to the metastatic state, the expression of these proteins declines. The increased abundance of four distinct proteins directly correlates with the loss of the metastatic phenotype: two of the four proteins are associated with isolated cellular membranes (36kD, pl 5.7; 22kDa, pl 5.6), on protein fractionates with the cytoplasm (65kD, pl 6.2), and one protein is enriched in the nuclei fraction (32kD, pl 5.8). These data indicate that computer‐assisted analysis of highly sensitive, large‐format, 2D‐PAGE can be used to identify specific proteins in subcellular compartments that are candidates for negative regulators of the metastatic process. © 1993 Wiley‐Liss, Inc.
Nature of DNA sequences at the attachment regions of genes to the nuclear matrix
Authors: Boulikas Teni
Published: May 2005

Matrix‐attached regions (MARs) have been demonstrated to nest origins of replication and transcriptional enhancers. A set of 13 rules is proposed aimed at facilitating the classification of a DNA sequence as a matrix attachment region. These rules, which were deduced from a study of known MARs from other genes and some others identified in our laboratory, are (1) potential origin of replication are MARs; (2) the major class of MARs seclude clusters of AT‐rich motifs and may harbor topoisomerase II binding and cleavage sites; (3) the AT‐rich class of MARs may comprise the DNA sequence motifs ATTA and ATTTA representing core binding sites of homeotic proteins, implying that MARs may participate in the differential activation of origins of replication and in gene switch during development; (4) the habitat of MARs may include mass binding sites for protein transcription factors; even weak factor binding sites may lead to the formation of tight protein‐DNA supramolecular structures; (5) MARs may contain intrinsically curved DNA; one type is oligo(dA) stretches of 3 to 7 nucleotides spaced every 10.5 nucleotides; (6) a class of MARs may contain kinked DNA, formed by CA, TG, and TA dinucleotides at distances of 5 or 10.5 nucleotides from their centers; the same dinucleotides, known to be abundant in protein recognition sites, may be overrepresented in a special class of MARs; (7) the AT‐rich core of MARs may be flanked, at one or both sides, by sequences that can adopt the left‐handed or triple‐helical DNA structure; these include TG, TA, GC repeats and polypurine or polypyrimidine stretches; (8) palindromic (dyad symmetry) sequences, able to form cruciform structures when the DNA is under torsional strain may be found within MARs, and more so when the MAR is also an origin of replication; (9) transcriptional enhancers may be MARs; (10) a class of MARs may be composed of stretches of GA‐rich DNA alternating with CT‐rich stretches, 5–50 nucleotides long; (11) a class of MARs may be enriched in TG boxes, usually 6–12 nucleotides long, such as TGTTTTGGGG; this type of MAR occurs in the 3′‐untranslated region of several genes, builds up the chromosome telomeres, and is highly recombinogenic; (12) a small fraction of Alu sequences might have MAR activity. This might depend on the number and distance from one another of DNA sequence motifs representing protein binding sites; and (13) MARs may coincide with the DNAse I hypersensitive sites of chromatin.

It is proposed here that MAR sequences can provide markers for mapping and sequencing the human, and other, genomes. Furthermore, it is proposed that large scale random cloning of MARs might advance our knowledge on the nature of DNA sequences that are used for the initiation of DNA replication, as transcriptional enhancers and as borders between chromatin domains.
Influence of electromagnetic fields on endochondral bone formation
Authors: Ciombor Deborah McK. , Aaron Roy K.
Published: May 2005

Endochondral ossification is a basic physiological process in limb development and is central to bone repair and linear growth. Factors which regulate endochondral ossification include several biophysical and biochemical agents and are of interest from clinical and biological perspectives. One of these agents, electric stimulation, has been shown to result in enhanced synthesis of extracellular matrix, calcification, and bone formation in a number of experimental systems and is the subject of this review. The effects of electric stimulation have been studied in embryonic limb rudiments, growth plates, and experimental endochondral ossification induced with decalcified bone matrix and, in all these models, endochondral ossification has been enhanced. It is not known definitively whether electric fields stimulate cell differentiation or modulate an increased number of molecules synthesized by committed cell population and this is a fertile area of current study.
The identification of a naturally occurring cell surface growth inhibitor related to a previously described bovine sialoglycopeptide
Authors: Fattaey Heideh K. , Enebo Daniel J. , Moos Philip J. , Johnson Terry C.
Published: May 2005

