bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance of zinc oxide single‐crystal semiconductor gas sensors in an experimental mine roadway
Authors: Hurts Nicholas W. , Jones T. Alwyn , Mann Brenda
Published: December 2004

The performance of zine oxide gas large‐scale experiments in an experimental underground mine roadway has been investigated. The sensors were in a simple prototype instrument and the response to products evolved from heated coal wood and PVC conveyor belt was determined. The response was measured as a function of sensor distancef from the combustion products, the ventilation rate in the roadway and the mass of coal dust used. Limited analuysis of the roadway air was also carried out. The results are compared with those obtained in laboratory studies1 and suggest that the sensors respond mainly to inorganic gases, e.g. CO, H2, H2S, HCl, and in the case of wood, acetic vapour at concentrations of about 1ppm. The experiments confirm the potential of the sensors as a means of detecting heating involving coal, wood and PVC conveyor belt.
Application of oxygen‐consumption calorimetry to non‐combustibility testing
Authors: Ahonen Antti , Weckman Henry , Yli‐Penttilä Martti
Published: December 2004

The feasibility of oxygen‐consumption calorimetry to testing of non‐combustibility of building materials is studied. The reasons for the ambiguities of the traditional test method are qualitatively discussed. The construction of the gas collecting system added to the conventional test apparatus is described and the necessary formulae for calculating the oxygen consumption and the simultaneously measured conventional test variables are compared. The results clearly suggest that oxygen‐consumption calorimetry is a consistent and powerful method for determining the non‐combustibility of building materials.
Evaluation of furniture‐fire hazard using a hazard‐assessment computer model
Authors: Bukowski Richard W.
Published: December 2004

The Center for Fire Research Fire (Toxic) Hazard‐Assessment computer model was used to evaluate the potential for hazard reduction by the modification of the combustion properties of upholstered furniture items in a residential occupancy. The potential benefits of these modifications are compared with the effects of variations in room size and construction to determine if they would be realized across a range of housing sizes and types. The results demonstrate the greatest benefit by the reduction of the mass loss (burning) rate of the item regardless of room size and even if the means used to reduce the burning rate results in an increase in smoke production and material toxicity. These results are intended as an indicator of potentially beneficial directions for further research and should not be taken as conclusive evidence of fact without experimental verification.
Combustion products of polymeric materials. IV—testing of toxicity hazard
Authors: Blahu̇šková A. , Mácha J. , Markoš A. , Procházková Z. , Štefl B. , Michal J.
Published: December 2004

A method based on exposure of mice in the CAB 4.5 smoke chamber for the evaluation of the biological toxicity of the products of flame and smoldering combustion of polumeric materials has been developed. A toxicity index, characterizing the danger from the products of combustion of the test polymer under the given conditions, was proposed on the basis of values of the concentration of carboxyhaemoglobin in the blood of the test animals, attaining charateristic critical limits (lethal limit) and LC50 values.
Chemical ‘fingerprint’ studies of fire atmosphere
Authors: Fardell P. J. , Murrell Janet M. , Murrell J. V.
Published: December 2004

Large‐scale fire experiments have been carried out in a special test rig to study the chemical nature of fire atmosphere using a ‘fingerprint’ sampling and analysis method. Four polymeric materials—wood, polypropylene, polymethyl methacrylate and polystyrene foam—were used separately as fuel, with both high and low ventilation. In addition to oxides of carbon the fingerprints yielded typically between twenty and forty different chemical compounds including several (e.g. aldehydes) which could contribute significantly to the irritancy of the fire gases. The change in nature of the fingerprint gases with the different ventilation conditions, different polymeric materials and with different stages of the same fire is discussed, together with the toxicological significance of the results.
Fire and rice husk particleboard
Authors: Bhatnagar Subodh Kumar
Published: December 2004

Experimental investigations were made to assess the fire behaviour of rice husk particleboard, a potential substitute for wood particleboards. Releveant fire properties, namely non‐combustibility, ignitability, fire propagation index, surface spread of flame classification, specific optical density of smoke generated, flammability and flame penetration were determined in accordance with existing standard methods. In this paper the salient results obtained are discussed.
Evaluating ignition data using the flux time product
Authors: Shields T. J. , Silcock G. W. , Murray J. J.
Published: December 2004

