bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat transmission in fire test furnaces
Authors: Sultan M. A. , Harmathy T. Z. , Mehaffey J. R.
Published: December 2004

This theoretical and experimental study, complementary to that discussed in Reference 1, examines those factors that affect the ‘test efficiency’ in fire resistance testing. Convective heat transfer in the test chamber may become an essential part of the heat transmission process if, due to small furnace size or non‐luminous furnace gases, the radiative transmission is not sufficiently intense. The standard technique of measuring and controlling the temperature conditions in the furnace chamber, though not in line with strict scienctific practices, appears to provide insurance against poor test result. A calibration procedure is described for checking the adequacy of fire resistance test facilities and some solutions presented for improving the test efficiency.
ISO ignitability test and proposed criteria
Authors: Bluhme D. A.
Published: December 2004

Having characterized the current classification of building materials in Scandinavia the use of the International Standards Organization ignitability test for classifying coverings, linings and materials is discussed. Further evidence for the assumption of a linear function for 1/tig (where tig is the time for sustained surface ignition) versus irradiance was gained for a number of materials, mainly wood‐based. This has been utilized in forecasting the ignitability behaviour in the new test by correlation of classes. Through definition of two borderline segments a simple table giving proposed criteria for tig is obtained.
Experimental observations of the effect of pressure and buoyancy on cellulose co‐current smoldering
Authors: Newhall J. L. , Fernandez‐Pello A. C. , Pagni P. J.
Published: December 2004

An experimental study has been performed to determine the potential effect of buoyancy on the rate of propagation of a co‐current smolder reaction through a porous solid fuel, and the range of flow velocities where buoyancy effects are significant. In the co‐current smolder reaction, the fuel and oxidizer enter the reaction zone from the same direction. In the present experiments this is accomplished by initiating the reaction at the top of the fuel bed, α‐cellulose packed at a void fraction 0.85, so that the smolder wave propagates downward opposing an upward forced flow of air. Since in a stratified density field, buoyancy is proportional to the product of gravity and density difference, buoyancy can be controlled by varying either the gravity vector or the gas density. In this study the latter method is followed, varying gas density through the ambient pressure at which the experiments are performed. The smolder velocity is measured for air flow rates varying from 0.2 to 6 gm−2s−1 at constant ambient pressures of 0.6, 0.8 and 1 atm. The results show that for flow rates larger than 1 gm−2s−1 the smolder velocity increases linearly with the air flow rate but is independent of pressure. The reaction peak temperature is weakly dependent on flow rate and independent of pressure. For the present experimental conditions the effect of buoyancy is only observed at very low air flow rates. The mechanisms by which it affects the smolder process appears to be by altering the transport of air to the reaction zone from upstream and downstream of the reaction.
The behaviour of commercially important diisocyanates in fire conditions. Part 2: Polymeric diphenyl methane‐4,4′‐diisocyanate (pmdi)
Authors: Marlair G. , Prager F. H. , Sand H.
Published: December 2004

The behaviour of polymeric diphenyl methane‐4,4′‐diisocyanate (PMDI) is described when examined in a laboratory small‐scale test for its reaction to fire (ease of ignition; heat release and toxic gas production). Full‐scale real fire scenarios have also been staged to predict events if (1) drumstock PMDI and (2) sizeable pools of liquid PMDI become enveloped in a fire. PMDI requires a stimulus (e.g. heat) before it will ignite from an applied flame. It then burns in a self‐sustaining manner for a few minutes, during which main emissions take place. Then a polymerization reaction begins, producing a low density non‐burning residue, which progressively dampens down the burning events by blanket action. Residues of 30–80% sample weight were recorded. The major toxic gas produced is carbon monoxide, though free isocyanate is to be expected in the early stages of the fire, and hydrogen cyanide could be important, especially in well‐developed fire conditions. Firefighters should therefore wear full protective clothing and fresh‐air breathing equipment. Events when drums of PMDI are exposed to fire depend heavily on the characteristics of the containers, with some rupture steps proceeding with considerable violence. Drumstock PMDI should be stored separately from easily ignitable materials.
Fire behaviour of sisal short fibers reinforced gypsum
Authors: Hernández‐Olivares F. , Oteiza I. , De Villanueva L.
Published: December 2004