A 66‐kDa sialoglycoprotein has been identified as the parental membrane molecule of an earlier described sialoglycopeptide (SGP), an 18‐kDa molecule released by protease treatment of intact bovine cerebral cortex cells that was shown to be a potent inhibitor of cellular proliferation. The 66‐kDa parental sialoglycoprotein (p‐SGP) was purified approximately 2,400‐fold, to apparent homogeneity, from bovine cerebral cortex cell membranes by its release during incubation with 3 M NaCl, preparative isolelectric focusing and lectin affinity chromatography. Although a membrane‐associated molecule, the p‐SGP appeared to be tightly bound to the cell membrane, since it was not released during incubations in the absence of 3 M NaCl. Incubation of the membrane preparations with 3 M urea proved to be too harsh, and the antigenicity required to follow the purification of the p‐SGP was abolished. Analyses by SDS–PAGE, under reducing and nonreducing conditions, suggested that the p‐SGP membrane component was a single polypeptide without subunit structure. The p‐SGP was shown to be structurally related to the SGP fragment by immunoblots with IgG raised to the SGP inhibitor, and functionally related to the SGP by its ability to inhibit Swiss 3T3 proliferation at concentrations strikingly similar to that previous measured with the SGP fragment.
Signal transduction pathways and cellular intoxication with Clostridium difficile toxins
Authors: Shoshan Maria Caspar , Fiorentini Carla , Thelestam Monica
Published: May 2005

In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin‐induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4‐bromophenacylbromide. Agents affecting certain G‐proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C‐dependent pathway, but not pertussis toxin‐sensitive G‐proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.
Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast‐like cells
Authors: Tokuda Haruhiko , Kotoyori Jun , Suzuki Atsushi , Oiso Yutaka , Kozawa Osamu
Published: May 2005

We investigated the effects of vitamin D3 on the signaling pathways by prostaglandin E2 (PGE2) in osteoblast‐like MC3T3‐E1 cells. The pretreatment with 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3), an active form of vitamin D3, significantly inhibited cAMP accumulation induced by 10 μM PGE2 in a dose‐dependent manner in the range between 1 pM and 1 nM. This effect of 1,25‐(OH)2D3 was dependent on the time of pretreatment up to 8 h. 1,25‐(OH)2D3 also inhibited the cAMP accumulation induced by NaF, a GTP‐binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25‐(OH)2D3 significantly inhibited PGE2‐induced IP3 formation in a dose‐dependent manner between 10 pM and 1 nM. However, 1,25‐(OH)2D3 had little effect on NaF‐induced IP3 formation. The pretreatment with 24,25‐dihydroxyvitamin D3, an inactive form of vitamin D3, affected neither cAMP accumulation nor IP3 formation induced by PGE2. These results strongly suggest that 1,25‐(OH)2D3 modulates the signaling by PGE2 in osteoblast‐like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE2 receptor and GTP‐binding protein, probably Gi2.
Characterization of atrial natriuretic peptide degradation by cell‐surface peptidase activity on endothelial cells
Authors: Frost Stephen J. , Whitson Peggy A.
Published: May 2005

Atrial natriuretic peptide (ANP) is a fluid‐regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I‐ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I‐ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 ± 60 nM and Vmax of 35 ± 14 pmol of ANP degraded/10 min/105 cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0–8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I‐ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell‐surface degradation of ANP was demonstrated when 125I‐ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin‐1, or substance P. The C‐terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell‐surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation‐independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.
Double immunofluorescent staining of α2,6 sialyltransferase and β1,4 galactosyltransferase in monensin‐treated cells: Evidence for different Golgi compartments?
Authors: Berger E. G. , Grimm K. , Bächi T. , Bosshart H. , Kleene R. , Watzele M.
Published: May 2005

β1,4 galactosyl‐ and α2,6 sialyltransferase (gal‐T EC 2.4.1.22 and sialyl‐T EC 2.4.99.1) sequentially elongate and terminate complex N‐glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi‐disturbing agents. Polyclonal, peptide‐specific antibodies to human sialyl‐T expressed as a β‐galactosidase‐sialyl‐T fusion protein in E. coli were developed and applied together with mABs to human milk gal‐T.

