bluuebunny/crossref_metadata_2025 huggingface dataset:
An approximate integral model for the burning rate of a thermoplastic‐like material
Authors: Quintiere James , Iqbal Naeem
Published: December 2004

An approximate integral model is formulated and solved to describe the pyrolysis or burning rate of a thermoplastic‐like material. A constant temperature gasification process is assumed to occur at the solid–atmosphere interface. The preheating ignition problem is also solved by a matching integral method. The ignition problem leads to a solution involving a non‐linear algebraic equation, but the gasification problem yields an exact solution provided the convective heat transfer coefficient is unaffected by the fuel mass loss or blowing effect. The results are compared to numerical solution in the literature and show good agreement. Comparisons with experimental data for PMMA are limited.
Experimental studies on the sensitivity of fire detectors
Authors: Chow W. K. , Wong William C. W.
Published: December 2004

Experimental studies were performed to assess the sensitivity and response times of four types of fire detectors: ionization smoke, optical smoke, infra‐red and rate of heat rise detectors. The detectors were installed in a fire chamber and subjected to ten types of fire under a controlled environment. Based on the experimental results, correlations between the fire properties and the response times of the detectors were found. Data collected on the predicted fire detector response time were analyzed by a statistical method. Those empirical expressions would be important for practising engineers in designing fire‐detection systems and setting responses for detectors in buildings of different uses.
The application of magnesium hydroxide as a fire retardant and smoke‐suppressing additive for polymers
Authors: Hornsby Peter R.
Published: December 2004

The use of magnesium hydroxide as a fire‐retardant additive in polymers is discussed in terms of its particle characteristics, thermal properties and factors influencing the mechanism of action. Consideration is also given to its role in reducing smoke evolution during polymer combustion.
Factors affecting reliability of light‐framing wood members exposed to fire—a critical review
Authors: Lau Peter W. C. , Barrett J. D.
Published: December 2004

This paper discusses the application of reliability‐based design concepts to the design of light structural framing wood members for fire safety. In reliability‐based designs we seek to ensure with high confidence that the member as designed will survive a ‘severe’ fire for a specified period. Reliability analysis expresses this confidence in a probabilistic context that allows different designs to be compared. The reliability of members loaded in tension has been formulated as a function of applied load, the member cross‐section size and initial strength, charring rate and strength degradation rate, of which the latter two are probably the most significant. These two factors may not be treated in the same manner as has been established for heavy timber design because of differences in material behaviour. The factors constituting the reliability function are discussed and their dependence on temperature is examined.
An overview of the fire retardant chemicals industry, past—present—future
Authors: Green Joseph
Published: December 2004

An overview of the fire retardant chemicals industry in the United States is given starting with an historical review of the chemical producers in about 1970. Marketing concepts for these specialty chemicals is discussed by customer types for each category of fire retardant. The fire retardants discussed are bromine and chlorine compounds, nonhalogen and halogenated phosphates and antimony oxide. The paper contains a section in how to select a fire retardant. Markets are discussed by fire retardant type and by polymer type. The current and future trends section discusses toxicity issues with bromine fire, retardants, non‐halogen fire retardants, smoke, processability and thin walled parts, and market projections.
Studies on magnesium hydroxide in polypropylene using simultaneous TG‐DSC
Authors: Larcey P. A. , Redfern J. P. , Bell G. M.
Published: December 2004

A simultaneous TG‐DSC system (STA‐625) has been used to investigate the suitability of using magnesium hydroxide (MH) as a flame retardant and smoke suppressant in polypropylene (PP) formulations. Several MH/PP formulations were examined at differing concentrations. The presence of MH in the system greatly alters the thermal degradation character of PP. This work forms the first in a series of application notes using various Theometric Scientific Thermal Analysis instruments.
An adaptive multigrid tool for elliptic and parabolic systems
Authors: Jones A. C. , Jimack P. K.
Published: December 2004

This paper describes a multigrid tool for use with adaptively refined meshes in two dimensions. A standard full approximation storage (FAS) multigrid scheme is modified to maintain efficient performance on non‐uniform grids arising as a result of local mesh refinement. This is achieved by allowing hanging nodes to exist but ensuring that at each stage of the FAS algorithm the solution is projected into the continuous space in which the hanging node values are interpolants of their coarse grid parent values. A non‐linear, elliptic, finite element example is provided to demonstrate the efficiency of the proposed algorithm. However, the method is also suitable for linear and non‐linear parabolic problems. Copyright © 2004 John Wiley & Sons, Ltd.
Contact damage in brittle coating layers: Influence of surface curvature
Authors: Qasim Tarek , Bush Mark B. , Hu Xiaozhi , Lawn Brian R.
Published: December 2004

