bluuebunny/crossref_metadata_2025 huggingface dataset:
Cation exchangers on a sugar‐beet pulp base. Application for decontaminating radioactive waste water
Authors: Langenhorst W. Th. J. P. , Tels M. , Vlugter J. C. , Waterman H. I.
Published: May 2005

A cation exchanger suitable for decontaminating low and medium radioactive waste water was sought. Regeneration being considered undesirable, the exchange material had to be cheap and readily obtainable. Sugar‐beet pulp, a weakly acidic cation exchanger, satisfies these conditions. Its capacity is about 0·62 mg eq. per gram of dry matter. In order to study the selectivity of the sugar‐beet pulp exchanger, the equilibrium curve of the reaction

equation image

was determined.

This curve was found to have the shape of the hyperbola proposed by Waterman and Weber for the characterization of the course of simultaneous reactions. Sugar‐beet pulp adsorbs the salts of the alkaline earth metals selectively in the presence of both Na+ and La+++. Sugar‐beet pulp was used to decontaminate a solution containing 140BaCl2 and 140LaCl3 and having an activity of about 10−2 μc/ml. The results were satisfactory.

By treating sugar‐beet pulp with formaldehyde and hydrochloric acid, the amount of water bound to the pulp is very much decreased. By this treatment a cation exchanger is produced having a capacity per unit volume about six times greater than that of sugar‐beet pulp. The capacity of this exchanger is about 0·5 mg eq. per gram of dry matter. The selective behaviour of sugar‐beet pulp treated with formaldehyde is similar to that of unmodified sugar‐beet pulp.

By treating sugar‐beet pulp with formaldehyde, hydrochloric acid and dilute sulphuric acid, a cation exchanger with very good mechanical and filtration properties is obtained. This exchanger has a capacity of about 1·3 mg eq. per gram of dry matter.

It seems possible to employ the cation exchangers obtained by treatment of sugar‐beet pulp with either formaldehyde and HCl or formaldehyde, HCl and H2SO4 also for purposes other than the removal of radioactive cations from water.
The recovery of staphylococcal enterotoxin from bacterial culture supernatants by ion exchange
Authors: Bergdoll M. S. , Sugiyama H. , Dack G. M.
Published: May 2005

A method has been developed for the recovery of enterotoxin from bacterial culture supernatants of staphylococcus strain S‐6 by use of the ion‐exchange resin Amberlite XE‐64 (IRC‐50). The enterotoxin from 200 ml of bacterial culture supernatant diluted with one volume of water and adjusted to pH 6 can be removed with 1 g of resin pretreated with 0·02 M sodium phosphate, pH 6·2. The enterotoxin is recovered from the resin with 0·2 M sodium phosphate, pH 6·2, with subsequent precipitation with EtOH at −10°C, dialysis, and freeze drying. Partially purified preparations containing 1 emetic dose per 50μg were obtained.
A microcompartmentation analysis of intermediate leakage response to substrate excess in a membrane‐bound bifunctional enzyme: Local control of hydroxyprogesterone channeling efficiency during cytochrome P450XVII‐catalysed androgen biosynthesis
Authors: Kühn‐Velten W. Nikolaus
Published: May 2005

Evidence is presented for the first time that the cytochrome P450XVII°Catalysed androgen formation from progesterone (P) in rat testicular microsomal membranes represents a metabolic sequence that exhibits the ability of intrinsic regulation of intermediate transfer and product formation efficiency. Exposure of this system, which catalyses a hydroxylation and oxidative cleavage reaction sequence, to increasing P concentration results in a decreased specific retention of the putative intermediate, 17α‐hydroxyprogesterone (HP) in the membrane compartment, and in a decreased HP conversion to androgens in favour of increasing HP transfer into the extramembrane space. This behaviour results in a decreased ratio of product vs. intermediate formation rates, which is interpreted as a partial “uncoupling” of the normal hydroxylation and cleavage reaction sequence catalysed by P450XVII. A similar pattern can likewise be observed in isolated testicular Leydig cells after exposure to increasing P concentrations under more physiological continuous‐flow conditions. Further calculations indirectly indicate that the specific retention of HP in the membrane compartment can partially be attributed to its specific association with the P450XVII during catalysis. The results strongly suggest the existence of a local “channel” that becomes more leaky and therefore less effective if loaded with high influx rates. This pattern may be related to significant but incomplete competition of exogenously entering P and endogenously formed and transiently bound HP for oxygen attack at the P450XVII active site.
Neutral endopeptidase from nuchal ligament of fetal calves
Authors: Johnson Alice R. , Gray Lynn D. , Youngblood Elky , Sullivan James
Published: May 2005