The fire behaviour of sisal short fibers reinforeed gypsum in laboratory tests is described Specially designed testing equipment that is easily available has been implemented in this work to analyze the fibers under load and their composite fire performance.
Developing rational, performance‐based fire safety requirements in model building codes
Authors: Bukowski Richard W. , Babrauskas Vytenis
Published: December 2004

The technical and philosophical basis for performance‐based assessment of building fire performance is reviewed. A strategy for the evolution of a performance code is described. Current efforts toward the development of performance codes in the USA and Japan are reviewed. Recommendations for critical steps necessary to advance the development and acceptance of performance codes are presented. The table of contents of the Japanese risk methodology for assessing ‘Article 38 equivalencies’ is included in an appendix.
Fire conditions for smoke toxicity measurement
Authors: Gann Richard G. , Babrauskas Vytenis , Peacock Richard D. , Hall John R.
Published: December 2004

This paper identifies those fire conditions most often present when smoke toxicity is the cause of death. It begins with a review of the evidence that smoke‐inhalation deaths are in the majority in fire fatalities in the United States. Next, there is an analysis of the evidence from the national fire experience showing the connection between post‐flashover fires and smoke‐inhalation deaths. Third is a presentation of real‐scale fire test results demonstrating that post‐flashover conditions are necessary to produce enough smoke to cause smoke‐inhalation deaths in the cases where they actually occur. The fourth component is a sampling of results from computer simulations of fires, affirming and broadening the results from the fire tests. It is concluded that smoke‐inhalation deaths occur predominantly after fires have progressed beyond flashover. This conclusion then provides a focus for smoke toxicity measurement in particular and fire hazard mitigation in general.
Correlation between cone calorimeter data and time to flashover in the room fire test
Authors: Östman Birgit A.‐L. , Tsantaridis Lazaros D.
Published: December 2004

Correlations based on linear regressions between data as time to ignition and heat release in the cone calorimeter and time to flashover in the room fire test have been developed. They are a further development of an earlier approach which has been modified and extended to a wider range of surface linings. The correlations apply so far only to surface linings on both walls and ceilings. When the density of the linings as a simplified measure of the thermal inertia is included, the correlations are improved significantly.

The new correlations are based on data readily available from the cone calorimeter test at one heat flux level, 50 kWm−2. The correlation coefficient for the basic relationship, including the density of the linings, is now 0.98 when applied to the 13 linings investigated earlier. This is slightly better than the previous study, in which the best correlation coefficient was 0.96. When applied to 28 linings, the correlation coefficient remains about the same (0.97).

Very similar regression equations have been obtained when analysing only 13 products and all 28. This is a strong indication of the general predictive capacity of this approach. The inclusion of other data such as thickness of linings or mass loss during fire does not improve the correlation coefficients. The approach is quite straightforward and simple. However, it has provided a useful prediction which is also valid for an extended range of linings.
Reduced combustion efficiency of chlorinated compounds, resulting in higher yields of carbon monoxide
Authors: Schnipper Anette , Smith‐hansen Lene , Thomsen Erling Sonnich
Published: December 2004

A series of experiments involving the combustion of inherently fire‐retarded compounds has been performed using the DIN 53 436 furnace. Six chlorinated compounds comprising four pesticides, an organic solvent, and polyvinyl chloride (PVC) have been investigated with the focus on the combustion efficiency expressed as the CO2/CO ratio in the fire effluent. It was found that increased chloride content in a compound resulted in decreased CO2/CO ratio. The correlation was found to be \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {{\rm CO}_{\rm 2} /{\rm CO}} = 0.61 \times ({\rm Cl/C)}^{- {\rm 1}} + 0.13 $\end{document}. The results indicate higher concentrations of CO in the fire effluent from chlorinated compounds than from wood. Together with a release of HCI this could result in an increased toxic potency. The overall toxic hazard may or may not be increased, since (inherently) fire‐retarded materials tend to have an improved fire performance.
Fire‐retardant performance of some surface coatings for naval ship interior applications
Authors: Brown J. R. , Mathys Z. , Riddell S. Z. , Wake L. V.
Published: December 2004