In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl‐T and gal‐T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal‐T followed by sialyl‐T; in the reassembled Golgi apparatus sialyl‐T and gal‐T were co‐localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal‐T positive Golgi elements while sialyl‐T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal‐T and sialyl‐T remained co‐localized. Treatment with chloroquine affected Golgi structure less than monensin and led to condensation of gal‐T positive and to slight enlargement of sialyl‐T positive structures.

Sequential recovery from BFA of gal‐T and sialyl‐T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.
Phorbol ester activation of functional rat protein kinase C β‐1 causes phenotype in yeast
Authors: Riedel Heimo , Hansen Hans , Parissenti Amadeo M. , Su Lihe , Sheih Hui‐Ling , Zhu Jianwei
Published: May 2005

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC β‐1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium‐ and phospholipid‐dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC β‐1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC β‐1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC β‐1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Protein product of the somatic‐type transcript of the Hoxa‐4 (Hox‐1.4) gene binds to homeobox consensus binding sites in its promoter and intron
Authors: Wu Ke , Wolgemuth Debra J.
Published: May 2005

The murine Hoxa‐4 gene encodes a protein with a homeodomain closely related to those produced by the Antennapedia‐like class of Drosophila genes. Drosophila homeodomain proteins can function as transcription factors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger consensus sequence, such as CAATTAA. The in vitro synthesized protein product of Hoxa‐4 was shown to bind to a subset of restriction fragments of the Hoxa‐4 gene itself as determined by gel retardation experiments. Direct examination of the sequences of the fragments bound by Hoxa‐4 protein revealed the presence of four regions containing the core ATTA motif. Two regions contained sequences of the CAATTAA class and were located ∼ 1 kb upstream from the putative somatic Hoxa‐4 promoter and within the intron. Two additional binding sites containing the consensus target sequence involved in autoregulation of Drosophila Deformed gene were identified: one immediately downstream of the putative embryonic transcription start site and one within the intron, respectively. Specific binding of the in vitro produced Hoxa‐4 protein to oligonucleotides corresponding to these sequences was observed in gel retardatin assays. The same results were obtained with Hoxa‐4 protein produced in a Baculovirus expression system. Experiments using oligonucleotides containing base substitutions in positions 1, 3, 4, and 5 in the sequence CAATTAA showed severely reduced binding. The use of truncated mutant Hoxa‐4 proteins in gel retardation assays and in transient co‐transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the Hoxa‐4 gene has the potential to auto‐regulate its expression by interacting with the homeodomain binding sites present in the promoter as well as in the intron.
Multitude of inverted repeats characterizes a class of anchorage sites of chromatin loops to the nuclear matrix
Authors: Boulikas Teni , Kong C. F.
Published: May 2005

In order to understand the nature of DNA sequences that organize chromatin into domains or loops, we have cloned the nuclear matrix DNA (1.7% of the total DNA) from human myelogeneous leukemia cells in culture. Nuclear matrix is formed by interactions between specific stretches of DNA of about 0.1 to 5.0 kb with protein transcription factors, nuclear enzymes, and structural proteins. Nuclear matrix is believed to be the exclusive nuclear microenvironment in which initiation of DNA replication, transcription, and repair take place. The matrix attachment regions (MARs) of DNA have transcriptional enhancer activity, harbor the origins of replication of the human genome, and define the borders between neighbouring chromatin loops. In this study we report the sequence of the human MAR fragment 19.2 of a size of 542 bp. Hum. MAR 19.2 is composed of TG‐, CA‐, CT‐, and GA‐rich blocks and shows 8 perfect and imperfect inverted repeats. Thus, we have identified a novel class of MARs with sequence characteristics divergent from the AT‐rich class of MARs. The inverted repeats of the 19.2 sequence might be stabilized into their cruciform configuration by torsional strain and by specific transcription/replication protein factors. This MAR might function in the initiation of replication of the flanking chromatim domain and in the regulation of the transcriptional activity of the gene(s) that reside in this domain.
Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: Hypophosphatasia fibroblast study
Authors: Fedde Kenton N. , Michel Michelle P. , Whyte Michael P.
Published: May 2005