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphora midon (1 μM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.
Kinetic and molecular species analyses of mitogen‐induced increases in diglycerides: Evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine
Authors: Raben Daniel M. , Pessin Melissa S. , Rangan Leela A. , Wright Timothy M.
Published: May 2005

A wide variety of agonist‐induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: (a) the kinetics of diglyceride generation, (b) the absolute mass levels, and (c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist‐induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist‐dependent. High concentrations of α‐thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet‐derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist‐dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with α‐thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet‐derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.
Oncogene amplification correlates with dense lymphocyte infiltration in human breast cancers: A role for hematopoietic growth factor release by tumor cells?
Authors: Tang Ruoping , Kacinski Barry , Validire Pierre , Beuvon Frédéric , Sastre Xavier , Benoit Patrick , dela Rochefordière Anne , Mosseri Véronique , Pouillart Pierre , Scholl Susy
Published: May 2005

One hundred six primary breast cancer samples were analysed for c‐erbB2, int‐2, and c‐myc gene amplification. Surgically confirmed nodal involvement was observed in 42%. Level of gene amplification was studied by Southern and/or slot blottechniques. Amplified c‐erbB2 gene sequences were present in 21.5% of all samples. Int‐2 was amplified in 13.1% and c‐myc was amplified in 10.3%. In a non‐parametric test (Kruskal‐Wallis) a strong negative association was found between high levels of c‐erbB2 amplification and absence of estrogen receptor (ER) (P = .0009) or progesterone receptor (PR) (P = .011) expression. No correlations were found between all or high levels of amplification of each oncogene separately or combined with T, N, grade, multifocality of tumor, or associated carcinoma in situ. There was a trend approaching statistical significance for patients with c‐erbB2 amplifications to have positive lymph nodes at surgery (P = 0.09). A somewhat surprising finding however was a very strong association between oncogene amplification and dense lymphocyte infiltration of the tumor (P = .05). This correlation is even stronger when only high levels of amplification are considered, either for each oncogene separately (P = .0048) or in combination (P = .007). We propose that malignant cell cytokine production may help explain this observation.
Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: Mediation by cyclic AMP
Authors: Egan John J. , Gronowicz Gloria , Rodan Gideon A.
Published: May 2005

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X‐100 (1%) nonextractable proteins. After 2–5 minutes, PTH caused a rapid and transient decrease of 50–60% in polymerized actin and myosin associated with the Triton X–100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose‐dependent with an IC50 of about 1 nM, and was partially inhibited by the (3–34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin‐2/AM (10 μM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (Pl 4.9) myosin light chain in cells preincubated with [32P]‐orthophosphate. The phosphorylation of this protein decreased within 2–3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A‐23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine‐conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.
Characterization of latent recombinant TGF‐β2 produced by chinese hamster ovary cells
Authors: Lioubin Mario N. , Madisen Linda , Marquardt Hans , Roth Richard , Kovacina Kristina S. , Purchio A. F.
Published: May 2005

Latent recombinant transforming growth factor‐β2 (LrTGF‐β2) complex has been purified from serum‐free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF‐β2 (414) precursor. Under neutral conditions, LrTGF‐β2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro‐region sequences. Acidification of LrTGF‐β2 resulted in the release of mature 24 kDa TGF‐β2 from the high molecular weight pro‐region‐containing complex, suggesting that TGF‐β2 was non‐covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF‐β2. Protein sequence analysis of the purified TGF‐β2 pro‐region indicated that signal peptide cleavage occured between ser(20) and leu(21). The pro‐region, which previously was found to contain mannose‐6‐phosphate, bound to the mannose‐6‐phosphate receptor. Proteolytic cleavage of mature TGF‐β2 from pro‐TGF‐β2 was inhibited by monensin and chloroquin suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF‐β2 precursor.
Inhibition of virus‐induced cell fusion by apolipoprotein A‐I and its amphipathic peptide analogs
Authors: Srinivas R. V. , Rui Zheng , Owens R. J. , Compans R. W. , Venkatachalapathi Y. V. , Gupta K. B. , Srinivas S. K. , Anantharamaiah G. M. , Segrest J. P.
Published: May 2005