Fracture from indentation by a hard sphere on bilayer systems composed of curved brittle coating layers on compliant polymeric substrates is investigated, in simulation of dental crown structures. Glass plates 1 mm thick are used as representative of enamel/crown layers, and epoxy filler substrates as representative of support dentin. Specimens with curved surfaces are prepared by pressing the glass plates onto steel sphere dies with radius of curvature down to 4 mm, to reflect common occlusal geometries. The influence of curvature on the conditions to initiate and propagate subsurface “radial” cracks, widely believed to be the principal failure mode in ceramic‐based dental crowns, is studied. Finite element calculations are used to evaluate stress states in the specimens. It is shown that surface curvature can play an important role in the radial crack evolution, initially by inhibiting initiation but subsequently, in the case of convex curvature, by strongly enhancing propagation to failure. Implications concerning the design of ceramic‐based dental crowns are considered. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 73B: 179–185, 2005
Selective in vitro transcription of chloroplast genes
Authors: Gruissem Wilhelm , Narita Jonathan O. , Greenberg Bruce M. , Prescott David M. , Hallick Richard B.
Published: December 2004

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA‐dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA‐sized products have been detected after transcription. The tRNA‐sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA‐sized products have been identified from transcription of a trnY1‐trnH1‐trnM1‐trnE1‐trnW1‐trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast‐extract can recognize a polycistronic tRNAVal‐tRNAAsn‐tRNAArg precursor and process it into tRNA‐sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5′ flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.
Pyrazolopyrimidine metabolism in Leishmania and Trypanosomes: Significant differences between host and parasite
Authors: Marr J. Joseph
Published: December 2004

The pathogenic hemoflagellates of the genera Leishmania and Trypanosoma are major causes of human disease in the tropical and subtropical areas of the world. In general, the agents used to treat diseases caused by these organisms are toxic and not suitable for administration to the millions of people infected. Investigations over the past several years have shown that there are several major differences between man and these protozoans with respect to purine metabolism. The differences appear to offer promise for the development of effective chemotherapeutic compounds. These organisms do not synthesize purines de novo, as does man. They are able to concentrate pyrazolopyrimidines within the cell and metabolize them as purines through the salvage pathways, ultimately incorporating them into nucleic acids. This does not occur in mammals. The pyrazolopyrimidine base allopurinol, which has served as a prototype, is activated by a phosphoribosyltransferase to the ribonucleotide. The ribonucleotide is aminated to the 4‐amino‐pyrazolopyrimidine ribonucleotide and subsequently phosphorylated to the triphosphate form and incorporated into RNA. The pyrazolopyrimidine ribonucleosides formycin B and allopurinol ribonucleoside are activated through a nucleoside phosphotransferase. The resulting ribonucleotide is aminated and incorporated into RNA as described above. These metabolic peculiarities occur not only in the forms of these parasites which are found in the insect vectors but also in the intracellular forms which are pathogenic in man. The differences in the enzymology and metabolism of purines which exist in the genera Leishmania and Trypanosoma offer excellent opportunities for chemotherapeutic exploitation.
Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K‐12
Authors: Sutcliffe Joyce A. , Foulds John D.
Published: December 2004

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli KI strain to an E coli K‐I2 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to “authentic” protein K when chymotryptic peptides of 125I‐labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.
Alterations in the transport and processing of Rous sarcoma virus envelope glycoproteins mutated in the signal and anchor regions
Authors: Wills John W. , Hardwick J. Marie , Shaw Karen , Hunter Eric
Published: December 2004

The env gene of Rous sarcoma virus codes for two glycoproteins which are located on the surface of infectious virions. Subcloning of these coding sequences in the place of the late region of SV40 DNA has allowed the expression of a normally glycosylated, functionally active glycoprotein complex on the surface of monkey cells. Through the use of site‐directed mutagenesis, the role of specific amino acids in the signal peptide, signal peptidase cleavage site, and membrane anchor region have been investigated. Amino‐terminal mutations have shown that deletion of the signal peptidase cleavage site along with one or two amino acids of the hydrophobic signal peptide results in the synthesis of an unglycosylated. uncleaved, and presumably cytoplasmically located precursor. Nevertheless, changing the signal peptidase cleavage site from ala/asp to ala/asn does not block the translocation of the glycoprotein across the membrane or the action of the peptidase. At the other end of the molecule, carboxy‐terminal mutations have shown that the deletion of the hydrophobic membrane anchor region is not sufficient for the secretion of the truncated glycoprotein. Interpretations of these results based on recent models for protein transport and secretion are discussed.
Pigment‐protein complexes of purple photosynthetic bacteria: An overview
Authors: Thornber J. Philip , Cogdell Richard J. , Pierson Beverly K. , Seftor Richard E. B.
Published: December 2004