A major impasse to understanding the physiologic role(s) of alkaline phosphatase (ALP) is uncertainty as to its natural substrates. Various in vitro studies have led other investigators to suggest that ALP functions as a plasma membrane phosphoprotein phosphatase, consistent with our demonstration of ecto‐topography of ALP in a variety of cell types. Thus, we compared the phosphorylation of plasma membrane proteins from control fibroblasts to those from profoundly ALP‐deficient fibroblasts of hypophosphatasia patients. Fibroblasts from 3 controls and 3 hypophosphatasia patients (ALP activity < 4% of control) were biosynthetically labeled with 32Pi for 2 h. 32P incorporation into total trichloracetic acid (TCA)‐precipitable material was not significantly different in control and patient cells. Plasma membranes were prepared from these cells by hypotonic shock, solubilized, and subjected to two‐dimensional (2‐D) gel electrophoretic separation. Video densitometric analysis of silver‐stained 2‐D gels failed to reveal any consistent difference in the protein profile between patient vs. control fibroblasts (i.e., unique species, altered pls, or increased abundance). Autoradiography of individual 2‐D gels demonstrated 63 plasma membrane phosphoproteins with molecular weights ranging from 15 to 152 kDa and predominantly acidic pls. Although several of these phosphoproteins appeared to have had donor‐specific labeling, none was unique or especially abundant in the hypophosphatasia group. Thus, in ALP‐deficient fibroblasts, normal incorporation of 32P into total cellular protein and into all identifiable plasma membrane phosphoproteins indicates that ALP does not modulate the phosphorylation of plasma membrane proteins.
Cytokines: Molecular keys to homeostasis, development, and pathophysiology
Authors: Evans Charles H.
Published: May 2005

Identification and definition of the role and diversity of action of cytokines, the regulatory proteins including lymphokines, monokines, interleukins, interferons, and a variety of other growth factors produced by virtually every nucleated cell into the body and having pleiotropic regulatory effects on hematopoietic and many other cell types [Thompson, 1991], are in the forefront of biomedical research. The explosive development of cytokine research is reflected by the inclusion of “cytokine,” first introduced as a term in 1974 to recognize that lymphokines could be produced by other than lymphoid cells in 4,267 biomedical articles published by the end of 1992. From the initial reference to cytokines in 1974 to inclusion in 45 articles in 1985, the doubling of citations each succeeding year, together with 45% of all the citations since 1974 occurring in 1992 alone, attests to the continued rapid expansion of this area of biological research. The important roles of cytokines in many physiological processes and pathophysiological conditions, although largely descriptive in nature, were recognized during the early development of this field of investigation [Balkwill and Burke, 1989; Cohen et al., 1974]. Current and future directions, as highlighted by the series of Prospect articles featuring areas as diverse as embryologic, extracellular matrix, bone, hematologic, and neurologic development and function as well as in the organism's response to foreign organisms in this issue of the journal, are focused on broadening our understanding of the positive and negative influence of cytokines in normal and abnormal differentiation and development and on the molecular pathways underlying cytokine action. The broad areas of cellular biochemistry represented by the Prospects, moreover, emphasize the synthesis and convergence of cytokine research towards an understanding of the critical controls in development and homeostatsis.
Cytokines in the differentiation of Th1/Th2 CD4+ subsets in leishmaniasis
Authors: Reiner Steven L. , Locksley Richard M.
Published: May 2005