Apolipoprotein A‐I (apoA‐I), the major protein component of serum high‐density lipoproteins (HDL), was found to inhibit herpes simplex virus (HSV)‐induced cell fusion at physiological (∼ 1 μM) concentrations, whereas HDL did not exert any inhibitory effect. Lipid‐associating, synthetic amphipathic peptides corresponding to residues 1–33 (apoA‐I[1–33]) or residues 66–120 (apoA‐I[66–120]) of apoA‐I, also inhibited HSV‐induced cell fusion, whereas a peptide corresponding to residues 8–33 of apoA‐I (apoA‐I[8–33]), which fails to associate with lipids, did not exert any inhibitory effect. These results suggest that lipid binding may be a prerequisite for peptide‐mediated fusion inhibition. Consistent with this idea, a series of lipid‐binding 22‐amino‐acid‐residue‐long synthetic amphipathic peptides that correspond to the amphipathic helical domains of apoA‐I (A‐I consensus series), or 18‐residue‐long model amphipathic peptides (18A series), were found to exert variable levels of fusion‐inhibitory activity. The extent of fusion‐inhibitory activity did not correlate with hydrophobic moment, hydrophobicity of the nonpolar face, helix‐forming ability, or lipid affinity of the different peptides. Peptides in which the nonpolar face was not interrupted by a charged residue displayed greater fusion‐inhibitory activity. Also, the presence of positively charged residues at the polar‐nonpolar interface was found to correlate with higher fusion‐inhibitory activity.
Implications and concepts of polyamine‐nucleic acid interactions
Authors: Feuerstein Burt G. , Williams Loren D. , Basu Hirak S. , Marton Laurence J.
Published: May 2005

Modeling, x‐ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left‐handed Z and the right‐handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.
Cytokine induction in HTLV‐I associated myelopathy and adult T‐cell leukemia: Alternate molecular mechanisms underlying retroviral pathogenesis
Authors: Tendler Craig L. , Greenberg Steven J. , Burton Jack D. , Danielpour David , Kim Seong‐Jin , Blattner William A. , Manns Angela , Waldmann Thomas A.
Published: May 2005

The human T‐cell lymphotropic virus type I (HTLV‐I) is capable of inducing a variety of host cellular genes including many of the cytokines responsible for immune regulation and osteoclast activation. This derangement in cytokine expression may contribute to the panoply of disease states associated with HTLV‐I infection such as the adult T‐cell leukemia (ATL) and HTLV‐I associated myelopathy/tropical spastic paraparesis (HAM/TSP). We wished to determine if there was a correlation between the expression of an array of cytokines and the diverse clinical manifestations of ATL and HAM/TSP. Utilizing the techniques of specific mRNA amplification by the polymerase chain reaction (PCR) as well as Northern blotting, we analyzed the ex vivo mRNA expression of γ‐interferon (IFN‐γ), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and transforming growth factor‐β, (TGF‐β1) in the peripheral blood of HAM/TSP and ATL patients as well as asymptomatic seropositive carriers. IFN‐γ, TNF‐α, and IL‐1β transcripts were up‐regulated in patients with HAM/TSP and seropositive carriers when compared to their levels in ATL and normal controls. In contrast, the ATL patients constitutively expressed higher levels of TGF‐α1 mRNA than HAM/TSP and seropositive carriers. In addition, TNF‐α and IL‐1β serum levels were elevated in HAM/TSP, but not in ATL patients nor seropositive carriers. However, the circulating leukemic cells from ATL patients secreted increased levels of TGF‐β1 protein into the culture medium than T‐cells derived from HAM/TSP patients. Collectively these results suggest that induction of IFN‐γ, TNF‐alpha;, and IL‐β in HAM/TSP may initiate an inflammatory cascade with subsequent events leading to immune mediated destruction of the central nervous system in these patients. Expression of osteoclast activators such as TNF‐α and IL‐β is not associated with hypercalcemia in ATL. Finally, impaired cellular and humoral immune responses present in ATL, but not in HAM/TSP, may be related to elevated levels of TGF‐β1 produced by the leukemic cells. These differences in retroviral‐induced host cytokine expression in ATL and HAM/TSP suggest alternate roles in disease pathogenesis.
The nucleus: A black box being opened
Authors: van Driel Roel , Humbel Bruno , de Jong Luitzen
Published: May 2005

