bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of solid char residues after thermal degradation of polystyrene, PVC and polyamide—Part II
Authors: Ševěček P. , Stužka V.
Published: December 2004

Structural residues of the original polymer remain in the char portion of degraded polymer depending on the temperature and duration of the thermal degradation and the temperature. Elementary analysis and infrared spectroscopy have been used for the identification of char residues.
The use of the ISO/TC92 test for ignitability assessment
Authors: Hu XiFang , Clark Ferrers R. S.
Published: December 2004

The ignitability of solids, including fire‐retardant‐containing polystyrene, is reported using results of a small‐scale thermal radiation exposure test (a modified ISO ignitability test procedure). Additional information is provided from the results of exposure to convective heating and from oxygen index determinations. The use of a permanent sample mask and smaller samples than described in the ISO procedure proved convenient. The ISO procedure was found useful for determining the response of ignition time to changes in radiant flux. Good agreement was found with the analysis of Quintiere and Harkleroad for most samples but not with polystyrene, with and without halogen fire retardant. The increase in ignition resistance with fire retardant concentration suggested by the oxygen index is not always consistent with the ignition delay times under radiative or convective assault.
Effect of environmental conditions on smoke propagation
Authors: McIlhagger R. , Hill B. J. , Brown A. J.
Published: December 2004

This paper examines the influence of oxygen and environmental temperature on the generation of smoke from burning polystyrene sheet. The experimentation was performed using a Stanton Redcroft HFTA linked to a Stanton Redcroft FTB Smoke Box. The results showed that different forms of smoke were formed depending upon the environmental conditions. For example, in oxygen‐enriched atmospheres and at elevated environmental temperatures, as in spacecraft fires, large smoke particles were formed with a lower level of obscuration. This phenomenon was accompanied by higher rates of burn and high fire temperatures. At lower oxygen contents, as in real‐life fire conditions, close to the Critical Oxygen Index, finer smoke particles were formed which gave a higher level of obscuration. The time to maximum obscuration was comparatively long.
The combustion of cigarette paper
Authors: Baldry P. J. , Cullis C. F. , Goring D. , Hirschler M. M.
Published: December 2004

Studies have been carried out of the thermal decomposition and combustion of cigarette paper with heating rates and final temperatures similar to those encountered during the smoking of cigarettes. Measurements have been made in particular of the amounts of carbon monoxide formed from the paper, both on its own and when it forms part of a cigarette. The extent of formation of char from cigarette paper decreases as the heating rate is increased, but carbon monoxide yields pass through a maximum at a heating rate of ca 60° s−1, due to the competing effects of temperature on the several different mechanisms by which carbon monoxide is produced. Measurements on cigarette papers impregnated with a wide range of inorganic and organic salts show that all the additives used tend to decrease carbon monoxide yields at low heating rates but to increase them quite sharply at very high ones. With cigarettes containing impregnated papers the concentration of carbon monoxide is increased in both the mainstream and sidestream smoke. Possible reasons for the behaviour observed with the various individual salts are discussed. Certainly, on the assumption that the results obtained with cigarettes are a good reflection of those on paper alone it seems unlikely that treatment of cigarette paper can ever reduce the amounts of carbon monoxide formed under cigarette smoking conditions, for all additives affect char formation in such a way that there is an overall increase in carbon monoxide yields.
One‐dimensional mathematical modelling of enclosure fire dynamics
Authors: Gupta A. K. , Kumar Surendra , Singh Bani
Published: December 2004

This paper presents a one‐dimensional mathematical model of enclosure fire dynamics. The model is based on the fundamental equations of mass, momentum and energy and uses the top hat velocity and temperature profiles for the movement of plume gases. A computational algorithm is also developed to solve the set of transient model partial differential equations. In addition, the explicit equations have been derived for computing the steady state velocity and temperature profiles. These derivations are based on the assumption that the plume width follows the relationship given by Eqn (12). Further, these relationships help to identify a dimensionless parameter, Frs, known as source Froude number, which has a significant effect on the behaviour of the plume gases. It may be noted that this assumption is only applicable for the case of Frs = 1. The plume behaviour is studied with reference to three cases of source Froude numbers, i.e. Frs < 1, Frs = 1, Frs > 1.
Flame spread properties of forest products. Comparison and validation of prescribed Australian and North American flame spread test methods
Authors: Gardner W. D. , Thomson C. R.
Published: December 2004