A minireview of antenna and reaction center pigmenl‐protein complexes of purple bacteria is presented. Advances in our knowledge of their structure and composition during the past 3 yr are emphasized and some new thoughts are introduced.
Genetic control of resistance to murine malaria
Authors: Stevenson Mary , Lemieux Suzanne , Skamene Emil
Published: December 2004

Strain variation in the level of resistance to malaria was investigated in inbred mice after infection with Plasmodium chabaudi. Following intraperitoneal infection with the typing dose of parasitized erythrocytes, mice of 11 inbred strains could be separated using survival time as the criterium into resistant and susceptible groups. Genetic analysis of F1 hybrid and backcross progeny derived from one of the most resistant (B10.A) and from the most susceptible (A/J) strains as parents suggested that host resistance in this strain combination was genetically controlled by a dominant, non‐H‐2‐linked, autosomal gene or closely linked genes. Analysis of the mechanisms of resistance to P chabaudi showed (1) phenotypic expression of the resistance gene was apparent within 6 days of infection as a significant difference between resistant and susceptible mice in the level of parasitemia; (2) the level of host NK cell activity was not related to the level of host resistance to malaria; (3) compared with susceptible A/J mice, resistant B1O.A hosts had an augmented erythropoietic response during the course of malaria as well as during phenylhydrazine‐induced anemia and (4) treatment with BCG or P acnes resulted in an equal degree of protection, measured by parasitemia and survival, in both resistant and susceptible mice.
The effect of phenformin and other adenosine triphosphate (ATP)‐lowering agents on insulin binding to IM‐9 human cultured lymphocytes
Authors: Vigneri Riccardo , Maddux Betty , Goldfine Ira D.
Published: December 2004

In the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM‐9 human cultured lymphocytes. After a 24‐hr preincubation, phenformin induced a twofold increase in specific 125I‐insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I‐insulin binding and energy metabolism in IM‐9 cells was investigated. The phenformin‐induced increase in insulin binding to IM‐9 cells was related to a time‐ and dose‐dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2‐deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM‐9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.
Role of membrane potential in protein folding and domain formation during secretion in escherichia coli
Authors: Copeland Bruce R. , Landick Robert , Nazos Penelope M. , Oxender Dale L.
Published: December 2004

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine‐isoleucine‐valine binding protein and the leucine‐specific binding protein are synthesized as precursors with 23 amino acid N‐terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine‐isoleucine‐valine binding protein, it and the closely related leucine‐specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N‐terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans‐positive side of the membrane, thus providing an additional role for the transmembrane potential.
The role of membrane lectins in dictyostelium discoideum aggregation as ascertained by specific univalent antibodies against discoidin I
Authors: Cano Amparo , Pestaña Angel
Published: December 2004

Antibodies against pure discoidin I have been used as a tool to ascertain the role of this lectin in aggregation of Dictyostelium discoideum. Discoidin I is widely expressed over the cell surface of aggregation‐competent AX‐2 cells, as ascertained by indirect immunofluorescence with specific (antidiscoidin I) antibodies. Univalent antidiscoidin I antibodies (Fab fragments) inhibit the aggregation‐specific intercellular adhesion of D discoideum AX‐2 cells in an in vitro assay. This inhibition depends on antibody concentration and cell density; a 50% inhibition of cell aggregation was obtained at antidiscoidin I Fab concentration of 4.5 mg/ml and 1 × 106 cells/ml. Aggregation and morphogenesis on solid support is also effectively inhibited when AX‐2 cells are starved in the presence of antidiscoidin I Fab fragments. The inhibition of morphogenesis is also dose dependent and more effective than in the in vitro assay. No inhibition of aggregation either in the in vitro assay or on morphogenesis on solid support was observed with preimmune Fab fragments at any of the concentrations tested (up to 9.6 mg/ml).
Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells
Authors: Minetti Maurizio , Ceccarini Marina , Maria Anna , Di Stasi M.
Published: December 2004

