bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the fire‐retardant properties of zinc hydroxystannate and antimony trioxide in halogenated polyester resins using the Cone Calorimeter method
Authors: Cusack P. A.
Published: May 2005

An investigation of the burning behaviour of a series of halogenated polyester resin formulations has been carried out using the Cone Calorimeter technique. The data obtained clearly indicate that zinc hydroxystannate (ZHS) is more effective than antimony trioxide (Sb2O3) in reducing the average and peak rates of heat release in a chlorinated resin and in decreasing the peak rate of heat release in a brominated resin. The decreases in average heat release rate in the brominated resin are similar for ZHS and Sb2O3. Hence, at an addition level of 2 phr (parts per hundred of resin), ZHS reduces the average heat release rates of a chlorinated and a brominated resin by 41% and 26%, respectively, and the peak heat release rates of the same resins by 38% and 39%, respectively. The degree of smoke suppression exhibited by ZHS in these Cone experiments is significantly greater than that given by Sb2O3. In general, the correlation between Cone Calorimeter data and results obtained using other laboratory fire tests (UL‐94, LOI) is poor, except in the case of smoke density measurements, where the correlation with NBS Smoke Box data is remarkably good. No correlation is observed between the measured values of CO and CO2 output by different test methods, although it is now generally considered that small‐scale fire tests cannot be used to predict gaseous concentrations in real‐fire situations.
Communication: Retainer frame effects on Cone calorimeter results for building products
Authors: Tsantaridis Lazaros , Östman Birgit
Published: May 2005

The Cone Calorimeter has been used for fire testing of different products on a small scale for almost a decade. However, the number of users has been increasing in the last two to three years. More experimentation with the apparatus has led to practical questions. One of these is about the use of a retainer frame which has been used mainly for building products, but very few direct comparisons have been presented.
Combustion behaviour of polyurethane flexible foams under Cone Calorimetry test conditions
Authors: Vanspeybroeck R. , Van Hees P. , Vandevelde P.
Published: May 2005

The first part of this study focuses on the effect of cone calorimeter test variables on polyurethane flexible foam properties such as ignitability, heat release rate, effective heat of combustion and mass loss. Three of the main commercial foam types were used, i.e. conventional slabstock foams, high‐resilience slabstock foams and all‐MDI (methylene diphenyldiisocyanate) moulded foams. A decrease in heat flux (down to 40%) with increasing distance from the conical heater was measured. As a consequence, results were found to depend to a large extent on the thickness and the melting behaviour of the foam samples. To achieve a sufficiently constant and uniform heat flux exposure, sample thickness had to be limited to 25 mm. In addition, repeatability was found to be good under various conditions, with percentage standard deviations for effective heat of combustion, peak rate of heat release and mass loss below 10%. Levels of radiant flux above 25 kW m−2 were found to be very severe to test flexible polyurethane foams. Under such conditions, foams that show large differences in combustion performance in small‐scale flammability tests performed almost identically in the cone calorimeter. In the second part of this study the effects of foam variables, such as foam type, density and melamine content, are defined. These effects were clearly pronounced at radiant flux levels of 15–25 kWm−2. Density was found to be the key variable in controlling ignition resistance. In addition, high‐resilience slabstock foams and all‐MDI moulded foams performed better than conventional slabstock foams of the same density. Melamine addition resulted in a delay of ignition for all three foam types and an incomplete combustion, decreased heat release and effective heat of combustion in HR‐slabstock and all MDI moulded foams. However, melamine is not effective as a heat sink in conventional slabstock foams. The different performance of the foam types under study can be explained by a different melting behaviour.
Comparison of Nordtest FIRE 007, CEN draft proposal (Radiant Panel) and Cone Calorimeter methods in the fire testing of floor coverings
Authors: Tomann Johanna
Published: May 2005

Different types of floor coverings were tested using three different methods: the Cone Calorimeter (ISO 5660), the Nordtest floor covering test NT FIRE 007 and the German Radiant Panel Method (DIN 4102 Teil 14). The results of the comparisons between both flame spread and smoke production are given. Some correlation is found between the results of the Cone Calorimeter and the NT FIRE 007. Between other methods, no correlations seem to exist.
Surface temperature measurements on burning wood specimens in the Cone Calorimeter and the effect of grain orientation
Authors: Urbas Jože , Parker William J.
Published: May 2005