The fire‐retardant performances of various surface coatings for naval ship interior applications have been evaluated by a number of small‐scale fire test methods. The coatings were eveluated as topcoats by cone calorimetry and limiting oxygen index methods and as primer/topcoat systems by radiat heating, direct flame impingement and reverse panel heating procedures. The coatings investigated were (1) a solvent‐based epoxy polyester, (2) a solvent‐based fireretardant alkyd,(3) a solvent‐based chlorimated alkyd and (4) a water‐based acrylic. In terms of the measured parameters, the chlorinated alkyd has superior fire properties while the epoxy polyserter, the fire‐retardant alkyd and parameters, the chlorinated alkyd has superior fire properties while the epoxy polyester, the fire‐retardant alkyd and the acrylic have significat disadvantages in relation to fire propagation. The relatively low amount of hydrogen chloride (∼ 0.3%—mass per initial mass) evolved by the chlorinated alkyd during cone calorimetric combustion and its outstanding fire‐retardant properties have resulted in it being considered acceptable for naval ship interior applications.
Use of the ARGOS fire risk analysis model for studying chinese restaurant fires
Authors: Chow W. K.
Published: December 2004

The fire risk in Chinese restaurants in Hong Kong is analyzed using the ARGOS fire risk analysis model developed at the Danish Institute of Fire Technology. A sample size of fifteen Chinese restaurants with different floor areas and fire load densities is considered. Fire simulations are performed for two cases by assuming a PU foam furniture fire occurring in the dining hall and a kerosene fire in the kitchen. Correlations are derived between the floor area and the predicted maximum hot gas temperature, the corresponding smoke layer interface height and the cost of damaged stock in the restaurants. The effectiveness of fire protection systems including sprinkler systems and smoke vents in controlling the fire is also discussed.
Essential oil composition of Hypericum richeri Vill. from Italy
Authors: Ferretti G. , Maggi F. , Tirillini B.
Published: December 2004

The essential oil composition of aerial parts of Hypericum richeri Vill. from Italy was analysed by GC and GC–MS. Fifty‐one components of the essential oil were identiﬁed, representing 98.5% of the oil. The most abundant components were (Z)‐β‐ocimene (19.5%), n‐nonane (13.8%), β‐bisabolene (8.7%), and (E)‐β‐ocimene (8.0%). Copyright © 2004 John Wiley & Sons, Ltd.
Genomic organization of variant surface glycoprotein genes in Trypanosoma brucei procyclic culture forms
Authors: Parsons Marilyn , Nelson Richard G. , Stuart Kenneth , Agabian Nina
Published: December 2004

The production of the variant surface glycoprotein coat of bloodstream form African trypanosomes ceases after conversion to the procyclic form. In the bloodstream stage alternate expression of different variant surface glycoprotein genes is responsible for the antigenic variation that occurs during relapse infections in the mammalian host. We have examined procyclic stage populations, derived from different bloodstream variant antigen types, for the two types of genomic alterations associated with variant surface glycoprotein genes in the bloodstream stage. Transcriptional activation of some variant antigen genes is accompanied by the generation of a new copy of the gene, the expression‐linked copy. We find that the expression‐linked copy is maintained after conversion to procyclic form, indicating that the presence of an expression‐linked copy is not sufficient for the expression of a surface coat. Sequences 3′ to other variant surface glycoprotein genes show expression‐independent variation in bloodstream stage trypanosomes. The same genes showed variation between procyclic populations of different origin, and between procyclics and their bloodstream parent. These data are discussed in light of observations on the sequence of variant antigen expression after cyclic transmission.
Herbicide‐quinone competition in the acceptor complex of photosynthetic reaction centers from rhodopseudomonas sphaeroides: A bacterial model for PS‐II‐herbicide activity in plants
Authors: Stein Randall R. , Castellvi Alicia L. , Bogacz Jerome P. , Wraight Colin A.
Published: December 2004