Until recently our knowledge about the structural and functional organization of the cell nucleus was very limited. Recent technical developments in the field of ultrastrcutural analysis, combined with ongoing research on the properties of the nuclear matrix, give new insight into how the nucleus is structured. Two types of observations shape our ideas about nuclear organization. First, most nuclear functions (replication, transcription, RNA processing, and RNA transport) are highly localized within the nucleus, rather than diffusely distributed. Moreover, they are associated with the nuclear matrix. Second, chromatin is organized in discrete loops, bordered by nuclear matrix attachment sequences (MARs). Each loop may contain one or several genes. The arrangement of chromatin in loops has profound consequences for the regulation of gene expression.
Reversible inhibition of osteoclastic activity by bone‐bound gallium (III)
Authors: Blair Harry C. , Teitelbaum Steven L. , Tan Hong‐Lin , Schlesinger Paul H.
Published: May 2005

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7–12 μM Ga(III) reduces calcium release from bone, but surprisingly, 10–100 μM Ga3+; added to isolated avian osteoclasts did not reduce their degradation of L‐(5‐3H)‐proline bone. 3H‐proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) > 100 μM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67 Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 μM gallium from 500 μI of tissue culture medium, with steady state at > 24 h. Osteoclasts on bone inhibited gallium binding capacity ∼ 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum‐containing medium than in phosphate‐buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre‐bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 μg of bone pre‐incubated with 1 ml of 1 μM Ga3+, with 10 pmoles Ga3+/μg bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/μg bone, resorption was essentially zero. In contrast, pre‐treatment of bone with [Ga3+] as high as 15 μM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below ∼150 pg/μg bone acts for a limited time and does not permanently damage the cells. We conclude that bone‐bound Ga(III) from medium concentrations < 15 μM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] > 50 μM.
1,25‐Dihydroxycholecalciferol modulates 3H‐Thymidine Incorporation in FRTL5 Cells
Authors: Ongphiphadhanakul Boonsong , Ebner Susana A. , Fang Shih Lieh , Lombardi Antonio , Baran Daniel T. , Braverman Lewis E.
Published: May 2005

1,25‐dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H‐thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10−11 and 10−9 M exerted no effect on 3H‐thymidine incorporation. However, at 10−7 M, 1,25(OH)2D3 slightly enhanced 3H‐thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H‐thymidine incorporation induced by calf serum in a dose‐dependent manner. 1,25(OH)2D3 also enhanced 3H‐thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA‐induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA‐induced 3H‐thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H‐thymidine incorporation induced by TSH in a dose‐dependent manner. This effect of 1,25(OH)2D3 on TSH‐induced 3H‐thymidine incorporation may be, in part, due to post‐cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H‐thymidine incorporation induced by Bu2cAMP without affecting the TSH‐induced increase in cAMP. The stimulatory effect of insulin on 3H‐thymidine incorporation, a cAMP‐independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H‐thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.
Collagen increases the synthesis of membrane‐associated proteoglycans produced by sertoli cells
Authors: Rodriguez J. Pablo , Minguell Jose J.
Published: May 2005

Sertoli cells in culture produce two isoforms of proteoglycans which are found in the culture medium and associated with the cell membrane. The amount of both types of proteoglycans increased when Sertoli cells were plated on type 1 collagen‐coated dishes as compared to uncoated dishes. The effect is due to an increase in the synthesis of proteoglycans rather than a diminished rate of degradation of these molecules. The collagen substrate also affects the distribution of these macromolecules; an increase in the amount of membrane‐associated proteoglycans occurs at the expense of the proteoglycans released to the culture medium. © 1992 Wiley‐Liss, Inc.
Leukoregulin, A T‐cell derived cytokine, upregulates stromelysin‐1 gene expression in human dermal fibroblasts: Evidence for the role of AP‐1 in transcriptional activiation
Authors: Mauviel Alain , Kähäri Veli‐Matti , Uitto Jouni , Kurkinen Markku , Evans Charles H.
Published: May 2005

Leukoregulin(LR), a product of activated T‐cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455–1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin‐1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose‐ and time‐dependent elevation of stromelysin‐1 mRNA levels, the maximum enhnacement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/ reporter gene construct containing 1.3 kb of 5′ flanking DNA of the human stromelysin‐1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This inhancement was abolished by a single base substitution in the AP‐1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP‐1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP‐1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin‐1 gene expression by LR is mediated by AP‐1 regulatory elements which are necessary, but not sufficient, for gene response. © 1992 Wiley‐Liss, Inc.
cAMP‐Dependent protein kinase modulates expression and subcellular localization of Dolichos biflorus agglutinin binding sites in renal epithelial cells
Authors: Amsler Kurt
Published: May 2005