Surface temperatures were measured on dry Douglas fir sapwood specimens during Cone Calorimeter tests using thermocouples and an infra‐red pyrometer. Good agreement between the thermocouples and the pyrometer was obtained when (1) the emissivity was assumed to be 1.0 and (2) the thermocouples were in good contact with the surface and were not located in the proximity of a fissure. The major fissures were normal to the grain of the wood and the volatiles vented through the fissures. Char oxidation in the region between the vertical fissures resulted in higher surface temperatures.
Sensitivity of the Room/Corner test to variations in the test system and product properties
Authors: Kokkala Matti A.
Published: May 2005

The effects of variations in the ISO 9705 Room/Corner Test equipment and product properties are discussed. Previous experimental results are reviewed and some new results from the EUREFIC fire research programme are reported. The effects considered include the room dimensions, the burner size, the heat output of the burner and the stand‐off distance of the burner. The error in the measured peak rate of heat release caused by the finite response time of the oxygen consumption calorimeter due to mixing of the exhaust gases is analyzed in detail. The effect of material properties determined with the cone calorimeter are evaluated by employing models simulating the Room/Corner Test.
An Intermediate‐Scale Calorimeter for building materials and assemblies
Authors: Shaw James R. , Urbas Joe
Published: May 2005

A new Intermediate Scale Heat Release Rate Test Method is being developed primarily for testing assemblies in the vertical mode. Using the oxygen consumption calorimetry methodology and exhaust collection systems that already exist in many laboratories in the US and around the world, the apparatus is very economical to install. The sample size lends itself to reptitive testing in a short period of time. Sample preparation is generally quick and easy. Further development of the method should progress now that the method has been introduced at ASTM and will be introduced formally into ISO in the near future. There has been a favorable response by several laboratories who are considering construction of the apparatus. Additional assemblies tests are already underway and additional data will be presented in the near future.
A new bioactive bone cement: Its histological and mechanical characterization
Authors: Nishimura Naomi , Yamamuro Takao , Taguchi Yasushi , Ikenaga Minoru , Nakamura Takashi , Kokubo Tadashi , Yoshihara Satoru
Published: May 2005

We have developed a bioactive bone cement using CaOSiO2P2O5CaF2 glass powders and ammonium phosphate solution, and investigaed ist histological and mechanical characteristics in vivo. A bone defect was drilled in the proximal metaphysis of the rat tibia and filled with the bioactive bone cement in paste form or polymethylmethacrylate (PMMA) bone cement in the dough state. The cements were allowed to harden in situ. Histological examinationdemonstrated direct bonding between the new cement and bone by 4 weeks. The bioactive bone cement did not degrade up to 24 weeks postimplantation. The inflammatory reaction to the bioactive bone cement was less intense than the reaction induced by PMMA. Changes in the mechanical properties of the cement in vivo were studied by implanting hardened cylindrical specimens of both types of cement into the hindlimb muscles of ras of rats for 12 weeks. The compressive strength of the bioactive cement increased significantly after implantation, and reached 68 MPa in 1 week and 73 MPa in 4 weeks. These values were comparable to those of PMMA, and were maintained up to 12 weeks after implantation. This bioacive bone cement hadens in situ within a few minutes with negligible rise of temperature and can be easily handled as a paste for filling bone cavities of different shapes. In addition, this cement has good osteoconductive and bone bonding potential and fairly high mechanical strength. Therefore, this new cement could be used both as a bioactive bone cement and bone defect filler.
Collagen‐immobilized hydrogel as material for lamellar keratoplasty
Authors: Kobayashi Hisatoshi , Ikada Yoshito , Moritera Takeshi , Ogura Yuichiro , Honda Yoshihito
Published: May 2005

A transparent poly(vinyl alcohol) hydrogel disc covalently immobilized with Type I collagen, fibronectin, and an adhesive oligopeptide and preseeded with corneal epithelial cells was implanted in rabbit cornea using a technique of lamellar keratoplasty, and the tissue response was studied by light and electron microscopy. Covalent immobilization of type I colagen on the surfaceof hydrogel was found to support growth and adhesion of the corneal epithelium in vitro. the in vivo experiment showed that cell seeding could prevant infection of the hydrogel. However, the implanted disc was rejected from the host cornea by epithelial downgrowth and the exchange between the seeded cells and host corneal epithelia was not observed until 14 days after surgery. The rejection might have occurred because of unsatisfactory fixation of the hydrogel to the host cornea.
The theory of scaling up laboratory data for the sedimentation type centrifuge
Authors: Ambler Charles M.
Published: May 2005