A select group of herbicides that inhibit photosystem II also act at the acceptor side of the reaction center (RC) from the photosynthetic bacterium Rhodopseudomonas sphaeroides, with much the same relative specificity as in plants. These include the triazines and some phenolic compounds. The proposal that herbicides inhibit the electron transfer from the primary quinone (QA) to the secondary quinone (QB) by competing for the secondary quinone binding site—the B‐site— [5], is tested here with terbutryn, the most potent of the triazines. Competition between terbutryn and ubiquinone (Q‐10) was observed using the kinetics of the back‐reaction as a measure of inhibition. The model includes binding equilibria before and after flash activation. The binding constants for the preflash (dark) equilibria, for reaction centers in 0.14% lauryl dimethylamine‐N‐oxide (LDAO), were K = 0.8 μM terbutryn, K = 2 μM Q‐10; both are detergent‐concentration dependent. After flash activation, binding equilibrium is not fully restored on the time scale of the back‐reaction because terbutryn unbinds slowly. This gives rise to biphasic decay kinetics from which koff for terbutryn was estimated to be 3 sec–1. Titrations of the rate of the slow back reaction indicated that the post‐flash equilibrium is less sensitive to inhibitor, in a manner that is independent of the much stronger binding of the semiquinone, Q, and indicative of a direct effect of the redox state of QA on the affinity of the B‐site for ligands. However, the effects on K and K could not be separated: either K > K or K < K. Some triazine‐resistant mutants have been isolated and are described. All appear to be herbicide binding site mutants. Whole cells and photosynthetic membrane vesicles (chromatophores) exhibit a 10–50‐fold increase in resistance to triazines due, in large part, to an increase in the rate of unbinding (koff). The modifications of the binding site appear to diminish the affinity of the B‐site for ubiquinone as well as terbutryn. It is concluded that bacterial RCs are a useful model for the study of herbicide activity and specificity.
Inhibition of erythrocyte membrane shape change by band 3 cytoplasmic fragment
Authors: Carter David P. , Fairbanks Grant
Published: December 2004

The ATP‐dependent transformation of crenated white human erythrocyte ghosts into smoothed disc and cup forms is inhibited by the soluble 40–45‐kilodalton (kDa) cytoplasmic portion of the major transmembrane protein, band 3. The band 3 fragment was prepared by chymotryptic treatment of inverted vesicles stripped of peripheral proteins. When present at ≥0.2 mg per mg membrane protein (ie, ≥2 mol fragment per mol endogenous band 3), the fragment significantly reduced the rate of shape change but did not alter the proportion of membranes that were ultimately converted into smoothed forms (>90%). The inhibitory activity of the fragment could not be attributed to contamination of the fragment preparation by actin or proteolytic enzymes. ATP‐independent shape transformation was not inhibited. The band 3 fragment may compete with endogenous, intact band 3 for an association with the spectrin‐actin network required for ATP‐dependent smoothing of crenated membranes.
A requirement for cholesterol and its structural features for a human macrophage‐like cell line
Authors: Esfahani Mojtaba , Scerbo Louis , Devlin Thomas M.
Published: December 2004

The lipid requirements of a human macrophagelike cell line were studied. The cells grew only about one generation in a medium supplemented with delipidated serum; during the growth the cholesterol content of the cells was depleted. Growth was restored by including in the medium serum lipids subjected to alkaline hydrolysis or cholesterol. The extent of growth was dependent on cholesterol concentration. No growth was obtained with 5‐cholestene, 5‐cholesten‐3‐one, cholesteryl chloride, coprostanol, β‐sitosterol, orstigmasterol. Very limited growth occurred with cholesterol methylether, epicholesterol, or β‐cholestarol. Therefore, for optimal growth of these cells there is a stringent requirement for the structural features of cholesterol, which include a 3‐βOH group, a Δ5 ‐double bond, a trans ring A/B configuration, and freedom of the side chain from bulky groups. This stringency far exceeds what was previously reported for other cells. Of the six sterols that failed to support growth at all, five were incorporated into cells moderately to extensively. This suggests that assembly of a functional membrane is impaired when these sterols are used as substrates for growth.
Kinetic analyses of the membrane‐bound alkaline phosphatase activity of hymenolepis diminuta (cestoda: Cyclophyllidea) in relation to development of the tapeworm in the definitive host
Authors: Pappas Peter W.
Published: December 2004