Previous studies demonstrated that, upon attaining confluence, a clone of the renal epithelial cell, LLC‐PK1, expressed progressively binding sites for the lectin Dolichos biflorus agglutinin (DBA) at the apical cell surface. Activation of cAMP‐dependent protein kinase enhanced surface expression dramatically. The goal of this study was to define the process leading to surface expression of DBA binding sites and to investigate further the role of cAMP‐dependent protein kinase in modulating surface expression. Both subconfluent and confluent cells exhibited intracellular DBA binding sites (50–70% of total cellular binding sites) in a perinuclear vesicular compartment which was disrupted by Brefeldin A treatment. Both total cellular content and the proportion of DBA binding sites at the cell surface increased modestly after confluence was attained. A 48 h treatment of cells with 1‐methyl‐3‐isobutyl xanthine, a phosphodiesterase inhibitor, dramatically increased the level of cellular DBA binding sites as well as the proportion of DBA binding sites at the cell surface. Analysis of two mutants of this cell line suggests that the effect of 1‐methyl‐3‐isobutyl xanthine requires cAMP‐dependent protein kinase activity but is not due to cAMP‐dependent protein kinase‐mediated activation of gene transcription.
Increase in epidermal growth factor receptor and its mRNA levels by parathyroid hormone (1‐34) and parathyroid hormone‐related protein (1‐34) during differentiation of human trophoblast cells in culture
Authors: Alsat Eliane , Haziza Jocelyne , Scippo Marie‐Louise , Frankenne Francis , Evain‐Brion Danièle
Published: May 2005

Human cytotrophoblasts in culture aggregate and fuse to form syncytiotrophoblasts. This process is associated with an increase in epidermal growth factor receptor (EGFR) expression [Alsat et al.: J Cell Physiol 154:122–128, 1993]. Recent studies have demonstrated the presence of parathyroid hormone‐related protein (PTHrP) in the human uterus and placenta. This led us to study the effect of PTH (1–34) and PTHrP (1–34) on the expression of EGFR during this differentiation process. Both peptides induced a concentration‐dependent increase in EGF binding, with a maximal effect at the physiological concentration of 1 nM. EGFR protein level assessed by cross‐linking and immunoblotting and EGFR biological activity assessed by measuring its EGF‐induced autophosphorylation were increased 2‐ and 2.5‐fold, respectively, when cells were treated for 24 h with 0.1 μM PTHrP or PTH compared to control cells. This effect was time‐dependent with a maximum at 3 h of treatment. This treatment also increased trophoblast cell EGFR mRNA levels, suggesting transcriptional regulation of the EGFR. To ascertain whether activation of protein kinase C (PKC) or protein kinase A (PKA) is involved in this PTH effect, we determined EGFR protein level and EGFR autophosphorylation after exposure of cells to PKA inhibitor and PKC inhibitor, alone or together with the peptide. The presence of a PKC inhibitor blocked a further increase in EGFR number by PTH, while PKA inhibitor had no effect. These results show that PTH and PTHrP increase the synthesis of EGF receptors which are strongly expressed in syncytiotrophoblasts and suggested that these peptides might be involved in human placental development.
Bone morphogenetic protein inhibits differentiation and affects expression of helix‐loop‐helix regulatory molecules in myoblastic cells
Authors: Murray Samuel S. , Murray Elsa J. B. , Glackin Carlotta A. , Urist Marshall R.
Published: May 2005

Bone morphogenetic protein (BMP) reproducibly induces chondrogenesis and osteogenesis when implanted into skeletal muscle. The exact identity of the cell that responds to BMP is not known. Furthermore, controversy exists regarding the possibility that myoblastic cells may transdifferentiate to chondrocytes and osteoblasts under the influence of BMP. We have therefore, undertaken studies on the effects of BMP on differentiation in L6 and C2C12 cells, two rodent myoblastic cell lines. To gain insights into the mechanisms of action of BMP, we have studied the effects of BMP on the levels of expression of the four known myogenic determination genes: myogenin, Myo D, herculin, and myf‐5. BMP inhibited myogenesis in myoblastic cells. Convincing evidence of transdifferentiation of myoblasts to chondrocytes or osteoblasts was not seen. BMP inhibited the expression of all four myogenic determination genes.
Estrogen receptor‐negative breast cancer cells transfected with the estrogen receptor exhibit increased RARα gene expression and sensitivity to growth inhibition by retinoic acid
Authors: Sheikh M. Saeed , Shao Zhi‐Ming , Chen Jian‐Chyi , Hussain Arif , Jetten Anton M. , Fontana Joseph A.
Published: May 2005