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryoheniolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryoheniolysis of red blood cells shows critical temperatures at 7°C and 19°C. In hypotonic solution, the osmotic resistance increases near 10°C and levels off above 20°C. EPR studies of red blood cell membrane of a 16‐dinyloxyl stearic acid spin label show, in the 0–50°C range, the presence of three thermotropic transitions at 8, 20, and 40°C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryoheniolysis and reduce the effect of temperature on hypotonic hemolysis. 16‐Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8°C transition. Both the 8°C and the 20°C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.
Calmodulin, a calmodulin acceptor protein, and calcimedins: Unique antibody localizations in hamster sperm
Authors: Moore Pamela B. , Dedman John R.
Published: December 2004

A calmodulin acceptor protein has been identified in isolated hamster caudal sperm by immunofluoresence and Western transfer techniques. The protein shows a localization in sperm heads identical to calmodulin. Fluorescence of both calmodulin and the acceptor protein are lost by treatment with MgCl2, conditions which release the acrosome. These results are consistent with the proposed function of calmodulin in a sperm function.
Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases
Authors: Blum Jacob J. , Hayes Alvernon
Published: December 2004

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris‐0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin‐activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin‐activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin‐activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the “light” 30S dynein ATPase fractions are more activated than the “heavy” 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin‐activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.
Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane
Authors: Weber Jakob , Warden Dean Allan , Semenza Giorgio , Diedrich Donald F.
Published: December 2004

Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein‐depleted ghosts, or detergent‐treated ghosts. D‐glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein‐depicted ghosts, the selective Solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC‐720 extracts could be prepared in which the glucose transporter retained activity for days at 4°C. These extracts were applied to affinity chromatography matrices of phloretin‐Agarose, prepared by coupling phloretinyl‐3′‐benzylamine (PBA) to CH‐Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4. 5, from the Affigel affinity medium.
Membrane‐cytoskeleton interactions and the regulation of chemotactic peptide‐induced activation of human granulocytes: The effects of dihydrocytochalasin B
Authors: Jesaitis Algirdas J. , Tolley James O. , Painter Richard G. , Sklar Larry A. , Cochrane Charles G.
Published: December 2004

When N‐formyl chemotaclic peptides bind to granulocyte receptors at 37°C they rapidly form a high‐affinity ligand‐receptor complex whose coisolation with cyto‐skeletal residues of Triton X‐100–extracted cells is under cellular control [Jesaitis et al: J Cell Biol 98:1378, 1984]. Experiments were performed to investigate the significance of this coisolation. When the granulocytes were preincubated with dihydrocytochalasin B (dhCB) for 10 min at 37°C and then stimulated with 50 nM N‐formyl‐Met‐Leu‐[3H]Phe, the rate of uptake of the radioligand by the cells was inhibited. Colocalization of the retained peptide with the Triton X‐100 fraction of these cells was also reduced relative to this fraction of the untreated cells. This inhibition was apparent before the onset of FMLP endocytosis. The inhibition was 50% effective at 0.25 μg dhCB/ml. Maximal inhibition (80–90%) occurred at doses of dhCB > 1 μg/ml. The 90% retention of two plasma membrane markers by the cytoskeleton was marginally affected. These results support the hypothesis that coisolation of the high‐affinity receptor‐peptide complexes with granulocyte cytoskeletons occurs because of specific association of the complexes with the cytoskeleton at the cell surface. In addition, since these events precede internalization, they suggest that formation of the association between the ligand‐receptor complex and cytoskeleton may be necessary for ligand‐receptor endocytosis. Experiments were also performed to evaluate other functional consequences of cytoskeletal disruption on chemotactic peptide‐stimulated functions. f‐Met‐Leu‐Phe stimulation of O production was potentiated due to prolongation of and increase in the rate of O production. This potentiation has the same dose dependency as the inhibition of receptor modulation. The possible relationship of these various functions is discussed.
Activators of protein kinase C and 5‐azacytidine induce IL‐2 receptor expression on human T lymphocytes
Authors: Depper Joel M. , Leonard Warren J. , Drogula Cynthia L. , Krönke Martin , Waldmann Thomas A. , Greene Warner C.
Published: December 2004

Resting human T lymphocytes do not express receptors for interleukin‐2, but expression is rapidly induced by exposure to PHA. After maximal expression 2–3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL‐2 receptor expression. Moreover, 5‐azacytidinc, which inhibits cytosine methyltransferase, and hydroxy‐urea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL‐2 receptor expression can be altered in vitro, and that IL‐2 receptor number, in combination with IL‐2 secretion, may contribute to the regulation of IL‐2–dependent immune responses.
Transferrin receptor induction is required for human B‐lymphocyte activation but not for immunoglobulin secretion
Authors: Neckers L. M. , Yenokida G. , Trepel J. B. , Lipford E. , James S.
Published: December 2004