The specific activities of the alkaline phosphatase (APase), type I phosphodiesterase and 5′‐nucleotidase activities associated with the brush‐border plasma membrane of the tapeworm, Hymenolepis diminuta, decrease significantly as the tapeworm grows and matures. Kinetic analyses of the APase activity associated with membrane preparations from whole 6‐, 12‐, and 18‐d‐old H diminuta, and individual pieces of 18‐d‐old H diminuta cut into ten pieces of equal length, failed to demonstrate qualitative changes in the APase activity. Therefore, the decreased specific activities are apparently due to changes in the ratios of enzymatically active to enzymatically inactive membrane proteins (ie, quantitative changes in the membrane proteins) which occur as the tapeworm grows.
Initiation of proliferative events by human α‐thrombin requires both receptor binding and enzymic activity
Authors: Carney Darrell H. , Stiernberg Janet , Fenton John W.
Published: December 2004

To determine the role of thrombin high‐affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active γ‐thrombin stimulates ion fluxes without binding to high‐affinity sites, whereas proteolytically inhibited DIP‐α‐thrombin which binds to high‐affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP‐α‐thrombin and γ‐thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native α‐thrombin. This combinatory effect is dose‐dependent for both γ‐thrombin and DIP‐α‐thrombin in the same concentration range as α‐thrombin alone. Thus, these same concentrations of α‐thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native α‐thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.
Acidification of endocytic compartments and the intracellular pathways of ligands and receptors
Authors: Yamashiro Darrell J. , Maxfield Frederick R.
Published: December 2004

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor‐mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin‐coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0–5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferring, and the penetration of diptheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex‐of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin‐containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.
Modulation of type α transforming growth factor receptors by a phorbol ester tumor promoter
Authors: Davis Roger , Like Betsy , Massagué Joan
Published: December 2004

Epidermal growth factor (EGF) and an EGF‐like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37°C induces a complete loss of high‐affinity (Kd = 35–50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half‐maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF /eTGF receptors. The EGF/ eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor‐promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type α transforming growth factors, eTGF and EGF.
The frequency of bone marrow cells that bind erythropoietin
Authors: Weiss Tania L. , Kung Charles K. H. , Goldwasser Eugene , Meier Paul
Published: December 2004

The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1–2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.
Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors
Authors: Hofmann Cecilia , Crettaz Marco , Bruns Pamela , Hessel Patricia , Hadawi Gina
Published: December 2004

Madin‐Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites [Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin‐stimulated incorporation of [14C]glucose into glycogen, nor insulin‐induced uptake of radiolabeled α‐aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface‐labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipilates were analyzed on SDS‐gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells were incubated first with serum containing antibodies to the insulin receptor and then with 125I‐protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin‐like activity were tested on MDCK cells. The insulino‐mimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [13H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin‐insensitive, However, since insulin‐associated responses could be elicited by some insulin mimickers, the post‐receptor limb of response in MDCK cells was apparently intact.
Increased turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (DB/DB) mice
Authors: Kadle Ranjana , Raizada Mohan K. , Fellows Robert E.
Published: December 2004

The turnover of surface insulin receptors in fibroblastic cultures from genetically diabetic (db/db) mice and nondiabetic (m/m) littermates has been determined by combining a heavy isotope density shift technique with cross‐linking of insulin to surface receptors. Our results indicate that the surface insulin receptors turn over faster in diabetic cells than in nondiabetic cells. In addition, fewer receptors are incorporated into the plasma membrane per hour in diabetic cells than in nondiabetic cells. It is possible to propose a model to account for the altered expression of surface insulin receptors in diabetic cells on the basis of abnormalities of receptor incorporation and turnover.
The cell attachment determinant in fibronectin
Authors: Pierschbacher M. D. , Hayman E. G. , Ruoslahti E.
Published: December 2004