We and others have shown previously that retinoic acid (RA) selectively inhibits the growth of estrogen receptor (ER)–positive human breast carcinoma (HBC) cells and ER‐negative cells are refractory to RA inhibition of growth. The ER‐negative cells inherently express lower levels of RARα and retinoic acid response element (RARE)–mediated RA‐induced CAT activity. In this study we report that when ER‐negative MDA‐MB‐231 cells were transfected with the ER gene they not only expressed higher levels of RARα and RARE‐mediated RA‐induced CAT gene expression, but their growth was now inhibited by RA. Estrogen enhanced RARα gene expression not only in established ER‐positive cell lines but also in ER‐transfected MDA‐MB‐231 cells. The estrogen effect appears to be direct and at the gene transcription level since it did not alter the stability of RARα mRNA and cycloheximide failed to block estrogen‐mediated enhancement of RARα gene expression. Our data strongly suggest that ER‐mediated enhancement of RARα levels plays an important role in RA inhibition of HBC growth. In addition, we also report here that HBC cells appear to express a unique isoform(s) of RARα which was detected only when the full‐length RARα cDNA was used as a probe; the RARα1 and RARα2 specific probes failed to hybridize with the HBC specific RARα message.
Tartrate resistant acid phosphatase activity in rat cultured osteoclasts is inhibited by a carboxyl terminal peptide (osteostatin) from parathyroid hormone‐related protein
Authors: Zheng M. H. , McCaughan H. B. , Papadimitriou J. M. , Nicholson G. C. , Wood D. J.
Published: May 2005

A carboxyl‐terminal peptide sequence (“osteostatin”) from parathyroid hormone related protein has been shown to have an inhibitory effect on osteoclastic bone resorption—an action opposite to its amino‐terminal sequence. In this study, we proposed that inhibition of osteoclastic bone resorption by osteostatin was associated with reduction of tartrate resistant acid phosphatase (TRAcP) activity in osteoclasts. Our results have indicated that osteostatin reduced TRAcP activity in a dose dependent manner. This effect of osteostatin was both sensitive (half maximal effect approximately 5 × 10−13 M) and potent (maximum inhibition approximately 50% of control). In the first 90 min of treatment, however, reduction of TRAcP activity was erratic but became persistent and progressive when the time course was extended. Moreover, throughout the experimental period the levels of TRAcP activity in the culture medium had fallen significantly. It appears that osteostatin has a biphasic effect on TRAcP activity, inhibiting its secretion and either suppressing its synthesis or increasing its degradation. In addition, osteostatin induced rapid cellular retraction of both human and rat cultured osteoclasts, which was morphologically distinct from that produced by calcitonin.
Specific region of the c‐myc promoter is responsive to electric and magnetic fields
Authors: Lin Hana , Goodman Reba , Shirley‐Henderson Ann
Published: May 2005

The level of c‐myc transcripts is increased in cells exposed to extremely low frequency (elf) electromagnetic (EM) fields at 60 Hz. The aim of the present experiments was to determine if regulatory regions upstream of the c‐myc gene modulate the response to EM fields. DNA upstream of P1 of both mouse and human c‐myc genes was transfected into cells as CAT constructs. The presence of DNA 5′ to the human or mouse myc genes results in increased expression of CAT following 20 min exposures of cells to 60 Hz elf EM fields. Specific portions of the human upstream DNA were deleted and introduced into cells. The region responsive to EM fields is located between −353 and −1,257 relative to the P1 promoter.
Nuclear matrix and the regulation of gene expression: Tissue specificity
Authors: Getzenberg Robert H.
Published: May 2005

Tissue specific regulation of gene expression by a single transcription factor or group of transcription factors cannot be explained simply by DNA sequence alone. For example, in the same animal a particular transcription factor is capable of interacting with DNA in the nucleus of many different cell types, resulting in unique gene expressions despite the presence of a similar genome in all cells. Historically, these differences in response to a single type of factor within target tissues in the same animal have been suggested to occur through different alterations in chromatin structure. Recent, data has demonstrated that combination of hormones and transcription factors working together may cooperatively play a role in the regulation of gene expression [Pearce and Yamamoto (1993): Science 259:1161–1165]. However, the molecular mechanisms of this tissue specific regulation of gene expression still remains largely unexplained. Current evidence suggests that in different cell types the interplay between the specific three‐dimensional organization of the genome and the structural components of the nucleus, the nuclear matrix, may accomplish the regulation of specific gene expression. © 1994 Wiley‐Liss, Inc.
Nucleosome positioning and gene regulation
Authors: Lu Quinn , Wallrath Lori L. , Elgin Sarah C. R.
Published: May 2005