The mathematical theory of the sedimentation type of centrifuge is developed in terms of unhindered settling of a single particle that has reached equilibrium velocity. From this is derived the sigma value for several of the different forms of sedimentation type centrifuge which is the calculated equivalent area of a settling tank theoretically capable of doing the same amount of work in a unit gravitational field. The limitations controlling the accuracy of scale up between centrifuges of similar geometry and between centrifuges of substantially different geometry are discussed.
Control of foam during fermentation by the application of ultrasonic energy
Authors: Dorsey Arthur E.
Published: May 2005

An ultrasonic generator was tested at 3 frequencies for its effects on Serratia marcescens during fermentation. The generator controlled foam adequately at 26, 29, and 34 kc/sec at medium aeration rates up to 0·6 ft3/min. Final viable counts were in the same range as those for cultures grown using lard oil as a foam control agent. At 34 kc/sec the final viable counts were slightly higher than those of cultures with lard oil.

No harmful effects were evident when large volumes of cultures were exposed to ultrasonic energy during growth.
Electronic computer solution for the MPN equation used in the determination of bacterial populations
Authors: Norman R. L. , Kempe L. L.
Published: May 2005

The utility and the practical precision of the Most Probable Number method for determining bacterial populations is currently restricted by a lack of adequate tables of solved examples or alternatively by an easy and quick solution to individual problems. Where an electronic computer is available, these restrictions need not apply. A computer programme and example solution for tabulated data and individual problems are presented in this paper.
Studies on large‐scale methods for propagation of animal cells
Authors: Rightsel Wilton A. , McCalpin Helen , McLean I. William
Published: May 2005

A new strain of cells, designated as PD–MEK–I, and derived from the kidneys of a rhesus monkey embryo, has been isolated and utilized as a prototype for mass propagation in suspended cell cultures, using techniques similar to those developed for antiobiotic fermentations. The use of the large‐volume units for cultivation of tissue cells has been applied. These units are useful for preparing masses of cells for studies of cellular antigens, virus assays, antitumour cytotoxicity studies, and other biologically active materials, as well as seed for continuing cultures. In addition, other methods of roller bottle cultures, shaker flask cultures, spinner cultures, and disposable 1‐litre prescription bottles, have been modified so that over 35 different human and animal cell lines are being maintained in continuous culture. Any one of these procedures may be modified for preparing and handling large numbers of replicate cultures as well as for maintaining stock seed cultures. The characteristics of growth of cells utilizing these methods are similar to those encountered in microbial populations and the principles of antibiotic fermentation have been applied appropriately.
Analytical control of the biological oxidation in the production of Δ1,4‐androstadiene‐3,17‐dione
Authors: Mizsei Antal , Szabó Antal
Published: May 2005

A rapid method for the assay of Δ1,4‐androstadiene‐3, 17‐dione has been worked out for use in the control of its production by biological oxidation. The method consists of a single selective extraction step, a specific colour reaction and a photometric measurement. Based upon the analyses of samples taken at frequent intervals, the oxidative process can be stopped at its optimum production level. Thus insufficient transformation and over‐oxidation can both be avoided. The time needed for one analysis is 20 minutes.
The prediction of oxygen transfer rates in the presence of mould mycelium
Authors: Solomons G. L. , Weston G. O.
Published: May 2005

An empirical relationship of oxygen transfer rate and mould viscosity is discussed, which leads to equations for predicting the OTR of mould fermentations.
Spray‐drying of tomato puree, groundnut and soyabean proteins and sugar–gelatine solutions
Authors: Evans G.
Published: May 2005

Tomato puree, groundnut and soya protein, and sugar‐gelatine solutions were successfully dried in a Scott pilot plant spray‐drier. The tomato puree did not need the addition of a carrier provided that the air plenum of the drier was suitably modified. Spray‐drying did not change the colour of tomato puree and the protein solutions, the viscosity characteristics of the protein solutions, or the nutritive value of the proteins. The conditions for the successful spray‐drying of sugar‐gelatine solutions were rather critical. The temperature in the spray‐drier had the major effect on the quality of the powders. Published data on spray‐drying do not provide a basis for predicting the drying behaviour of different products; experiments on the plant are essential.
Ion‐exchange chromatography of the streptomycin complex
Authors: Pereira J. N.
Published: May 2005