A protocol based on the flux time product (FTP)1 is used to analyze ignition data obtained from the Cone Calorimeter under an impressed flux in the range 20–70 KWm−2 for different orientations and modes of ignition for conditioned cellulosic materials. The mean, maximum and minimum ignition times are depicted graphically by orientation and mode of ignition. Flux time products, FTP indices, critical irradiances and estimates of the convective heat loss associated with a change in specimen orientation are derived using the mean time‐to‐ignition data. It is demonstrated that consideration of the thermal thickness of a specimen may not be necessary when the proposed FTP methodology is utilized to determine valid correlations between the time‐to‐ignition and the incident radiant flux.
Note: Effect of flame retardant treatment on energy of pyrolysis/combustion of wood cellulose
Authors: Garba B. , Eboatu A. N. , Atta‐Elmannan M. A.
Published: December 2004

The effect of potassium aluminium sulphate, (hereafter referred to as alum) on the energy of pyrolysis/combustion of wood cellulose was investigated using thermogravimetric analysis (TGA). It was observed that the energy of pyrolysis of all the materials employed increases with flame retardant loading. Hence, alum functions as flame retardant (FR) by a complex process that entails the dehydration, condensed phase and vapour phase mechanisms. An attempt is made to explain this fact.
Fire‐endurance testing of building constructions containing wood members
Authors: Richardson L. R. , Onysko D. M.
Published: December 2004

In the later part of 1992 the National Research Council of Canada (NRC), in collaboration with a number of industry and governmental partners, commenced a research program to quantify sound‐transmission‐class and fire‐resistance ratings for gypsum‐board protected wood‐frame and steel‐frame building assemblies. The ‘partners’ include Forintek Canada Corp. acting on behalf of Canada's wood products industry. Although thousands of fire‐resistance tests have been carried out over the past century, many of which were on wood‐frame constructions, three interrelated issues arose in regard to the wood studs used in these tests: moisture content, strength, and magnitude of the load superimposed on the walls that the studs would be required to withstand during the fire tests. This paper discusses those issues.
Experimental and analytical protocol for ignitabiilty of common materials
Authors: Dietenberger Mark A.
Published: December 2004

In this study, a protocol was developed to increae accuracy, generality and efficiency when determining piloted ignition properties. A new procedure for calibrating the radiative and convective heat flux protiels on exposed speciments, such as Douglas‐fir plywood, has been implemented for the lateral ignition and flame spread test (LIFT) apparatus. The boundary conditions needed for heat transfer anylysis are made unambiguous by including a simple, direct measure f surface emissivity. A new aluminum foil shutter improves accuracy for measuring ignition time. A recently developed theroy of ignitanility provides a formula to account for the transition form thick to thin thermal behaior, allowing specimens of finite thicknesses and a fuln range of test irradiances.
Note: Effect of some halogenated flame retardants on the thermal characteristcs of selected tropical construction materials
Authors: Garba B. , Abdularahman F. W. , Obaseki M. E.
Published: December 2004

The effects of some halogenated flame retradants (FR), sodium bromide and ammonium cupric chloride dihydrate (ACCD) on the thermal behaviour of roofing palm (Borassus aethiopun) and rattan palm (calamus deeratus) were studied. Their ignition time, flame propagation rate and afterglow time were investigated. No ignition occurred on the materials treated with 0.4M ACCD and no ignition was observed on roofing palm treated with 0.8M sodium bromide. Flame propagation rate and afterglow time were drastically reduced. Gravimetric analysis showed that these retardants acted by vapour phase and condensed phase mechanisms. An increase in char formation was also noted as a result of this treatment in all cases.
Flammability testing of pure and flame retardant‐treated cotton fabrics
Authors: Hshieh Fu‐yu , Beeson Harold D.
Published: December 2004