Recent genetic and biochemical studies have revealed critical information concerning the role of nucleosomes in eukaryotic gene regulation. Nucleosomes package DNA into a dynamic chromatin structure, and by assuming defined positions in chromatin, influence gene regulation. Nucleosomes can serve as repressors, presumably by blocking access to regulatory elements; consequently, the positions of nucleosomes relative to the location of cis‐acting elements are critical. Some genes have a chromatin structure that is “preset,” ready for activation, while others require “remodeling” for activation. Nucleosome positioning may be determined by multiple factors, including histone–DNA interactions, boundaries defined by DNA structure or protein binding, and higher‐order chromatin structure. © 1994 Wiley‐Liss, Inc.
Multiple functions of dynamic histone acetylation
Authors: Davie James R. , Hendzel Michael J.
Published: May 2005

Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear functions, including replication, transcription, and RNA splicing. It is becoming increasingly evident that nuclear processes are localized to distinct regions in the nucleus. For example, transcriptionally active genes and RNA transcripts are found in discrete transcription foci. Current evidence suggests that nuclear matrix–bound transcriptionally active DNA sequences are in nucleosomes with dynamically acetylated histones. Histone acetylation, which precedes transcription, alters nucleosome and chromatin structure, decondensing the chromatin fibre and making the nucleosomal DNA accessible to transcription factors. Histone acetyltransferase and histone deacetylase, which catalyze this rapid acetylation and deacetylation, are associated with the internal nuclear matrix. We hypothesize that these enzymes play a role in maintaining the association of the active chromatin domains with the internal nuclear matrix at sites of ongoing transcription. © 1994 Wiley‐Liss, Inc.
Microenvironment revisited: Time for reappraisal of some prevailing concepts of cancer metastasis
Authors: Fodstad Øystein , Kjønniksen Inge
Published: May 2005

The development and biological characteristics of tumor metastasis are influenced by multiple cell and host‐associated factors. To study these factors experimentally, the necessity of choosing adequate in vivo model systems for human tu mor metastasisis emphasized. Recent research has provided results that highlight the role of the microenvironment in determining important characteristics of the metastatic cells, including their degree of differentitation and sensitivity to drugs. Furthermore, evidence is presented as background for discussing the general validity of the notion of clonal selection of metastatic, and whether the metastatic phenotype is acquired through the last of a series of mutational events occurring during tumor progression.
Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones
Authors: Ferreira Luciano R. , Norris Kathleen , Smith Timothy , Hebert Carla , Sauk John J.
Published: May 2005

Hps47, Grp78, have been implicated with procellagen maturation events. In particular Hps47 has been shown to blind to nascent procellagen α1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collgen has previsously been suggested to mechanism. The early association of Hps47 with procollagen and its relatively late relese suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hps47. Herein, we examined how these events occurs in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the releses of Hps47, Grp78, and Grp94 from maturing procollange is delayed. Thus, in cell experiencing metabolic stress, newly synthesized procollagen unable to property fold became stable bound to a complex of molecular chaperones. In that Hps47, Grp78, and Grp98 could be recovered with nascent procollagen and as oligomer in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.
Heparan sulfate primed on β‐D‐xylosides restores binding of basic fibroblast growth factor
Authors: Miao Hua‐Quan , Fritz Timothy A. , Esko Jeffrey D. , Zimmermann Joseph , Yayon Avner , Vlodavsky Israel
Published: May 2005

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA‐745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low‐ and high‐affinity cell surface receptors. Exposure of pgsA‐745 cells to β‐D‐xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β‐D‐xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10–30 μM) of estradiol‐β‐D‐xyloside and naphthyl‐β‐D‐xyloside, but not by cis/trans‐decahydro‐2‐naphthyl‐β‐D‐xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside‐primed heparan sulfate in restoration of bFGF‐receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside‐primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside‐primed heparan sulfate and was abolished in cells that were exposed to 0.5–1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low‐affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin‐binding growth promoting factors.
Studies on the turnover of exogenous mannose‐terminal glucocerebrosidase in rat liver lysosomes
Authors: Murray Gary J. , Oliver Katharine L. , Jin Fu‐Sheng , Brady Roscoe O.
Published: May 2005