Various studies have revealed the presence of several maltolproducing compounds in streptomycin preparations. These previous studies have utilized the techniques of counter‐current distribution, column chromatography, paper chromatography, and electrophoresis. A system of cation‐exchange chromatography has been reported which separates, at least partially, six components of the streptomycin complex. This technique involves the use of a gradient elution procedure.

In addition to five maltol‐producing compounds, another member of the complex is reported. The possible significance of such a compound is discussed.
Redox potential changes in the 2‐keto‐L‐gulonic acid fermentation—I. Correlation between redox potential and dissolved‐oxygen concentration
Authors: Tengerdy Robert P.
Published: May 2005

The redox potential changes in the oxidative fermentation of L‐sorbose to 2‐keto‐L‐gulonic acid were investigated, using a mutant strain of Pseudomonas. The growth medium contained sorbose as the sole carbohydrate source. It was found that the redox potential curve of the fermenting system indicated qualitatively the existing oxygen demand of the culture. Increasing aeration rates led to higher oxygen demand and lower redox potentials. A comparison of the redox potential curves revealed that although the amount of dissolved oxygen directly affected the respiration of the culture, it did not necessarily mean respiratory insufficiency. The redox potential difference between the aerated and the deoxygenated (nitrogen flushed) fermentation broth was found to be linearly proportional to the logarithm of the dissolved‐oxygen concentration. This observation served as the basis of a new quantitative method of dissolved‐oxygen measurement, for which the name differential redoxymetry was proposed.
Redox potential changes in the 2‐keto‐L‐gulonic acid fermentation—II. Relationship between redox potential and product formation
Authors: Tengerdy Robert P.
Published: May 2005

In the investigation of the redox potential changes of the oxidative fermentation of L‐sorbose to 2‐keto‐L‐gulonic acid, it was found that the 2‐keto‐L‐gulonic acid formation proceeded favourably at low negative redox potential values. Low redox potentials were maintained in a continuous fermentation process in the chemostat, with a resulting high 2‐keto‐L‐gulonic acid formation rate. L‐Tyrosine and some of its analogues stimulated the 2‐keto‐L‐gulonic acid formation. The stimulation seemed to be bound to low redox potential values, and suggested the action of a direct oxidase‐type enzyme.
Interaction of growth factors during progression towards steroid independence in T‐47‐D human breast cancer cells
Authors: Daly Roger J. , King Roger J. B. , Darbre Philippa D.
Published: May 2005

When deprived of steroid in the long term, T‐47‐D human breast cancer cells lose estrogen sensitivity of cell growth. This loss of response results from an increased basal growth rate in the absence of steroid, not from a loss of estrogen‐simulated growth, and it occurs without any loss of estrogen receptor number or function. Growth factor gene expression and sensitivity have been investigated in this model system in an attempt to unravel the molecular mechanisms underlying the progression to steroid autonomy. The transition was accompanied by a decreased dependence on added serum and by a loss of the stimulatory effects of insulin and basic fibroblast growth factor, but also by an acquired sensitivity to stimulation by transforming growth factor‐β (TGF‐β). An increase in TGF‐β mRNA was detected following loss of steroid sensitivity. There was no increase in epidermal growth factor (EGF) receptor number. These findings are discussed in relation to current knowledge concerning the mechanisms by which estrogens stimulate breast cancer cell proliferation.
Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection
Authors: Lirette Ronald , Caradonna Sal
Published: May 2005

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post‐infection, while virus‐encoded dUTPase activity increases. Prelabeling of cells with 35S‐methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S‐methionine labeling during infection.

Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post‐translationally phosphorylated at serine residues. Pulse labeling of virus‐infected cells with 32P‐orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post‐infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P‐orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection.

A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild‐type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus‐infected cells.