A Controlled‐atomosphere cone calorimeter was used to investigate the burning of pure and flame retardant‐treated cotton fabrics. The condensed‐phase flame retardants used were Morguard (containing ammonium dihydrogen phosphate and diammonium hydrogen phoisphate) and Nochar (containing ammonium sulfate and a sodium salt). The fabrics were tested at 25 kW m−2 incident heat flux in environments containing 15–30% oxygen. The flame retardants increased the time to ignition, residue yield, and CO and CO2 yields. The flame retardants decreased the peak and average mass loss rates, the peak and average heat release rates, the effective heat of combustion at peak heat release rate, and the propensity to flashover. The effect of oxygen concentration on the burning of pure and flame retardant‐treated cotton fabrics has also been investigated. The flame retardants had better performance when the treated fabrics burned in the lower oxyge concentrations. The result of this study indicate that the controlled‐atmosphere cone calorimeter is a good tool for studying the effect of flame retardant and oxygen concentration on the burning of materials.
Numerical prediction of fire spread over vegetation in aribitrary 3D terrain
Authors: Costa A. M. , Pereiraal J. C. F. , Siqueira M.
Published: December 2004

Forest fires involve a wide range of unknown variables, e.g. wind flow over complex terrain, atmospheric stability, vegetation burning characteristics, location and fuel type, etc. The main objective of the present work is to incorporate the Rothermel simplified combustible model into a 3D unsteady flow solver appropriated for convective atmospheric flows over complex terrain. The released combustion energy is taken into account in the enthalpy equation. The unsteady byouant plume strongly influences the local wind speed direction and magnitude. A speical interactive coupling procedure was developed that links the calculated location of fire front and fire energy release to the momentum and energy transport for each time step. The general computer algorithm includes several other features such as the effect‐drag of trees on the momentum transport and the consequent modifications in the k and ε turbulence model employed. The results include the prediction of fire development in flat terrain and in a valley and over hills covered by vegetation. A parametric study was conducted to detect the influence of wind speed, vegetation and fuel content on burning area, burning speed and wind speed direction.
Combined haploid and insertional mutation screen in the zebrafish
Authors: Wiellette Elizabeth , Grinblat Yevgenya , Austen Matthias , Hirsinger Estelle , Amsterdam Adam , Walker Charline , Westerfield Monte , Sive Hazel
Published: December 2004

To identify genes required for development of the brain and somites, we performed a pilot screen of gynogenetic haploid zebrafish embryos produced from mothers mutagenized by viral insertion. We describe an efficient method to identify new mutations and the affected gene. In addition, we report the results of a small‐scale screen that identified five genes required for brain development, including novel alleles of nagie oko, pou5f1, ribosomal protein L36, and n‐cadherin, as well as a novel allele of the laminin g1 gene that is required for normal skeletal muscle fiber organization and somite patterning. genesis 40:231–240, 2004. © 2004 Wiley‐Liss, Inc.
Development of fungal mycelia as a skin substitute: Characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound‐healing process
Authors: Su Ching‐Hua , Liu Shu‐Hui , Yu Shi‐Yau , Hsieh Yi‐Ling , Ho Hsiu‐O , Hu Chung‐Hong , Sheu Ming‐Thau
Published: December 2004

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte‐SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 μg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37°C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 72A: 220–227, 2005
Biosynthesis of the photosynthetic membranes of rhodopseudomonas sphaeroides
Authors: Kaplan Samuel , Cain Brian D. , Donohue Timothy J. , Shepherd William D. , Yen Grace S. L.
Published: December 2004

The steady‐state biosynthesis of the photosynthetic membrane (ICM) of Rhodopseudomonas sphaeroides has been reviewed. At moderate light intensities, 500 ft‐c, preexisting ICM serves as the insertion matrix for newly synthesized membrane components. Whereas the bulk of the membrane protein, protein‐pigment complexes, and pigments are inserted into preexisting ICM throughout the cell cycle, phospholipid is transferred from outside the ICM to the ICM only at the time of cell division. Because the site of cellular phospholipid synthesis is the cytoplasmic membrane, these results infer that despite the physical continuity of cytoplasmic membrane and ICM, there must exist between these membranous domains a “barrier” to the free diffusion of cellular phospholipid. The cyclical alternation in protein to phospholipid ratio of the ICM infers major structural and functional alternations, such as changes in the protein to lipid ratio of the membrane, specific density of the membrane, lipid structure within the membrane, and the rate of cyclic electron flow. When biochemical studies are correlated with detailed electron microscopic investigations we can further conclude that the number of photosynthetic units within the plane of the membrane can vary by nearly a factor of two over the course of the cell cycle. The average physical size of the photosynthetic units is constant for a given light intensity but inversely proportional to light intensity. The distribution of photosynthetic unit size classes within the membrane can be interpreted as suggesting that the “core” of the photosynthetic unit (reaction center plus fixed antenna complex) is inserted into the membrane coordinately as a structural entity. The variable antenna complex is, on the other hand, inserted independent of the “core” and randomly associates with both old and new core complexes. Finally, we conclude that there is substantial substructure to the distribution of photosynthetic units within the ICM, ie, they are highly ordered and exist in a defined spatial orientation to one another.
Influence of mitoxantrone on nucleic acid synthesis on the T‐47D breast tumor cell line
Authors: Safa Ahmad R. , Chegini Nasser , Tseng Michael T.
Published: December 2004