Fibronectin possesses a domain that interacts with cell surfaces. The ability of fibronectin to promote cell attachment can be duplicated with a short amino acid sequence, glycyl‐L‐arginyl‐glycyl‐L‐aspartyl‐L‐serine, taken from that domain. The tripeptide Arg‐Gly‐Asp appears to be irreplaceable for maintenance of the activity of this peptide, wheareas the serine residue can be replaced with some, but apparently not all, possible residues. This recognition sequence, or a closely related sequence, is present in a number of proteins other than fibronectin that interact with cells. These proteins include collagens, fibrinogen, thrombin, a bacterial surface protein, and two viral proteins, as well as discoidin‐I, a protein implicated in the aggregation of Dictyostelium discoideum. A similar sequence is also repeated in some, but not all, fibronectin molecules, making it possible that some fibronectin molecules have more than a single cell attachment site. Synthetic peptides constructed from sequences taken from several of these other proteins have also been shown to promote cell attachment. The tripeptide sequence may, therefore, constitute an ancient cellular recognition mechanism common to many proteins.
Characterization of a membrane‐associated glycoprotein complex implicated in cell adhesion to fibronectin
Authors: Hasegawa Takayuki , Hasegawa Etsuko , Chen Wen‐Tien , Yamada Kenneth M.
Published: December 2004

We have characterized a 140‐kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two‐dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two‐dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co‐migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.
Interaction between Raf and Myc oncogenes in transformation in vivo and in vitro
Authors: Cleveland J. L. , Jansen H. W. , Bister K. , Fredrickson T. N. , Morse H. C. , Ihle J. N. , Rapp U. R.
Published: December 2004

3611 MSV, a raf‐oncogene‐transducing murine retrovirus, induces fibrosarcomas and erythroid hyperplasia in newborn mice after a latency of 4–8 wk. In contrast, new recombinant murine retroviruses carrying the myc oncogene (J‐3, J‐5 construct viruses) do not induce tumors before > 9 wk. A combination of both oncogenes in an infectious murine retrovirus (J‐2) induces hematopoietic neoplasms in addition to less prominent fibrosarcomas and pancreatic adenocarcinoma 1–3 wk after inoculation. The hematologic neoplasms consist of immunoblastic lymphomas of T and B cell lineage and erythroblastosis. If animals were inoculated with a variant of the J‐3 virus, which induces altered foci in cultures of NIH 3T3 cells, carcinoma developed in the pancreas with a 2–6 mo latency. In parallel to the synergistic action of both oncogenes on hematopoietic cells in vivo, we find that raf‐oncogene‐induced transformation of bone marrow cells in culture is enhanced by the addition of myc, which by itself does not transform these cells when grown in standard media. We conclude that concomitant expression of raf and myc oncogenes in hematopoietic and epithelial cells alters their respective transforming activities. The contribution of v‐myc in this synergism was examined by use of a series of recombinant murine retroviruses capable of expressing the avian v‐myc to study the effect of altered myc expression on hematopoietic/lymphoid cells. With either interleukin 3‐ or interleukin 2‐dependent cell lines, introduction of the recombinant viruses abrogated the requirement for IL 3 or IL 2 for growth, and associated with this was the suppression of c‐myc expression. The findings suggest that myc is a component in the signal transduction pathway for IL 3 and IL 2 and support an autoregulatory mechanism of c‐myc expression. In contrast to v‐myc, expression of v‐raf primary lymphoid hematopoietic cells has an immortilizing function without abrogating the requirement for IL 3 for growth. This suggests that v‐raf and v‐myc affect different components of growth regulation, as, for example, commitment (v‐myc ) and cell cycle progression (v‐raf).
The present status of erythrocyte spectrin structure: The 106‐residue repetitive structure is a basic feature of an entire class of proteins
Authors: Speicher David W.
Published: December 2004