These studies indicate that cellular dUTPase activity is diminished in wild‐type HSV‐infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.
Interrelationships of platelet‐derived growth factor isoform‐induced changes in c‐fos expression, intracellular free calcium, and mitogenesis
Authors: Diliberto Pamela A. , Bernacki Susan H. , Herman Brian
Published: May 2005

Both increases in c‐fos proto‐oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet‐derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF‐induced increases in c‐fos proto‐oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose‐response relationships and mechanisms of growth factor‐induced changes in these two parameters as well as DNA synthesis. Concentration‐dependent increases in [Ca2+]i, c‐fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF‐induced increases in c‐fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin‐2, and TMB‐8. Under these conditions, PDGF‐induced DNA synthesis was blocked, whereas c‐fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF‐induced c‐fos expression was inhibited by 75–80%, while PDGF‐induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC‐independent component of PDGF‐stimulated c‐fos expression was found to be independent of increases in [Ca2+]i. These data suggest that (1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c‐fos proto‐oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, (2) PDGF‐stimulated increases in [Ca2+]i are not required for c‐fos expression, and (3) distinct pathways regulate PDGF‐induced c‐fos expression and mitogenesis, with c‐fos expression being substantially PKC‐dependent yet [Ca2+]i‐independent, while mitogenesis is [Ca2+]i‐dependent yet PKC‐independent.
Gene expression during endochondral bone development: Evidence for coordinate expression of transforming growth factor β1 and collagen type I
Authors: Bortell Rita , Barone Leesa M. , Tassinari Melissa S. , Lian Jane B. , Stein Gary S.
Published: May 2005

Subcutaneous implatation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro‐ and osteo‐inductive events which are directly comparable to those which occur in normal endochondral bone development. Although the morphological and biochemical sequence associated with endochondral bone formation in this model has been well characterized, to date little information is available as to the gene regulation by which these events occur. To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell‐growth‐regulated and tissue‐specific genes were examined by slot blot analysis and compared to the morphological changes occuring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c‐myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to (1) production of fibroprogenitor cells (day 3), (2) onset of bone formation (day 9), and (3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post‐implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7–10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor β1 (TGFβ) were compared to those of collagen types I and II; a direct temporal correlation of TGFβ mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGFβ and type I collagen mRNA levels is consistent with a functional role for TGFβ in extracellular matrix production during in vivo bone formation.
Iron regulation of ferritin gene expression
Authors: Munro Hamish N.
Published: May 2005

Ferritin is a ubiquitous iron‐storage protein found in the cells of animals, plants, molds, and bacteria which it protects from toxic intracellular levels of iron. Ferritin stores iron within a hollow protein shell formed by subunits of two types, H and L. The 5′ untraslated regions of the two subunit mRNAs contain an almost indentical 28‐nucleotide sequence which regulates translation by binding to a specific cell sap protein. When cell iron level is low, this repressor protein obstructs trnslation of stored ferritin mRNAs, whereas increased iron levels release this protein, thus permitting extensive ferritin subunit synthesis to respond rapidly. Similar motifs in the 3′ untranslated region of transferrin receptor mRNA interact with this protein to regulate breakdown of the mRNA and thus change the receptor population. Finally, transcription of the H and L genes can be independently increased by iron and other factors. In the case of iron, synthesis of the L‐mRNA is increased preferentially since ferritin shells with a preponderance of L‐subunits store iron more efficiently. Thus regulation of ferritin synthesis at the translational and transcriptional levels and by transferrin receptor mRNA abudance at the level of breakdown provide a coordinate mechanism for protecting cells against the effects of excess iron.
Analysis of DNA–protein complexes induced by chemical carcinogens
Authors: Costa Max
Published: May 2005

DNA–protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis‐platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis‐platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA–protein complex. An antiserum which was raised to chromate‐induced DNA–protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA–protein complexes formed by cis‐platinum, UV, formaldehyde, and chromate.