Mitoxantrone exerts growth inhibitory effects, suppresses [3H]‐thymidine as well as [3H]‐uridine incorporation, and induces ultrastructural alterations in T‐47D human breast tumor cells. At low concentration (10−9M) the drug induced little effect on cell proliferation; cell growth kinetics were inhibited at a concentration of 10−5M. [3H]‐thymidine and [3 H]‐uridine incorporation declined rapidly at the concentrations tested (10−9, 10−7, and 10−5 M), revealing a potent effect on metabolic activity of the cultured cells. The sharpest decline in DNA and RNA synthesis occurred within the first 2 hr of drug treatment. Serial ultrastructural examinations indicated definitive alterations in chromatin structure, disintegration of nucleolar components as early as 2 hr after drug treatment, and complete segregation of nucleolar components following 8‐hr exposure to concentrations of the drug between 10−5 and 10−7 M. A distinct increase in the density of mito‐chrondrial matrix was evident. The in vitro data presented in this report demonstrate the growth inhibitory and antimetabolic effects of mitoxantrone on human breast tumor cells and suggest that the drug may be a promising antitumor agent.
Sugar‐specific endocytosis of glycoproteins by Lewis lung carcinoma cells
Authors: Roche A. C. , Barzilay M. , Midoux P. , Junqua S. , Sharon N. , Monsigny M.
Published: December 2004

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing α‐D‐glucose residues: This binding is competitively inhibited by neoglycoproteins containing α‐D‐glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell‐associated neoglycoprotein was higher at 37°C than at 4°C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing α‐D‐glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar‐specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate‐binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and a neoglycoprotein containing α‐D‐glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.
Amphiphilic properties of the avian myeloblastosis virus major glycoprotein, gp 80
Authors: Marciani Dante J.
Published: December 2004

The major glycoprotein (gp 80) from avian myeloblastosis virus (AMV) displays significant lipophilic properties, as shown by its strong interactions with acetylated uncharged decylamino agarose in hydrophobic chromatography. In effect, release from binding was achieved only by the added presence of a polarity reducing agent (ethylene glycol) and the strong anionic detergent sodium dodecyl sulfate. The hydrophobic behavior of the glycoprotein, coupled to the high content of hydrophilic carbohydrates, indicates its amphiphilic character. Confirmation of the amphiphilic nature of the AMV gp 80 was obtained by charge shift electrophoresis and crossed hydrophobic interaction immunoelectrophoresis. In both instances, the electrophoretic behavior of the glycoprotein was dependent on the presence of detergents. The AMV gp 80 displays the properties of integral membrane proteins.
Modulation by retinyl acetate of microfilament bundle formation in C3H/10T1/2 cells
Authors: Mordan Lawrence J. , Hui Sek‐Wen , Bertram John S.
Published: December 2004

Retinyl acetate has been previously shown to inhibit carcinogen‐induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 μg/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol‐triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electrophoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells: it is hypothesized that these actions are related to the antineoplastic activity of retinoids.
The role of duplication in the expression of a variable surface glycoprotein gene of trypanosoma brucei
Authors: Young John R. , Turner Mervyn J. , Williams Richard O.
Published: December 2004