Spectrin, the major component of the erythroid membrane skeleton, is a long, asymmetrical rodlike protein that interacts with several other proteins to form a two‐dimensional membrane skeleton. Progress in several laboratories over the past few years including substantial partial peptide and nucleotide sequence determination has greatly enhanced our knowledge of the structrual properties of this large molecule (heterodimer = 465,000 daltons). The alpha and beta subunits are homologous with approximately 30% identity. They are aligned in an anti‐parallel side‐to‐side orientation with the amino‐ and carboxy‐termini near opposite physical ends of the molecule. The predominant structural feature elucidated from sequencing this large molecule is the nearly universal occurrence in both subunits of a single type of repetitive structure. The periodicity of this homologous structure is exactly 106 amino acid residues. As many as 36 homologous, but non‐identical, repeats exist and comprise more than 90% of the mass of the heterodimer. Each of these repetitive units is folded into a triple‐stranded structure that is highly helical. Peptide maps, antibody cross‐reactivity, peptide sequence analysis, and more recently nucleic acid sequences have defined several major properties of the erythroid molecule and related proteins in other tissues. Tissue‐specific spectrins have the same 106‐residue repetitive structure and show sequence homology to erythroid spectrin.
Demonstration of a relationship between talin and P235, a major substrate of the calcium‐dependent protease in platelets
Authors: Beckerle Mary C. , O'Halloran Theresa , Burridge Keith
Published: December 2004

Talin is a 225,000‐Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely opposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000‐Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high‐molecular‐weight proteins are cleaved to lower‐molecular‐weight forms. Here we demonstrate that talin is closely related to one of these platelet high‐molecular‐weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth‐muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190‐200kD polypeptide by a calcium‐activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one‐dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one‐dimensional partial peptide maps, we conclude that P235 is the platelet form of talin.
Isolation and characterization of nuclear lamina from ehrlich ascites tumor cells
Authors: Krachmarov Chavdar , Tasheva Bistra , Markov Dimitar , Hancock Ronald , Dessev George
Published: December 2004

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein‐DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two‐dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C. whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.
Ligand–receptor interactions: Detailed evaluation of occupancy‐dependent affinity
Authors: Faguet Guy B.
Published: December 2004

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy‐dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte–leukoagglutinin (LPHA) system, which closely mimics the IM9‐insulin model. Reliable estimates of total binding capacity (728 ng/106 cells) essential to our report were calculated from a wide database by the least‐squares model. At occupancies ≥ 0.085, receptors are associated with low and fixed affinity (1.5 × 106M−1), whereas at occupancies ⩽ 0.085, affinity is high and fixed (1.8 × 108M−1) or high but variable (1 × 107M−1 to 1.5 × 106M−1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor–ligand complex dissociation velocity over a wide range of occupancies (0.01–0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of ≅ 0.004 and is magnified as ligand binding to high affinity receptors increases up to ≅ 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand‐induced cell responses.
Actions of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP formation in chicken and rat osteoclasts
Authors: Nicholson G. C. , Livesey S. A. , Moseley J. M. , Martin T. J.
Published: December 2004

The effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast‐rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti‐cyclic AMP and FITC‐labelled goat anti‐rabbit IgG. Treatment of rat osteo‐clasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium‐regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.
Sponge aggregation factor: In situ localization by fluorescent monoclonal antibody techniques
Authors: Gramzow Monika , Dorn August , Steffen Renate , Müller Werner E. G.
Published: December 2004