Other work in this area, involving studying whether DNA–protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA–protein complexes, the stability and repair of these lesions, and characterization of DNA–protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA–protein complexes and these findings are also reviewed.
Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes
Authors: Bordoni R. , Fine R. , Murray D. , Richmond A.
Published: May 2005

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.
Interleukin‐1β‐ and tumor necrosis factor‐α‐independent monocyte stimulation of fibroblast collagenase activity
Authors: Tipton David A. , Pabst Michael J. , Dabbous Mustafa Kh.
Published: May 2005

To investigate the mechanism of cyclosporine (Cs)‐induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM‐LPS) that contained 665 pg/ml IL‐1β and 16 pg/ml TNFα and significantly (P < 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM‐LPS‐Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose‐dependent manner, with MCM‐LPS‐Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL‐1β and TNFα production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM‐LPS‐Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM‐LPS (no Cs). This suggested that factor(s) other than or in addition to IL‐1β and TNFα might be responsible for the stimulation of GN 23 collagenase activity. MCM‐LPS depleted of IL‐1β by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL‐1β and TNFα, when tested alone or together at levels found in the stimulatory MCM‐LPS and MCM‐LPS‐Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL‐1β and TNFα were not necessarily involved. Cs may alter the synthesis of other collagenase‐stimulating cytokines, accounting for the diminished ability of Cs‐treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.
Initiation of lymphocyte DNA synthesis
Authors: Coffman Frederick D. , Fresa Kerin L. , Cohen Stanley
Published: May 2005

The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat‐labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen‐stimulated lymphocytes after IL‐2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precurser which is present in resting cells. Nuclei from mitogen‐unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age‐related decline of immunity.

Quiescent lymphocytes lack ADR and synthesize an ADR‐inhibitory activity. The ADR inhibitor is a heat‐stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.
A novel suicide inhibitor strategy for antiparasitic drug development
Authors: Wang C. C.
Published: May 2005

DL‐α‐Difluoromethylornithine (DFMO), a suicide inhibitor of eukaryotic ornithine decarboxylase (ODC), has therapeutic activities against African trypanosomiasis. The Ki, value of DFMO for ODC of Trypanosoma brucei is somewhat higher than that for mouse ODC. The therapeutic efficacy of DFMO cannot therefore be attributed to a preferential inhibition of the parasite enzyme. The T. brucei gene encoding ODC was cloned and sequenced, and the derived amino acid sequence has 61.5% homology with that of mouse ODC, except that the C‐terminal 36 amino acids of the mouse enzyme are missing from the parasite enzyme. The cloned T. brucei and mouse ODC genes were expressed in ODC‐deficient Chinese hamster ovary cells (CHO) where the T. brucei enzyme was stable, but mouse ODC was unstable. Thus, the observed difference in intracellular stability is a property of the ODC protein itself, rather than of the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome ODC and the C‐terminus of mouse ODC also was rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxy‐terminus determine its stability.

The relatively slow turnover of the parasite enzyme constitutes the basis of selective antitrypanosomal action of DFMO. By this same token, many other proteins known to perform crucial functions in bacteria, fungi, protozoa, helminths, etc., also may have shorter half‐lives in the mammalian hosts than in parasites. Suicide inhibitors of these proteins may have desirable characteristics as good chemotherapeutic agents. This new approach could provide an additional strategy for controlling infectious diseases.
Killer polyamines?
Authors: Coffino Philip , Poznanski Ann
Published: May 2005

Mammalian cells can rapidly make large changes in their rate of polyamine biosynthesis in response to mitogenic and trophic signals. However, cultured cells seem to grow adequately as long as they are supplied a steady but unregulated supply of polyamines. This implies that complex and rapid changes in polymine synthesis serve a function in a special rather than a general biological context. We suggest that the appropriate context in which regulation of polyamines mediates crucial functions is the mammalian embryo and that one function of polyamines is to act as substrate in an oxidative pathway that arbitrates programmed cell death.
Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs, monensin, low temperature, and chloroquine
Authors: McAbee Douglas D. , Lear Mark C. , Weigel Paul H.
Published: May 2005

We studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for 125I‐asialo‐orosomucoid binding, to measure receptor activity, or 125I‐anti‐Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin‐permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub‐37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ⩽ 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin‐and chloroquine‐treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.
Structure‐function studies of heparin‐binding (acidic fibroblast) growth factor‐1 using site‐directed mutagenesis
Authors: Burgess Wilson H. , Shaheen Anne M. , Hampton Brian , Donohue Patrick J. , Winkles Jeffrey A.
Published: May 2005