Leishmania major infect only macrophages in the host, where they reside in endolysosomal compartments into which MHC class II molecules co‐localize. Experimental infection in mice has provided a useful model for the differentiation of Th1 CD4+ effector lymphocytes that are required for the generation of IFN‐γ that activates the macrophage to a microbicidal state. Genetically susceptible BALB/c mice aberrantly activate Th2 CD4+ effector cells that are ineffective in arresting infection. Increasing evidence suggests that, rather than discrete parasite antigens or MHC molecules, cytokines mediate the critical decision in the developmental switch to either the Th1 or Th2 effector phenotype.
Modulation of human microvascular endothelial cell bioenergetic status and glutathione levels during proliferative and differentiated growth
Authors: Mallery S. R. , Lantry L. E. , Laufman H. B. , Stephens R. E. , Brierley G. P.
Published: May 2005

During angiogenesis, formerly differentiated human microvascular endothelial cells (HMECs) return to a proliferative growth state. Many fundamental questions regarding HMEC function, such as how HMECs adapt to changes in bioenergetic requirements upon return to proliferative growth, remained unanswered. In this study, we evaluated whether modifications in HMEC bioenergetic profiles and glutathione (GSH) levels accompanied the cellular transition between differentiated and proliferative growth. To provide insight into the continuum of cellular adaptations that occur during this transition, we used a method recently developed in our laboratory that induces a state of morphological and functional predifferentiation in HMECs. Cellular morphology, in conjunction with flow cytometric DNA analyses and HMEC functional assays (the directed migration and intercellular association involved in microtubule formation) were employed to validate the HMEC culture state of growth. Analysis of the HPLC nucleotide profiles disclosed several findings common to all culture growth states. These uniform findings, e.g., cellular energy charges > 0.90, and highly reduced redox states, revealed that cultured HMECs maintain high rates of oxidative metabolism. However, there were also significant, culture growth state related differences in the nucleotide profiles. Proliferative HMECs were shown to possess significantly higher (relative to both large vessel endothelial cells, and differentiated HMECs) levels of GSH and specific nucleotides which were related with a return to the active cell cycle‐ATP, GTP, UTP, and CTP, and NADPH. Further, the nucleotide profiles and GSH levels of the predifferentiated HMECs were determined to be intermediate between levels obtained for the proliferative and differentiated HMECs. The results of this study demonstrate that the capacity to modulate their cellular bioenergetic status during growth state transitions is one of the adaptations that enable HMECs to retain a growth state reciprocity. In addition, our findings also show that HMECs, especially during the proliferative growth state, are biochemically distinct from endothelial cells harvested from large vessels, and therefore suggest that HMECs are the cells of choice to employ when studying diseases that affect the human microvasculature.
Organization and expression of H1 histone and H1 replacement histone genes
Authors: Doenecke D. , Albig W. , Bouterfa H. , Drabent B.
Published: May 2005

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433–449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1° and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.
Microtubular protein in its polymerized or nonpolymerized states differentially modulates in vitro and intracellular fluxes catalyzed by enzymes of carbon metabolism
Authors: Cortassa S. , Aon M. A. , Cáceres A.
Published: May 2005

The fluxes through HK/G6PDH and PK/LDH coupled‐enzymatic reactions were quantified in the presence of physiological concentrations (1–15 μM) of polymerized or non‐polymerized microtubular protein (MTP) from rat brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MTP mainly in the lower range of MTP concentration. At fixed MTP concentrations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and PEP, respectively, was detected. That dependence varied according to the polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (⩽ 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G‐actin (⩽ 1 mg/ml). In permeabilized Saccharomyces cerevisiae cells, the PK‐catalyzed flux was sensitive to microtubule disruption by nocodazole (15 μg/ml). The HK/G6PDH system was not affected by nocodazole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluorescence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed intracellularly. The experimental evidence is in agreement with the observed phenomenon of increase in fluxes in the enzymatic reactions assayed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supramolecular structures as a supraregulatory mechanism of synchronization of systemic cellular processes such as metabolic fluxes. © 1994 Wiley‐Liss, Inc.