Nineteen forest products in sawn board and panel form (including three fire retardant treated) have been tested to AS1530.3—Test For Early Fire Hazard Properties of Materials and ASTM E84—Standard Test Method for Surface Burning Characteristics of Building Materials. These tests are used in the building codes to regulate the flame spread properties of all combustible building materials in Australia and interior finsihes in North America, respectively. A correlation was established between Time to Ignition (AS1530.3) and Flame Spread Index (ASTM E84) for the forest products that had not been fire retardant treated. Room fire testing of eight of the forest products validaded Time to Ignition (AS1530.3) and Flame Spread Index (ASTM E84) as reliable test criteria for regulating the use of combustible materials.
The flammability of polyacrylonitrile and its copolymers IV. The flame retardant mechanism of ammonium polyphosphate
Authors: Zhang J. , Horrocks A. R. , Hall M. E.
Published: December 2004

Ammonium polyphosphate is shown to be an effective flame retardant for homopolymeric and selected fibre‐forming copolymers of acrylonitrile. Compared with other phosphates and phosphorus‐containing species, it significantly increases both limiting oxygen index and char levels. The mechanism of retardancy is shown to be both physical and chemical in character. When heated to 300°C, the polyphosphate melts and converts to polyphosphoric acid which acts as a physical barrier to surface polymer oxidation and promotes nucleophilic oligomerization of pendant, adjacent nitrile groups. A reduction in the activation energy of this first stage of acrylic polymer degradation suggests that the favoured decomposition route gives rise via oligomerization to char‐promoting precursors. Char analyses indicate an empirical formula of C30H13N7P2, which compares favourably with that of a proposed phosphorylated, polynuclear, aromatic heterocyclic structure.
Quantifying the ignition propensity of cigarettes
Authors: Ohelemiller Thomas J. , Villa Kay M. , Braun Emil , Rberhardt Keith R. , Harris Richard H. , Lawson J. Randall , Gann Richard G.
Published: December 2004

Research funded under the Fire Safe Cigarette Act of 1990 (United States Public Law 101–352) has led to the development of two test methods for measuring the ignition propensity of cigarettes. The Mock‐Up Ignition Test Method uses substrates physically similar to upholstered furniture and mattresses: a layer of fabric over padding. The measure of cigarette performance is ignition or non‐ignition of the substrate. The Cigarette Extinction Test Method replaces the fabric/padding assembly with multiple layers of common filter paper. The measure of perfomance is full‐length burning or self‐extinguishment of the cigarette. Routine measurement of the relative ignition propensity of cigarettes is feasible using either of the two methods. Improved cigarette performance under both methods has been linked with reduced real‐world ignition behavior; and it is reasonable to assume that this, in turn, implies a significant real‐would benefit. Both methods have been subjected to interlaboratory study. The resulting reproducibilities were comparable to each other and comparable to those in other fire test methods currently being used to regulate materials which may be involved in unwanted fires. Using the two methods, some current commercial cigarettes are shown to have reduced ignition propensities relative to the current best‐selling cigarettes.
Progressive optimization on unstructured grids using multigrid‐aided finite‐difference sensitivities
Authors: Catalano L. A. , Dadone A. , Daloiso V. S. E.
Published: December 2004

This paper proposes an efficient and robust progressive‐optimization procedure, employing cheap, flexible and easy‐to‐program multigrid‐aided finite‐differences for the computation of the sensitivity derivatives. The entire approach is combined with an upwind finite‐volume method for the Euler and the Navier–Stokes equations on cell‐vertex unstructured (triangular) grids, and validated versus the inverse design of an airfoil, under inviscid (subsonic and transonic) and laminar flow conditions. The methodology turns out to be robust and highly efficient, the converged design optimization being obtained in a computational time equal to that required by 11–17 (depending on the application) multigrid flow analyses on the finest grid. Copyright © 2004 John Wiley & Sons, Ltd.
Voltage‐dependent orientation of membrane proteins
Authors: Blumenthal Robert , Kempf Christoph , Van Renswoude Jos , Weinstein John N. , Klausner Richard D.
Published: December 2004

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane‐active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans‐positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans‐negative electrical potential, melittin appears to assume a transbilayer position.