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to espression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well‐characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accomodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.
Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves
Authors: Bennett John , Jenkins Gareth I. , Hartley Martin R.
Published: December 2004

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5‐bisphosphate carbox‐ylase/oxygenase (RUBISCO), and the light‐harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de‐etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40‐fold, at least 200‐fold, and about 25‐fold, respectively, while the total RNA content per apical bud increased only 3.5‐fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60‐, 100‐, and 200‐fold, respectively. The LHCP increased steadily in concentration during de‐etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24‐hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far‐red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far‐red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady‐state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.
Partial characterisation of the high and low molecular weight forms of P388D1‐derived interleukin 1
Authors: Prestidge R. L. , Koopman W. J. , Bennett J. C.
Published: December 2004

The murine macrophage‐derived cell line P388D1 secretes the lymphokine inter‐leukin 1 (IL‐1) when stimulated by a variety of agents. When stimulated by bacterial lipopolysaccharide (LPS) the cells release IL‐1 in both high and low molecular weight (m.w.) forms. The proportion of high m.w. IL‐1 is reduced when IL‐1‐containing supernatants are concentrated by ammonium sulfate precipitation subsequent to hollow‐fiber filtration. The high m.w. form can be converted to the low m.w. form by proteolysis, reduction and alkylation, or chromatography in a dissociating solvent. The low m.w. form remains as such, even when reconcentrated in fetal calf serum‐containing medium. The high m.w. form thus likely consists of a complex between low m.w. IL‐1 and another protein secreted by the P388D1 cell line.
Active proteinase inhibitors associated with human breast epithelial cells
Authors: Gendler Sandra J. , TőKés Zoltán A.
Published: December 2004

The major glycoproteins synthesized by human breast epithelial cells have been characterized [6,8]. The most consistently observed and prominent component in supernatants of organ cultures of breast surgical specimens and of MCF‐7 cells was gp 68 which has been immunologically identified as α‐1‐antichymotrypsin (Achy). In the present study we demonstrate that this glycoprotein can form an irreversible complex with chymotrypsin, which indicates that it is a functional inhibitor. The 14C‐glucosamine‐labeled gp 68 forms a stable, 88,000‐dalton. enzyme‐inhibitor complex with chymotrypsin. The molecule is secreted continuously for 9 days into a chemically defined, serum‐free medium. In addition to the de novo synthesized inhibitor, another component is adsorbed from fetal bovine scrum and subsequently released into serum‐free medium. This component also forms an irreversible, 88,000‐dalton complex with enzyme. The observations establish that two types of inhibitors are associated with human breast epithelial cells, one actively synthesized and the other derived from serum. Both of these molecules may have significant roles in stabilizing cell surface components and in protecting extracellular matrices from untimely degradation.
Dimethyl sulfoxide decreases specific EGF binding
Authors: Earp H. Shelton , Blaisdell Joyce , Lin Qixiong
Published: December 2004

Dimethyl sulfoxidc (DMSO) stimulates tyrosine phosphorylation of the hepatic EGF receptor in isolated membrane preparations. To determine whether DMSO affects EGF binding, primary cultures of rat hepatocytes were incubated with 1–10% DMSO for 30 min prior to the addition of 125I‐EGF. DMSO (1–2%) reduced specific 125I‐EGF binding; the effect was maximal (a 40–60% reduction) at 5–7.5% DMSO and was reversed by removing the DMSO. Scatchard analysis showed that the reduction in binding was due to a change in receptor affinity. The decrease in binding was not seen when other, slightly less polar, solvents (eg, acetone and ethanol) were tested. DMSO also reduced 125I‐EGF binding to purified rat liver plasma membranes. This reduction was seen in the absence of added ATP and in membranes that had been pretreated with TLCK, a tyrosine kinase inhibitor. Thus, completion of the receptor autophosphorylation reaction was not necessary to effect the change. The data arc consistent with a DMSO‐induced alteration of receptor conformation that rcversibly reduces receptor affinity.
Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:Sugar phosphotransferase system
Authors: Saier Milton H. , Grenier Frank C. , Lee Catherine A. , Waygood E. Bruce
Published: December 2004