Mannose‐terminal glucocerebrosidase prepared by exoglycosidase digestion of human placental glucocerebrosidase is reported effective in the treatment of patients with type 1 Gaucher's disease [Barton et al. (1991); N Engl J Med 324:1464–1470]. However, the amount of enzyme that is necessary for therapeutic effect is much higher than would be predicted from in vitro activity measurements. We have investigated the fate of infused enzyme following intravenous administration in Sprague‐Dawley rats. In this model system, the enzyme is rapidly cleared from the plasma compartment by receptor‐mediated endocytosis via the mannose‐specific receptor present on reticuloendothelial cells. Enzyme activity measured in rat liver biopsy specimens at various times post‐infusion revealed a rapid initial loss of approximately one‐half of the maximum delivered enzyme in the first hour followed by a slower decay with a half‐life of approximately 6–8 h. The loss in enzyme activity is paralleled by a loss in enzyme protein when analyzed by Western blots. There is no evidence for return of enzyme activity or inactive enzyme protein to the plasma. Incomplete integration into the lysosomal membrane was demonstrated by the use of differential extraction of purified rat liver lysosomes to distinguish between lumenal and membrane bound enzyme. Immunoelectron microscopy of rat liver following infusion of mannose‐terminal glucocerebrosidase confirmed localization of the delivered enzyme primarily within the lumen of the lysosomes of Kupffer cells and to a lesser extent associated with the lysosomal membrane. Enzyme activity was stable in isolated rat liver lysosomes preloaded with mannose‐terminal glucocerebrosidase and incubated in the absence or presence of ATP. Acidification of the lysosomes to pH 3 results in a rapid loss of enzyme activity and protein; however, the relationship between the in vitro loss and the loss in enzyme activity in intact liver is not clear. We conclude from these studies that rapid intracellular degradation of administered glucocerebrosidase is the prime factor responsible for the high dose required for effective treatment of Gaucher's disease.
Properties of blood‐contacting surfaces of clinically implanted cardiac assist devices: Gene expression, matrix composition, and ultrastructural characterization of cellular linings
Authors: Menconi Michael J. , Pockwinse Shirwin , Owen Thomas A. , Dasse Kurt A. , Stein Gary S. , Lian Jane B.
Published: May 2005

The development of implantable cardiac assist devices for prolonged circulatory support has been impeded by the problem of excessive thrombogenesis on the blood–prosthetic interface, with subsequent embolization. To overcome this obstacle, a ventricular assist device has been developed with textured blood‐contacting surfaces to encourage the formation of a tightly adherent, hemocompatible, biological lining. In this study, we applied molecular biological techniques, in conjunction with conventional ultrastructural and biochemical techniques, to characterize the biological linings associated with the blood‐contacting surfaces of 11 of these devices, which had been clinically implanted for durations ranging from 21 to 324 days. No clinical thromboembolic events or pump‐related thromboembolism occurred. Biological linings developed on the textured surfaces composed of patches of cellular tissue intermingled with areas of compact fibrinous material. In addition, islands of collagenous tissue containing fibroblast‐like cells appeared after 30 days of implantation. Many of these cells contained microfilaments with dense bodies indicative of myofibroblasts. RNA hybridization analyses demonstrated that the colonizing cells actively expressed genes encoding proteins for cell proliferation (histones), adhesion (fibronectin), cytoskeleton (actin, vimentin) and extracellular matrix (types I and III collagen). Linings, which never exceeded 150 μm in thickness, remained free of pathological calcification. Textured blood‐contacting surfaces induced the formation of a thin, tightly adherent, viable lining which exhibited excellent long‐term hemocompatibility.
Insulin receptor overexpression in 184B5 human mammary epithelial cells induces a ligand‐dependent transformed phenotype
Authors: Frittitta Lucia , Vigneri Riccardo , Stampfer Martha R. , Goldfine Ira D.
Published: May 2005

To determine the role of the insulin receptor overexpression in breast epithelial cell transformation, the 184B5 human breast epithelial cell line was transfected with human insulin receptor cDNA. In two cell lines transfected with and overexpressing human insulin receptors (IR) (223.8 and 184.5 ng IR/106 cells), but not in untransfected cells, insulin binding and tyrosine kinase activity were elevated, and insulin induced a dose‐dependent increase in colony formation in soft agar.