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogenstimulated T cells (probably B‐cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B‐cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA‐treated T‐cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen‐stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B‐cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B‐cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell‐derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.
Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture System
Authors: Herschman Harvey R. , Brankow David
Published: December 2004

Unlike 12‐O‐tetradecanoylphorbol‐l3‐acctate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.
Interaction of very low density lipoprotein with chicken oocyte membranes
Authors: Krumins Solveig A. , Roth Thomas F.
Published: December 2004

The interaction of hen 125I‐VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7–9.1 × 10‐8 M or 43.5–45.5 μg VLDL protein/ml. Binding capacity was 2.0 μg VLDL protein/mg membrane homogenate protein. The apparent rate constants were, k1 = 2.4 × l05 M‐1min‐1 and k2 = 2.1 × 10‐2 min‐1. Specific binding required the presence of divalent cations. Whereas binding was completely re‐stored after treatment with EDTA by the addition of Mn++, only 60% of binding was restored using Ca++.
Differential effects of polyamines on rat thyroid protein kinase activities
Authors: Levasseur Steven , Henricks Lorraine , Poleck Thomas , Friedman Yochanan , Burke Gerald
Published: December 2004

Ornithine decarboxylase, the rate‐limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic‐AMP‐dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6–2.4‐fold increase in cytosolic cyclic‐AMP‐independent (messenger‐independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger‐independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger‐independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid‐dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin‐regulated ornithine decarboxylase activity in thyroid.
Tubulin‐colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end
Authors: Bergen Lawrence G. , Borisy Gary G.
Published: December 2004

The effects of colchicine and tubulin‐colchicine complex (TC) on microtubule depolymerization were studied using the axoneme‐subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141–150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5–100 μM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4 : 1–1 : 1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC‐affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.
Spectrin involvement in a 40°C structural transition of the red blood cell membrane
Authors: Minetti Maurizio , Ceccarini Marina , Di Stasi Anna Maria M. , Petrucci Tamara C. , Marchesi Vincent T.
Published: December 2004

Proteins involved in a structural transition detected in red blood cell membranes at 40°C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40°C transition detected in ghosts by both a stearic acid spin label (16‐doxyl stearic) and a sulfhydryl‐specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25‐30°C and a massive transition with an onset temperature of 48 and 40°C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40°C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti‐4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1 : 1 molar ratio. The involvement in this transition of a localised spectrin domain(s) containing few exposed sulfhydryl groups is proposed.
Hydrophobicity and amphiphilicity in protein structure
Authors: Eisenberg David , Wilcox William , McLachlan Andrew D.
Published: December 2004

The importance of the hydrophobic interaction in stabilizing native protein structure has long been appreciated. However, more than other component forces, this one has resisted quantitative description. We present two approximate methods of assessing the hydrophobic component to the free energy of protein folding. Both are expressed in terms of what can be called hydrophobic moments of the protein. The first method is intended to yield an approximate value for the hydrophobic energy. This energy is calculated from a set of atomic coordinates in terms of the hydrophobicity (or 0th hydrophobic moment) of each amino acid residue and its accessibility or lack of it to aqueous solvent. The second method considers the first moment of the hydrophobicity of a group of residues, the hydrophobic moment. Segments of secondary structure in folded proteins tend to have hydrophobic moments that oppose each other. For example, α‐helices on the protein surface tend to have one hydrophobic face and one hydrophilic face, with the hydrophilic face out towards the solvent. This pattern of organization is often apparent from a computer model of the protein that shows the magnitude and direction of the hydrophobic moment of each segment of secondary structure. Examples are given for the incorrectly folded structures of Novotný et al [J Mol Biol 177:787, 1984] and for the correct structures to which they correspond.
Relationship between protease activity and a sialoglycopeptide inhibitor isolated from bovine brain
Authors: Sharifi B. G. , Bascom C. C. , Fattaey H. , Nash S. , Johnson T. C.
Published: December 2004

We have recently described the isolation and purification to homogeneity of a new Sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the Sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6–8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequence.
Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells
Authors: Krachmarov Chavdar , Iovcheva Christina , Hancock Ronald , Dessev George
Published: December 2004

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single‐stranded DNA and pulse‐labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited.

When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed.