These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside–negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.
A monoclonal antibody recognizes an epitope common to an avian‐specific nuclear antigen and to cytokeratins
Authors: Franklin Richard M. , Emmons Lyman R. , Emmons Rebecca P. , Kai Osamu , Oommen Anna , Pink J. Richard , Rijnbeek Anne‐Marie , Schnetzler Marianne , Tuderman Leena , Vainio Eeva
Published: December 2004

X3, a monoclonal antibody of unusual specificity, is described. This antibody reacts with one or more cytokeratin polypeptides and also reacts with an avian (chicken, quail) nuclear antigen that appears to be present in all cell types (chicken) tested, although with variable staining pattern and intensity. This antigen is distinct from the cytokeratins but does have an epitope in common with this class of proteins. It disappears from the nucleus during the early stages of cell division and reappears during anaphase as a granular cytoplasmic structure. In late telophase the antigen is relocated in the nucleus. This antigen, which we have designated as avian‐specific nuclear antigen (AVNA), is not associated with chromatin or ribonucleoproteins. From immunoblotting experiments on chicken fibroblast nuclei, AVNA is probably a complex composed of one or several polypeptides, one of which has a molecular weight of approximately 60 kD. The proteins were identified as nuclear matrix proteins rather than pore complex‐lamina proteins by immunoblotting experiments on the purified nuclear matrix of chicken erythrocytes. The major polypeptide had a molecular weight of 60 kD and the minor polypeptide a molecular weight of 69 kD.
Golgi/granule processing of peptide hormone and neuropeptide precursors: A minireview
Authors: Steiner Donald F. , Docherty Kevin , Carroll Raymond
Published: December 2004

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage‐site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues (trypsinlike), Leu‐Ala, Leu‐Ser, or Tyr‐Ala bonds (chymotrysinlike) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B‐like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B‐like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide “leftovers,” such as the proinsulin C‐peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relationships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects in converting proteases per se represent interesting areas for study in the search for disturbances in the production of neuroendocrine substances.
Linker mutagenesis in the gene of an outer membrane protein of escherichia coli, LamB
Authors: Bouges‐Bocquet B. , Villarroya H. , Hofnung M.
Published: December 2004

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in‐phase deletions or small insertions. Except for two in‐phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.
Mitochondrial membrane biogenesis: Characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase
Authors: McEwen Joan E. , Cumsky Michael G. , Ko Christine , Power Scott D. , Poyton Robert O.
Published: December 2004

A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation. This mutant, E4‐238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild‐type polypeptide (Mr = 12,500), the polypeptides from E4‐238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis‐acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4‐238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13‐511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid‐encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.
Rat liver membrane secretory component is larger than free secretory component in bile: Evidence for proteolytic conversion of membrane form to free form
Authors: Kloppel Thomas M. , Brown William R.
Published: December 2004

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component‐polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate‐polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory, component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component‐containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000‐dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC‐like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane‐associated secretory component.
Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases
Authors: Blum Jacob J. , Hayes Alvernon
Published: December 2004

Cilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5‐min extraction was generally insensitive to added calmodulin, whereas that obtained by 20‐hr extraction of the 5‐min extracted axonemes was activated by calmodulin, the activation being much larger in the “light” 14S fractions than in the “heavy” fractions. The 30S dynein ATPase obtained by a 5‐min extraction was generally activated over 1.6‐fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3‐fold. After further purification of the 5‐min extracted 30S dynein and of the 5‐min to 20‐hr‐extracted 14S dynein on DEAE‐Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro‐9‐[3‐(2‐hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms mat are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.
Electrophoretic analysis of the cytoplasmic and nuclear protein changes after induction of differentiation in WEHI‐3B myelomonocytic leukemia cells
Authors: Cooper Paul C. , Burgess Antony W.
Published: December 2004

In response to a differentiation factor (G‐CSF) the myelomonocytic leukemia cell line (WEHI‐3B(D+) differentiates to form mature macrophages and neutrophils. The effect of G‐CSF on WEHI‐3B(D+) differentiation was augmented by low concentrations (5 ng/ml) of actinomycin D. Quantitative binding of an antineutrophil serum was used to segregate the differentiated cells from the leukemic blast cells. Molecular markers of later myeloid differentiation were detected in myelocytes and macrophages purified from differentiating WEHI‐3B(D+ ) cells. To study the initial molecular processes associated with the initiation of WEHI‐3B(D+) cells to differentiation, the protein changes were analyzed using gel electrophoresis. Quantitative analysis of the fluorographs from the two‐dimensional (2D) electrophorograms of the 35S‐labeled proteins revealed major changes in the biosynthetic rates for 16 proteins within 5 hr: The biosynthesis of six proteins was increased and another ten proteins were synthesized at a reduced rate. Two of the proteins (17K and 36K daltons) were located in the nucleus. Pulse‐chase experiments indicated that protein turnover for these proteins was rapid but the degradation of four proteins was suppressed. At least six of the proteins (16K to 120K daltons) were acidic and were associated with the cytoplasm. Electrophoretic analysis of the 35S‐labeled proteins indicated that a 35K protein induced by G‐CSF was found in high abundance only in purified cells of intermediate differentiation (eg, myelocytes). Other proteins (eg, a very high molecular weight protein, and a 16K dalton protein) were obviously late markers of differentiated neutrophils or macrophages.
Rat liver 6‐phosphofructo 2‐kinase/ fructose 2,6‐bisphosphatase: A review of relationships between the two activities of the enzyme
Authors: El‐Maghrabi M. Raafat , Pilkis Simon J.
Published: December 2004

Both the synthesis and the degradation of Fru‐2,6‐P 2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6‐phosphofructo 2‐kinasc/fructose 2,6‐bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady‐slate concentration of fructose 2,6‐P2, an activator of 6‐phosphofructo 1‐kinase and an inhibitor of fructose 1,6‐bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors.

Recent evidence suggests that both reactions may proceed by two‐step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6‐P and fructose 2,6‐P2. A labeled phosphoenzyme is formed rapidly during incubation with [2‐32P]Fru‐2,6‐P2. The labeled residue has been identified as 3‐phosphohistidinc. However, it was not possible to demonstrate significant labeling of the enzyme directly from [γ‐32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E‐P is labile) and a fructose 2,6‐bisphosphatase site which is readily phosphorylated by fructose 2,6‐P2. Additional evidence in support of two active sites include: (1) limited proteolysis with thermolysin results in loss of 6‐phosphofructo 2‐kinase activity and activation of fructose 2,6‐bisphosphatase, (2) mixed function oxidation results in inactivation of the 6‐phosphofructo 2‐kinase but no affect on the fructose 2,6‐bisphosphatase, (3) N‐ethylmaleimide treatment also inactivates the kinase but docs not affect the bisphosphatase, and (4) p‐chlorom‐ercuribenzoate immediately inactivates the fructose 2,6‐bisphosphatase but not the 6‐phosphofructo 2‐kinasc. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine‐synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PU in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.
An improved purification method for cytoplasmic dynein
Authors: Pratt M. M. , Hisanaga S. , Begg D. A.
Published: December 2004

An improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)—the substitution of diethylaminoethyl (DEAE)‐cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphoceliulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/ Mg++ATPase = 0.8) and inhibition by sodium vanadate and erythro‐9‐2,3‐hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides ( > 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3–5 ml of packed eggs, a 20‐fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.
Evidence for transsynaptic regulation of neuronal cell surface heparan sulfate proteoglycan in developing rat superior cervical ganglion
Authors: Greif Karen F. , Trenchard Holly I.
Published: December 2004

The effect of neonatal deafferentation on the expression of a neuronal cell surface heparan sulfate proteoglycan (HeS‐PG) was investigated in the developing rat superior cervical ganglion. Two monoclonal antibodies, one directed against the core protein of HeS‐PG, and one to a determinant associated with a heparan sulfate side‐chain, were used to monitor postnatal increases of HeS‐PG by ra‐dioiminunoassay. Following neonatal deafferentation by section of the cervical sympathetic trunk, total protein per ganglion was slightly reduced at survival times of 7, 14, and 30 days. Expression of the core protein determinant on HeS‐PG was not altered in deafferented ganglia. In contrast, levels of side‐chain determinant were significantly reduced at 14 and 30 days. These results suggest that processing of HeS‐PG side‐chains by principal ganglionic neurons is partially regulated by transsynaptic influences during development. Transsynaptic regulation of neuronal development may be a more general process than was believed previously, with effects not limited to molecules associated with synaptic development.
Detection of mRNAs containing regulatory peptide coding sequences using synthetic oligodeoxynucleotides
Authors: Gozes Illana , Bodner Mordechai , Shani Yael , Fridkin Mati
Published: December 2004

To understand the regulation of the production of peptide hormones, it is vital to elucidate their biosynthetic pathways. We chose to study a major regulatory peptide, vasoactive intestinal peptide (VIP), a peptide possessing both neurotransmitter and neurohormone actions. To identify the specific peptide mRNA we are using, as hybridization probes, radiolabeled synthetic oligodcoxynucleotides with sequence complementary to the predicted peptide mRNA sequence. Employing this approach, we identified and partially purified a ∼ 1600‐base long mRNA containing VIP related sequences which can be translated in vitro into VIP‐immunoreactive polypeptides. Such mRNA was detected in normal VIP producing tissue (rat brain), as well as in a tumor producing VIP (human buccal tumor). This mRNA differs in size from a known VIP‐mRNA identified in human neuro‐blastoma cells, suggesting the possibility of different VIP‐mRNAs in different cell types.
Microcompartmentation of cAMP in wild‐type and memory‐mutant dunce strains of Drosophila melanogaster
Authors: Friedrich Peter , Solti Magda , Gyurkovics Henrik
Published: December 2004

The “enzyme‐probe” method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild‐type Canton‐S and memory‐mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleo‐tide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton‐S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.
Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma
Authors: Stevens Jeff W. , Oike Yasuteru , Handley Christopher , Hascall Vincent C. , Hampton Anne , Caterson Bruce
Published: December 2004

A ternary complex of hyaluronic acid‐binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid‐binding region has a molecular weight of ≅ 65,000 (HA‐BR65). N‐terminal amino acids in the complex were selectively l4C‐carbamylated. The resulting derivatized HA‐BR65 was isolated, and tryptic peptide maps were prepared and developed on two‐dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mr by ≅ 8,000 by SDS‐PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA‐BR65 to a polypeptide of ≅ 55,000 (HA‐BR55) which still retains the same N‐terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid‐binding region. Direct SDS‐PAGE analysis revealed one major intermediate with Mr ≅ 109,000 (HA‐BR109) as well as HA‐BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS‐PAGE gel at Mr ≅ 120,000 (HA‐BR120) and ≅ 140,000 (HA‐BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid‐binding region, and all of them contained the same N‐terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid‐binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.
Three classes of signalling molecules on B‐cell membranes
Authors: Corley Ronald B. , LoCascio N. J. , Ovnic Mariana , Arnold Larry W. , Pillai P. S. , Scott David W. , Haughton Geoffrey
Published: December 2004