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non‐sugar‐specific phospho‐transfer protein called Enzyme I, two small non‐sugar‐specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar‐specific Enzymes II or Enzyme II‐III pairs which are phosphorylated at the expense of phospho‐HPr or phospho‐FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose‐specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of (1) PTS protein sequence data; (2) structural analysis of operons encoding proteins of the PTS; (3) genetic regulatory mechanisms controlling expression of these operons; (4) enzymatic characteristics of the PTS systems; (5) immunological cross reactivities of these proteins; (6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; (7) the nature of the phosphorylated protein intermediates; (8) molecular weight comparisons among the different Enzymes II and Enzyme II‐III pairs; and (9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.
PDGF Stimulates transient phosphorylation of 180,000 dalton protein
Authors: Harrington Maureen A. , Estes John E. , Leof Edward , Pledger W. Jack
Published: December 2004

Cell‐free extracts of platelet‐derived growth factor (PDGF) treated, density‐arrested, quiescent BALB/c‐3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P‐40 solubilized cell preparations that had been incubated with [γ‐32P]ATP. When quiescent BALB/c‐3T3 cell cultures were incubated at 37°C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3‐min exposure of the intact cells to PDGF, which was maximal after 10–15 minutes and had diminished by 30–60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4°C: however, in contrast to PDGF exposure at 37°C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4°C. When cells exposed to PDGF at 4°C were transferred to 37°C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37° C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions.

The amount of the stimulation PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF‐stimulated phosphorylation activity in relation to the cell cycle and growth regulation.
The structure of a small collagenous fragment isolated from chicken hyaline cartilage
Authors: Mayne Richard , van der Rest Michel , Weaver Darrel C. , Butler William T.
Published: December 2004

In previous experiments, two collagenous fragments were isolated from pepsin digests of chicken hyaline cartilage and called the high molecular weight. (HMW) and low molecular weight (LMW) fractions [3]. In the present experiments, the chains of LMW were isolated after denaturation and subsequent reduction and alkylation of interchain disulfide bridges and were further fractionated by carboxymethyl‐cellulose chromatography. Four peaks were resolved during chromatography and were designated LMW 1, 2A, 2B, and 3. Amino acid analyses and peptide mapping after cleavage with trypsin, V8 protease, and cyanogen bromide showed that three genetically distinct chains must be present in LMW. Fractions 2A and 2B were very similar, but not identical, in structure. LMW 1, 2A plus 2B, and 3 were consistently isolated in approximately equal proportions, suggesting that the probable chain organization of LMW is [1][2A + 2B][3]. This suggestion was supported further by experiments that attempted to fractionate LMW by carboxymethyl‐cellulose chromatography after denaturation but without reduction and alkylation of interchain disulfide bridges. No fractionation of LMW was achieved, the single peak subsequently being shown to contain LMW 1, 2A plus 2B, and 3.
Stimulation of pp60c‐src tyrosyl kinase activity in polyoma virus–infected mouse cells is closely associated with polyoma middle tumor antigen synthesis
Authors: Bolen Joseph B. , Lewis Andrew M. , Israel Mark A.
Published: December 2004

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product. pp60c‐src. The results of our studies demonstrate that infection of mouse cells with wild‐type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c‐src tyrosyl kinase activity. The level of pp60c‐src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c‐src kinase activity was not observed in mouse cells infected with transformation‐defective strains of polyoma virus. In examining the kinetics of pp60c‐src kinase stimulation in mouse cells at various times following wild‐type polyoma virus infection, we found that the level of pp60c‐src kinase activity correlated directly with the synthesis of polyoma virus‐encoded tumor antigens. By comparing wild‐type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c‐src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.
Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: Protein crosslinking studies
Authors: Painter Richard G. , Gaarde William , Ginsberg Mark H.
Published: December 2004

When intact platelets are incubated at 37°C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton‐insoluble cytoskeleton [4]. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2‐deoxy‐D‐glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA‐induced anchorage of GPIIb and III to the Triton‐insoluble cytoskeleton is a passive process requiring clustering of GPIIb‐III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane‐rich fractions at 37°C could induce association of GPIIb and III with a sedimentable actin‐rich, Triton‐insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA‐treated cells. Adding DNAse I, an actin depolymcrizing agent, into the Triton extraction buffer inhibited the ConA‐induced sedimentation of GPIIb‐III and actin by 50% in the presence of Mg2+‐ATP. Treatment of ConA‐treated membranes with dimethyl‐3,3′‐dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton‐insoluble crosslinked species of discrete molecular weights. When these crosslinked species were analyzed by SDS‐PAGE in the presence of β‐mercaptoethanol, they were found to be composed of a 180–200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction.

Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly ( < 10% of the total input GPIIb and III) but that the 180–200 k dalton protein and actin content were similar to that seen with ConA‐treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb‐III ( ∼ 90–95% of the total) around a small fraction of IIb‐III that is associated with a submembranous cytoskeleton.
Binding of antibodies in liposomes to extracellular matrix antigens
Authors: Torchilin V. P. , Klibanov A. L. , Ivanov N. N. , Gluckhova M. A. , Koteliansky V. E. , Kleinman H. K. , Martin G. R.
Published: December 2004

We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (<2%). The binding of antibody‐containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome‐antibody complex with the antigen was estimated at 1–10 × 10−9 M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.
Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity
Authors: Zaremba Sam , Keen James H.
Published: December 2004

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle‐free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain‐coated vesicles by extraction with 0.5 M TRIS‐HCl followed by Sepharose CL‐4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr ∼ 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500‐dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+‐induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr ∼ 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr ∼ 50,000 and 16,500 bands. This treatment abolished the in vitro coat‐forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr ∼ 100,000 band. Disappearance of the Mr ∼ 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr ∼ 76,000 and 65,000. When the elastase‐treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr ∼ 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr ∼ 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000‐dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.
Studies with digitonin‐treated rat hepatocytes (nude cells)
Authors: Katz Joseph , Wals P. A.
Published: December 2004

Isolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1–2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90–95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment.

Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt‐free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15–20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy‐requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt‐free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.
Actin‐associated cell‐surface glycoprotein from ascites cell microvilli: A disulfide‐linked multimer
Authors: Jung Goeh , Andrews David M. , Carraway Kermit L. , Carraway Coralie A. Carothers
Published: December 2004

Isolated microvilli of the MAT‐Cl subline of the 13762 rat mammary adenocarcinoma contain a transmembrane complex composed of a cell surface, cytoskeleton‐associated glycoprotein (CAG), actin, and a 58,000‐dalton polypeptide (58K). The behavior of CAG has been studied by differential centrifugation and velocity sedimentation gradient centrifugation of detergent extracts of microvilli. CAG can be pelleted along with a fraction of the microvillar actin even in the presence of ionic detergents and under microfilament‐depolymerizing conditions. By velocity sedimentation analysis CAG in Triton/PBS extracts sediments as a large, hetero‐geneous species (sedimentation coefficient > 25S). In Sarkosyl and sodium dodecyl sulfate (SDS) the size and heterogeneity are somewhat reduced. In SDS CAG sediments as a 20S species in the absence of mercaptoethanol and as a 5S species in the presence of mercaptoethanol. These results indicate that CAG is a disulfide‐linked multimer in the microvillus membrane. We suggest that the stable multimeric structure of CAG permits it to act as the membrane association site for several microfilaments and plays an important role in the formation and stabilization of the microvillus structure.
Transforming growth factor (TGF) activity in human urine: Synergism between TFG‐beta and urogastrone
Authors: Twardzik Daniel R. , Sherwin Stephen A.
Published: December 2004

Human urine contains an acid and heat stable peptide with an apparent molecular weight of 8,000–10,000 that, in the presence of urogastrone (EGF), induces the anchorage‐independent growth of nontransformed cells in semisolid media. This nonmitogenic growth‐modulating activity does not compete with EGF for binding to EGF membrane receptor sites and can be resolved from EGF by high‐performance liquid chromatography. The urine‐derived growth factor has been purified more than 10,000‐fold and shares many biochemical properties with and is functionally related to the B class of TGFs isolated from transformed cells and nonneoplastic tissues. The low molecular weight anchorage‐independent growth‐, stimulating activity universally present in human urine is a result of the synergistic interaction of this urine‐derived TGF‐beta and urogastrone.