Comparison of microdosimetric simulations using PENELOPE and PITS for a 25 keV electron microbeam in water

The calculations presented compared the performances of two Monte Carlo codes used for the estimation of microdosimetric quantities: Positive Ion Track Structure code (PITS) and a main user code based on the PENetration and Energy Loss of Positrons and Electrons code (PENELOPE-2000). Event-by-event track structure codes like PITS are believed to be superior for microdosimetric applications, and they are written for this purpose. PITS tracks electrons in water down to 10 eV. PENELOPE is one of the few general-purpose codes that can simulate random electron-photon showers in any material for energies from 100 eV to 1 GeV. The model used in the comparison is a water cylinder with an internal scoring geometry made of spheres 1 microm in diameter where the scoring quantities are calculated. The source is a 25 keV electron pencil beam impinging normally on the sphere surface. This work shows only the lineal energy y and spectra graphical presentation as a function of y since for microdosimetry and biology applications, and for discussion of radiation quality in general, these results are more appropriate. The computed PENELOPE results are in agreement with those obtained with the PITS code and published previously in this journal. This paper demonstrates PENELOPE's usefulness at low energies and for small geometries. What is still needed are experimental results to confirm these analyses.     

Partial characterization of a prostaglandin-induced suppressor factor

Glass adherent splenic T cells, cultured in the presence of prostaglandin E₂ (10^?5M), were found to elicit a factor capable of nonspecifically suppressing PHA- and LPS-induced mitogenesis. Cells from C57B1/6J, Balb/C, and C3H/He mice were all capable of producing this suppressor factor, although some degree of variability in the response of cells from C3H mice to the factor was observed. The suppressor (designated prostaglandin-induced T-cell derived suppressor, PITS) was characterized biochemically and it was found that the activity was resistant to boiling, and treatment with RNase and DNase, yet was sensitive to treatment with proteinase K, trypsin, and Pronase. Further, PITS supernatants were found to contain at least two suppressors with approximate molecular weights of 35,000 (PITS_α) and 5000 (PITS_β). Results from experiments with cycloheximide-treated glass-adherent T cells indicate that prostaglandin E₂ may function by inducing the release rather than de novo synthesis of the PITS. These results indicate that the reported overall suppressive effect of prostaglandin E₂ on lymphocytes may in part be due to the release by certain T cells of a suppressive factor.     

The hemoglobins of the bullfrog Rana catesbeiana. The structure of the beta chain of component C and the role of the alpha chain in the formation of intermolecular disulfide bonds

The adult bullfrog Rana catesbeiana has two major hemoglobin components, B and C. Component C polymerizes by disulfide bond formation between tetramers but component B does not. The amino acid sequence of the first 112 residues of the beta chain of component C has been reported (Baldwin, T. O., and Riggs, A. (1974) J. Biol. Chem. 249, 6110-6118). We have completed the sequence of the beta chain of component C by determining the last 28 residues. This segment contains the 2 cysteinyl residues of the chain. Examination of models indicates that neither of these is in a readily accessible position for the formation of intertetramer disulfide bonds. Reactive sulfhydryl groups of the alpha chains are shown to be responsible for the initial formation of disulfide bonds between tetramers. The beta chains within the tetramers form disulfide bonds only when the hemoglobin molecules are subjected to prolonged incubation at 37 degrees C under oxygen. The beta chains of components B and C appear to be identical; the alpha chains are clearly quite different. This suggests that the alpha B and alpha C subunits interact in the association of the deoxygenated tetramers B and C to form what appears to be a BC2 molecule.     

Modification of beta-adrenoceptor signal transduction pathway by genetic manipulation and heart failure

The beta-adrenoceptor (beta-AR) mediated signal transduction pathway in cardiomyocytes is known to involve beta1- and beta2-ARs, stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of beta1- and beta2-ARs has been shown to increase heart function by increasing Ca2+ -movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of Gs-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of beta2-AR has also been shown to affect Gi-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. Beta1- and beta2-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate beta-AR indirectly through the involvement of beta-AR kinase (betaARK), beta-arrestins and Gbeta gamma-protein subunits. Genetic manipulation of different components and regulators of beta-AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of PARK in heart dysfunction and therapeutic role of betaARK inhibitors in the treatment of heart failure. Varying degrees of defects in the beta-AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in beta1-AR density, an increase in the level of G1-proteins and overexpression of betaARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of beta-AR signal transduction pathway in the failing heart. Thus different components and regulators of the beta-AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

Hepatocytes express abundant surface class I MHC and efficiently use transporter associated with antigen processing, tapasin, and low molecular weight polypeptide proteasome subunit components of antigen processing and presentation pathway

Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of K(b) and D(b) on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and beta(2)-microglobulin (beta(2)m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/beta(2)m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin(-/-) background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-gamma or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and beta(2)m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.     

Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGF beta), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGF beta and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGF beta to bFGF determines the magnitude of the Schwann cell response.     

Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts

Cell migration is essential for both organogenesis and tumor progression. Bone morphogenetic proteins (BMPs) are reported to be critical for not only bone formation but also tumor invasion. Here, we found that treatment with recombinant human BMP-2 (rhBMP-2) enhanced the haptotactic response of murine osteoblastic MC3T3-E1 and osteosarcoma Dunn cells to various extracellular matrix (ECM) components, including fibronectin, type I collagen, and laminin-1. Function-blocking antibody against integrin alpha5beta1 partially inhibited haptotaxis to fibronectin, suggesting that the response was propagated via these integrins. rhBMP-2 slightly increased the expression level of integrin beta1, and enhanced the speed of cell spreading on fibronectin, focal adhesion formation and phosphorylation of focal adhesion kinase (FAK) at Tyr397. By means of sucrose gradient flotation, incorporation of integrin beta1 in fractions of detergent (CHAPS) resistant membrane was increased when the cells were treated with rhBMP-2. Further, treatment with methyl-beta-cyclodextrin to deplete membrane cholesterol abrogated the effect of rhBMP-2 on haptotaxis, and exogenously added cholesterol reversed this inhibitory effect. Collectively, these results provide insights into the mechanism by which BMP signaling enhances cell migration by modulating fibronectin-integrin beta1 signaling via cholesterol enriched membrane microdomains, lipid rafts.     

Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin-kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin-kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ;soluble' RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin-kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.     

The murine DNA glycosylase NEIL2 (mNEIL2) and human DNA polymerase beta bind microtubules in situ and in vitro

8-oxoguanine DNA glycosylase (OGG1), a major DNA repair enzyme in mammalian cells and a component of the base excision repair (BER) pathway, was recently shown to be associated with the microtubule network and the centriole at interphase and the spindle assembly at mitosis. In this study, we determined whether other participants in the BER pathway also bind microtubules in situ and in vitro. Purified recombinant human DNA polymerase beta (DNA Pol beta) and purified recombinant mNEIL2 were chemically conjugated to fluorochromes and photosensitive dyes and used in in situ localization and binding experiments. Results from in situ localization, microtubule co-precipitation and site-directed photochemical experiments showed that recombinant human DNA Pol beta and recombinant mNEIL2 associated with microtubules in situ and in vitro in a manner similar to that shown earlier for another BER pathway component, OGG1. Observations reported in this study suggest that these BER pathway components are microtubule-associated proteins (MAPs) themselves or utilize yet to be identified MAPs to bind microtubules in order to regulate their intracellular trafficking and activities during the cell cycle.     

A neuronal cell line (PC12) expresses two beta 1-class integrins-alpha 1 beta 1 and alpha 3 beta 1-that recognize different neurite outgrowth-promoting domains in laminin

Integrins mediate neuronal process outgrowth on components of the ECM. Integrin alpha subunit-specific antibodies have been used to examine the roles of individual beta 1 integrins in attachment and neurite outgrowth by the neuronal cell line, PC12, in response to laminin and collagen. alpha 1 beta 1 and alpha 3 beta 1 were identified as the major beta 1 integrins expressed by PC12 cells. In functional assays, both alpha 1 beta 1 and alpha 3 beta 1 mediated PC12 cell interactions with laminin, whereas alpha 1 beta 1 alone mediated responses to collagen types I and IV. alpha 1 beta 1 and alpha 3 beta 1 were shown to recognize two different neurite-promoting sites in laminin: alpha 1 beta 1 interacted with the cross-region of laminin present in proteolytic fragments E1-4 and E1; alpha 3 beta 1 recognized a site in the long arm contained in laminin fragment E8. Thus, PC12 cells express two beta 1 integrins, which together function in attachment and neurite outgrowth on laminin and collagen. These integrins are candidates for mediating neurite outgrowth of sympathetic and other neurons in response to these ECM components.     

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.     

Beta2-glycoprotein I and atherosclerosis

Appreciation of the multifactorial nature of atherosclerosis requires a broad understanding of the mechanisms that underlie its pathogenesis. Autoimmune factors have recently been shown to be associated with the initiation and progression of atherosclerosis. In this context, modified lipoproteins were explored because of their de-novo occurrence within the vessel wall, and heat shock proteins are also being reported by several authors as triggers of autoimmune-like reactions that associate with atherosclerosis. Antiphospholipid antibodies in general and anti-beta2-glycoprotein I (beta2GPI) antibodies in particular have been shown to confer a procoagulant tendency in humans, either in the presence or the absence of the antiphospholipid syndrome. These findings and the ability of antibodies to beta2GPI to activate monocytes and endothelial cells led us to consider whether they are proatherogenic. In a series of studies it was shown that inducing an immune response to beta2GPI in atherosclerosis-prone mice accelerated atherosclerosis. We also demonstrated the abundance of beta2GPI in the atheroma, in conjunction with immunopotent cells. Moreover, when beta2GPI-reactive lymph node and spleen cells were transferred to LDL-receptor-deficient mice they promoted fatty streak formation, proving a direct proatherogenic role for beta2GPI-specific lymphocytes. Perhaps the most important implications of the existence of antigen-specific immune reactions within the atheroma is the ability to exploit them for the purpose of selective immunomodulation. Indeed, we have found that inducing immunological tolerance to beta2GPI by prior oral feeding with the antigen resulted in a significant reduction in the extent of atherosclerotic lesions. Thus, beta2GPI is a candidate player in the atherosclerotic plaque, and can possibly be employed as an immunomodulator of plaque progression.