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell‐dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of haptenbinding B cells, the other a B‐cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B‐cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T‐cell activation, the second as a signalling receptor for B‐cell excitation. On the CH12 surface, the I‐A molecule fulfills the former function, but T‐cell interactions with I‐A fail to result in B‐cell stimulation, suggesting that B‐cell la may limit helper T cell‐B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T‐cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B‐cell receptors, those that bind B‐cell stimulating factors.
Regulation of gonadotropin release, GnRH receptors, and gonadotrope responsiveness: A role for GnRH receptor microaggregation
Authors: Conn P. Michael , Rogers Deloris C. , Seay Sallie G. , Jinnah Hyder , Bates Michael , Luscher Diana
Published: December 2004

Gonadotropin‐releasing hormone (GnRH) stimulates luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) release from pituitary gonadotrope cells. Additional receptor‐mediated actions of the releasing hormone include homologous regulation of both the GnRH receptor and of cell responsiveness. While it is apparent that the release mechanism is Ca2+ mediated, it remains unclear how this receptor‐mediated action is integrated with regulation of the receptor and with cell responsiveness. It is the purpose of this review to describe the requirements for gonadotropin release as well as for receptor and response regulation in order to prepare an integrated model for these actions of the releasing hormone.
Comparisons of evolutionarily distinct fibronectins: Evidence for the origin of plasma and fibroblast cellular fibronectins from a single gene
Authors: Akiyama Steven K. , Yamada Kenneth M.
Published: December 2004

Plasma and fibroblast cellular fibronectins from three different species were compared for structural similarities and differences. Partial tryptic digestion of either human or chicken plasma and cellular fibronectins yields homologous protease‐resistant domains within a species but few homologies between species regardless of the source. Within a species, human or chicken plasma and fibroblast cellular fibronectins are immunologically indistinguishable as determined by the ELISA technique. There is limited immunological cross‐reactivity between species. Two‐dimensional tryptic peptide maps of fibroblast cellular and plasma fibronectins from the same species are also very similar: 85–95% of the spots on such maps comigrate. When peptide maps from different species are compared no more than 10% of the spots comigrate.

Three models for the genetic origin of cellular and plasma fibronectins in vertebrates are considered. A model in which both fibroblast cellular and plasma fibronectins arise from the same gene is the simplest that is consistent with the data.
Domain structure of neurofilament subunits as revealed by monoclonal antibodies
Authors: Angeletti Ruth Hogue , Trojanowski John Q. , Garden Martin , Schlaepfer William W. , Lee Virginia M.‐Y.
Published: December 2004

Monoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme‐linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin‐treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially computed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized “sidearm” domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.
Down‐regulation of gonadotropin and β‐adrenergic receptors by hormones and cyclic AMP
Authors: Fishman Peter H. , Rebois R. Victor , Zaremba Terrye
Published: December 2004

Loss of gonadotropin receptors in murine Leydig tumor cells and of β‐adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down‐regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist‐mediated receptor loss was rapid and almost total, down‐regulation by cyclic AMP was slower and less extensive. Down‐regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone‐mediated down‐regulation was preceded by desensitization of hormone‐stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down‐regulation. We conclude that down‐regulation of receptors in these cells is a complex process involving both cyclic AMP‐independent and ‐dependent events.
Immunopurification, characterization, and nature of membrane association of human melanoma‐associated oncofetal antigen gp87 defined by monoclonal antibody 140.240
Authors: Liao Shuen‐Kuei , Kwong Pak C. , Khosravi Mohammed J.
Published: December 2004

A melanoma‐associated oncofetal antigen, gp87 (a p97‐like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two‐step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde‐insolubilized MoAb 140.240 (ascites fluid) resulted in a 13‐fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr‐activated Sepharose 4B the bound material from the first step was further purified resulting in a 330‐fold purification with 90% recovery. SDS‐polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalaninc in gp87. We have also compared gp87 with two well defined antigens, HLA‐A,B,C (integral membrane protein) and “94K” melanoma/carcinoma–associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate‐buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP‐40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA‐A,B,C antigens, could only be extracted with NP‐40, strongly suggesting that gp87 is an integral melanoma cell component.
Matrix influence on the tumor cell stimulation of fibroblast collagenase production
Authors: Biswas Chitra
Published: December 2004