The heparin‐binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF‐1 can be dissociated from the receptor‐binding activities of the growth factor by site‐directed mutagenesis of a single lysine residue. Thus, the mutant HBGF‐1 has normal receptor‐binding activity and is capable of stimulating tyrosine kinase activity and proto‐oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto‐oncogene expression from the mitogenic response is observed when the human wild‐type HBGF‐1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild‐type or mutant HBGF‐1. Production of wild‐type induces a transformed phenotype, whereas over‐production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site‐directed mutagenesis of putative nuclear translocation sequences in the wild‐type protein do not affect mitogenic activity. Thus, the role of nuclear translocation in the mechanisms of action of HBGF‐1 remains unclear.
The construction of high efficiency human bone marrow tissue ex vivo
Authors: Emerson Stephen G. , Palsson Bernhard O. , Clarke Michael F.
Published: May 2005

The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM‐CSF and IL‐3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.
Long‐term marrow cultures: human and murine systems
Authors: Quesenberry Peter , Temeles Daniel , McGrath Helen , Lowry Philip , Meyer David , Kittler Ellen , Deacon Donna , Kister Kay , Crittenden Rowena , Srikumar Kotteazeth
Published: May 2005

The intramedullary control of marrow cell production has been a difficult area to approach experimentally. The introduction by Dr. Dexter and colleagues of long‐term stromal dependent culture systems for murine marrow and the adaptation of these systems to human marrow growth have allowed for in‐vitro studies of stromal dependent hemopoiesis. Despite some controversy in this area, most studies appear to show that adherent murine or human stromal cells are capable of producing a relatively large number of hemopoietic growth factors including G‐CSF, GM‐CSF, CSF‐1, IL‐6 and, at least by PCR analysis, IL‐3. Other work indicates that the most primitive hemopoietic cells which appear to be multifactor responsive adhere directly to these stromal cells presumably through mediation of various adherence proteins.

An early acting, multilineage factor termed hemolymphopoietic growth factor‐1 (HLGF‐1) has been isolated from a murine stromal cell line and may be identical to the recently described ligand for the c‐kit receptor. This may represent an important early survival/maintenance factor for stem cells in this system.

Studies on primitive stem cells, especially the high proliferative potential colony forming cell (HPP‐CFC), indicate that they are responsive to varying combinations of growth factors and that with increasing numbers of growth factors, as studied in serum‐free systems, decreasing concentrations of the factors may be biologically active.

These observations altogether suggest that intramedullary hemopoiesis may be regulated by the positioning of early multifactor responsive stem cells via adherent proteins in juxtaposition to synergistically acting combinations of grwoth factors attached to stromal cell surfaces or the extracellular matrix. In addition, selective production of different growth factors from different subsets of cells may create growth factor gradients and explain the spacial distribution of different cell types within the marrow cavity.
A role for osteocalcin in osteoclast differentiation
Authors: Glowacki J. , Rey C. , Glimcher M. J. , Cox K. A. , Lian J.
Published: May 2005

Specific cellular interactions with components of the extracellular matrix can influence cellular differentiation and development of many tissues. The extracellular matrix of bone is composed of organic constituents and a solid phase of calcium and inorganic phosphate (apatite). When implanted subcutaneously in rats, particles of bone matrix (BPs) recruit progenitors that differentiate into multinucleated cells with osteoclastic features. Because BPs deficient in osteocalcin, a bone matrix protein, were less efficient at promoting osteoclast formation than were normal BPs, we directly examined the influence of osteocalcin on osteoclast differentiation. We evaluated tissue responses to particles of synthetic crystalline apatite alone (Ap), having many of the features of native apatite of mature bone, or to apatite prepared with osteocalcin (Ap/OC), bovine serum albumin (Ap/BSA) or rat bone collagen (Ap/Col). Twelve days after subcutaneous implantation in normal rats, Ap, Ap/BSA, and Ap/Col particles generated a mild foreign body reaction with multinucleated cells in direct contact with the particles; these cells were negative for tartrate‐resistant acid phosphatase (TRAP) activity and lacked ruffled borders. In contrast, Ap particles containing approximately 0.1% osteocalcin were partially resorbed and they generated more multinucleated cells that were TRAP‐positive, were immunoreactive with an antibody against tartrate‐resistant purple acid phosphatase, and displayed ultrastructural features of active osteoclasts including ruffled borders and clear zones. These data support the hypothesis that osteocalcin may function as a matrix signal in the recruitment and differentiation of bone‐resorbing cells.