Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle

Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered--localization of diffusion restrictions--is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.     

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

Image fusion analysis of volumetric changes after interstitial low-dose-rate iodine-125 irradiation of supratentorial low-grade gliomas

The aim of this study was to compare the volumes of tumor necrosis, reactive zone and edema with the three-dimensional dose distributions after brachytherapy treatments of gliomas. The investigation was performed an average of 14.2 months after low-dose-rate (125)I interstitial irradiation of 25 inoperable low-grade gliomas. The prescribed dose was 50-60 Gy to the tumor surface. Dose planning and image fusion were performed with the BrainLab-Target 1.19 software. In the CT/ MRI images, the "triple ring" (tumor necrosis, reactive ring and edema) developing after the interstitial irradiation of the brain tumors was examined. The images with the triple ring were fused with the planning images, and the isodose curves were superimposed on them. The volumes of the three regions were measured. The average dose at the necrosis border was determined from the isodose distribution. For quantitative assessment of the dose distributions, the dose nonuniformity ratio (DNR), homogeneity index (HI), coverage index (CI) and conformal index (COIN) were calculated. The relative volumes of the different parts of the triple ring after the interstitial irradiation compared to the reference dose volume were the following: necrosis, 40.9%, reactive zone, 47.1%, and edema, 367%. The tumor necrosis developed at 79.1 Gy on average. The average DNR, HI, CI and COIN were 0.45, 0.24, 0.94 and 0.57, respectively. The image fusion analysis of the volume of tumor necrosis, reactive ring and edema caused by interstitial irradiation and their correlation with the dose distribution provide valuable information for patient follow-up, treatment options, and effects and side effects of radio therapy.     

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus-induced interferon (IFN)-alpha. They exhibit a lineage-negative phenotype but all express BDCA-4, which is homologous to the neuronal receptor neuropilin-1. Specific staining with anti-BDCA-4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN-alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage-positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti-BDCA-4 mAb also resulted in at least two-fold lower or reduced virus-induced IFN-alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN-alpha release induced by non-viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA-4 on PDC as demonstrated for IFN-alpha response to virus.     

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).     

IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.