The aggregation factor (AF) from sponges mediates a heterophilic interaction of homologous cells. Applying electron microscopical means, we succeeded only very rarely in identifying the 90 S AF particle in tissue sections from Geodia cydonium. By means of a fluorescent antibody technique, we have now localized the cell binding domain of the AF in situ. Previous studies in this laboratory have led to the identification of the 47‐kDa cell binding protein of the AF, using the monoclonal antibody (mab) 5D2‐D11 [Gramzow M, Bachmann M, Zahn RK, Uhlenbruck G, Dorn A, Müller WEG, J Cell Biol, 102:1344–1349, 1986]. This mab and mab 7D5, directed against a 92‐kDa protein in the AF complex, were chosen for the fluorescent studies. By using mab 5D2‐D11, the plasma membranes of cells from different regions in the sponge could be brightly stained. However, mab 7D5 reacted only very weakly with the sponge surfaces. By applying the immuno‐blotting technique it was furthermore demonstrated that the cell binding protein is present both in the associated form with AF complex and in a free state. Moreover, it was established that the 47‐kDa binding protein is not present in (1) homologous glycoconjugates, (2) lectin, or (3) collagen; these components are known to be involved in cell‐matrix interaction.
Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants
Authors: Verghese Margrith W. , Smith Charles D. , Snyderman Ralph
Published: December 2004

Leukocyte activation by Chemoattractants provides an important model to study the biochemical mechanisms of stimulus‐response coupling in these cells. Well‐defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high‐ and low‐affinity states which are regulated by guanosine di‐ and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although Chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane‐bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor‐transduction mechanisms. Stimulation of phagocytes by Chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([Ca2+]i) which appears to result from the production of inositol 1,4,5‐triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP‐ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT‐sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIPi in PMN membranes only in the presence of GTP. This receptor‐mediated hydrolysis of PIPi is not observed in plasma membranes prepared from PT‐treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT‐sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2‐diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.
Transforming growth factor‐α attenuates the acquisition of aromatase activity by cultured rat granulosa cells
Authors: Adashi Eli Y. , Resnick Carol E. , Twardzik Daniel R.
Published: December 2004

The effect of transforming growth factor‐α (TGFα) on granulosa cell differentiation, as assessed by the acquisition of aromatase activity, was evaluated in vitro by using a primary culture of rat granulosa cells. Harvested from immature, diethylstilbestrol‐treated rats, granulosa cells were cultured under serum‐free conditions for 72 hr in the presence of saturating concentrations (10−7M) of aromatase substrate androstenedione with or without the specified experimental agents. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen was negligible, remaining unaffected by treatment with TGFα; (10 ng/ml) by itself. Whereas treatment with follicle‐stimulating hormone (FSH) resulted in a substantial increase in the extent of aromatization, concurrent treatment with TGFα: (10 ng/ml) resulted in significant (P<0.05), yet reversible inhibition (78 ± 5.6%) of FSH action. Significantly, this effect of TGFα could not be accounted for by a decrease in cellular viability or plating efficiency nor by a decrease in the number of cells or their DNA content. Although independent of the FSH dose employed, the TGFα effect proved dose‐ and time‐dependent, with an apparent median inhibitory dose (EC50) of 0.33 ± 0.04 ng/ml, and a minimal time requirement of 48 hr. Capable of substantial inhibition of the forskolin‐stimulated accumulation of extracellular adenosine 3′, 5′ cyclic monophosphate (cAMP) and estrogen, TGFα had a measurable albeit limited effect on N6, 2‐′O‐Dibutyryladensine 3′:5′‐cyclic monophosphate‐supported estrogen production. Relative potency comparison revealed epidermal growth factor (EGF; EC50 = 0.24 ± 0.03ng/ml) and TGFα to be virtually equipotent as regards the attenuation of FSH‐stimulated estrogen biosynthesis. Taken together, our findings indicate that TGFα, like EGF, acting at subnanomolar concentrations, is capable of attenuating the FSH‐stimulated (but not basal) accumulation of estrogen. This effect of TGFα proved time‐ and dose‐dependent, involving virtually complete neutralization of FSH action at site(s) both proximal and distal to cAMP generation. As such, these findings provide yet another example of the remarkable qualitative and quantitative similarities between EGF and TGFα, thereby reaffirming the prospect that ligands of the EGF/TGFα receptor may play a modulatory role in the course of granulosa cell ontogeny.