Cocultures of rabbit fibroblasts and mouse B‐16 melanoma cells produce increased levels of collagenase against type I collagen. This stimulatory effect was also found when fibroblasts were cultured in conditioned media from tumor cells. However, the level of the stimulatory factor in conditioned media was influenced by matrix deposited by fibroblasts. Thus, conditioned media collected from mono‐layers of B‐16 plated on fibroblast matrix consistently showed high levels of the factor activity. The influence of the matrix on the level of the factor was not removed by treating the fibroblast matrix with collagenase or chondroitinase ABC and was not reproduced by collagen‐coated dishes.
Activation of type I collagen genes in cultured scleroderma fibroblasts
Authors: Vuorio Tuula , Mäkelä Jyrki K. , Vuorio Eero
Published: December 2004

Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of proα1(I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of proα1(I)collagen mRNAs relative to total RNA were two‐ to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of proα1(I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of non‐affected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state.
Evidence for p15 cleavage site in myc‐specific proteins of avian MC29 and OK10 viruses
Authors: Sovová Vlasta , Trávníček Miloslav , Hložánek Ivo , Černá Hana , Alitalo Kari , Vaheri Antti
Published: December 2004

Myc‐related proteins were precipitated from MC29 virus‐transformed cells (PR‐2) and from OK10 virus‐transformed cells (9C) by anti‐gag and anti‐myc sera. Immunoprecipitates were cleaved with the avian retroviral protease p15 and the cleavage products analyzed in SDS‐PAGE. Cleavage fragments of p110gag‐myc (product of MC29 virus) and p58myc (product of OK10 virus) showed the presence of a p15 cleavage site within the myc‐specific region. The site is missing in deletion mutants of MC29 virus.
Phosphorylation of a tropomyosin‐like (30 KD) protein during platelet activation
Authors: Bourguignon Lilly Y. W. , Field Seth , Bourguignon G. J.
Published: December 2004

In this study, we have used the tumor promoter 12‐o‐tetradecanoylphorbol‐13‐acetate (TPA), as well as its biologically inactive analogue 4α‐phorbol 12, 13‐didecanoate (4α‐PDD), to investigate platelet protein phosphorylation and its possible correlation with platelet activation. Our data show that TPA, but not 4α‐PDD, induces a preferential phosphorylation of a 30,000 dalton (30 KD) protein. This phosphoprotein is found to be physically associated with an actomyosin‐containing platelet cytoskeleton complex. Further analysis using both standard two‐dimensional gel electrophoresis and one‐dimensional urea‐SDS gel electrophoresis reveals that this 30 KD protein has several tropomyosin‐like properties. Most importantly, the degree of TPA‐induced phosphorylation of the 30 KD protein is directly proportional to the extent of platelet granule release and the shape change of the platelet, as well as to the degree of aggregation. We speculate that this phosphorylated tropomyosinlike protein may play a pivotal role in the regulation of actomyosin‐mediated platelet contractility, which has been previously implicated in a variety of platelet functions.
Molecular shape and self‐association of vinculin and metavinculin
Authors: Molony Leslie , Burridge Keith
Published: December 2004

Vinculin, a 130,000‐dalton protein localized to adhesion plaques, and metavinculin, a 150,‐000 dalton protein closely related to vinculin, have been studied using rotary shadowing and electron microscopy. Both proteins have globular head regions attached to rod‐shaped tail domains. Vinculin and metavinculin also both form complexes consisting of four to six individual molecules. These multimers are formed by head‐to‐head as well as tail‐to‐tail interactions.

Talin, another protein which has been localized to adhesion plaques and binds to both vinculin and metavinculin, has also been investigated using shadowing techniques. Talin is an elongated, flexible molecule in high ionic strength buffers, as shown here by rotary shadowing and negative stain electron microscopy.