Preparations of purified, high‐molecular weight, double‐stranded DNA contain variable amounts of fast‐sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation.

These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts.

Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average, length practically all DNA was released. The last 1–4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters.

At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA.

It was shown, however, that digestion of nuclear lamina‐DNA complex with EcoRI or Hae III led to the formation of DNA‐protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.

These results argue against the existence of direct tight bonds between DNA and nuclear lamina in vivo but demonstrate that such bonds can be generated under certain conditions in vitro.
Antipeptide antibodies directed against the carboxy‐terminal region of SV40 structural proteins VP2 and VP3
Authors: Streckert Hans‐Jürgen , Brüssow Harald , Sure Klaus , Werchau Hermann
Published: December 2004

Rabbits were immunized with a synthetic heptapeptide of the sequence Arg‐Asn‐Arg‐Ser‐Ser‐Arg‐Ser corresponding to the carboxy‐terminal region of the SV40 viral proteins VP2 and VP3. The raised antibodies recognize the viral proteins in enzyme‐linked immunosorbent (ELISA) and Western blot assay. Specificity of the antibodies were confirmed by competition experiments. The antibodies recognize VP2 and VP3 in infected cells by immunofluorescence and in subcellular fractions by ELISA. No interaction with virions was observed.
Differential expression of metastasis‐associated cell surface glycoproteins and mRNA in a murine large cell lymphoma
Authors: Nicolson Garth L. , LaBiche Ronald A. , Frazier Marsha L. , Blick Mark , Tressler Robert J. , Reading Christopher L. , Irimura Tatsuro , Rotter Varda
Published: December 2004

A metastatic variant cell subline of the Abelson virus‐transformed murine large lymphoma/lymphosarcoma RAW 117 has been selected in vivo ten times for liver colonization. Highly metastatic subline RAW117‐H10 forms greater than 200 times as many gross surface liver tumor nodules as the parental line RAW117‐P. Analysis of cellular proteins and glycoproteins indicates reduced expression of murine Moloney leukemia virus‐associated p15, p30, and gp70, and increased expression of a sialoglycoprotein, gp150, in the highly metastatic H10 cells. Northern analyses of oncogene expression suggested that mRNA of various oncogenes was expressed equally or not expressed in the RAW117 cells of differing metastatic potential. Differential gene expression was examined using a cDNA library of 17,600 clones established from poly A+ mRNA isolated from H10 cells. The cDNA library was screened by the colony hybridization technique using probes made from both RAW117‐P and ‐H10 cells. Approximately 99.5% of these cDNA clones were expressed identically in P and H10 cells. Of the few differentially expressed cDNA clones (approx. 150/17,600), one‐half of these were identified as Moloney leukemia virus sequences in a separate probing with a radiolabeled Moloney leukemia virus probe. The remainder of the differentially expressed mRNA detected by colony hybridization of the cDNA library were expressed at higher levels (approx. 1/6) or lower levels (approx. 1/3) in the highly metastatic H10 cells.
Partial purification of microsomal signal peptidase from hen oviduct
Authors: Baker R. Keith , Bentivoglio Gian Paolo , Lively Mark O.
Published: December 2004

Signal peptidase has been purified approximately 600‐fold from hen oviduct microsomes. Treatment of microsomes with ice‐cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P‐40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.
Effects of monensin on vesicular transport pathways in the perfused rat liver
Authors: Kloppel Thomas M. , Brown William R. , Reichen Juerg
Published: December 2004

In the rat hepatoctye, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I‐asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I‐ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I‐ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H‐leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA‐precipitable 3H‐protein appeared in bile about 20 min before TCA‐precipitable 3H‐protein appeared in the perfusate in monensin‐treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.
Molecular cloning of a tumor promoter‐inducible mRNA found in JB6 mouse epidermal cells: Induction is stable at high, but not at low, cell densities
Authors: Smith James H. , Denhardt David T.
Published: December 2004

From the mouse JB6 epidermal cell line C122 we have isolated a cDNA clone representing a 1.6‐kilobase mRNA, called 2ar, that exhibits a biphasic induction in response to 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). The first phase of induction in subconfluent cells is transient, peaking at 6 h after the addition of TPA and returning to noninduced levels by 24 h. When the cells reach plateau density, in the continued presence of TPA, this mRNA is reinduced and remains so upon continued exposure to the tumor promoter. Serum and certain growth factors also induce 2ar mRNA in serum‐deprived quiescent fibroblasts. In vitro nuclear “run‐on” transcription experiments indicate that the induction of 2ar mRNA is controlled at the transcriptional level.