Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Developmental regulation of dUTPase in Drosophila melanogaster

dUTPase prevents uracil incorporation into DNA by strict regulation of the cellular dUTP:dTTP ratio. Lack of the enzyme initiates thymineless cell death, prompting studies on enzyme regulation. We investigated expression pattern and localization of Drosophila dUTPase. Similarly to human, two isoforms of the fly enzyme were identified at both mRNA and protein levels. During larval stages, a drastic decrease of dUTPase expression was demonstrated at the protein level. In contrast, dUTPase mRNAs display constitutive character throughout development. A putative nuclear localization signal was identified in one of the two isoforms. However, immunohistochemistry of ovaries and embryos did not show a clear correlation between the presence of this signal and subcellular localization of the protein, suggesting that the latter may be perturbed by additional factors. Results are in agreement with a multilevel regulation of dUTPase in the Drosophila proteome, possibly involving several interacting protein partners of the enzyme. Using independent approaches, the existence of such macromolecular partners was verified.     

Effects of intermediate bound states in dynamic force spectroscopy

We revisit some aspects of the interpretation of dynamic force spectroscopy experiments. The standard theory predicts that the typical unbinding force f* is linearly proportional to the logarithm of the loading rate r when a single energy barrier controls the unbinding process. For a more complex situation of N barriers, it predicts at most N linear segments for the f* vs. log(r) curve, each segment characterizing a different barrier. Here we extend this existing picture using a refined approximation, provide a more general analytical formula, and show that in principle up to N(N + 1) / 2 segments can show up experimentally. As a consequence, the determination of the positions and even the number of the energy barriers from the experimental data can be ambiguous. A further possible consequence of a multiple-barrier landscape is a bimodal or multimodal distribution of the unbinding force at certain loading rates, a feature recently observed experimentally.     

A soluble carotenoid protein involved in phycobilisome-related energy dissipation in cyanobacteria

Photosynthetic organisms have developed multiple protective mechanisms to survive under high-light conditions. In plants, one of these mechanisms is the thermal dissipation of excitation energy in the membrane-bound chlorophyll antenna of photosystem II. The question of whether or not cyanobacteria, the progenitor of the chloroplast, have an equivalent photoprotective mechanism has long been unanswered. Recently, however, evidence was presented for the possible existence of a mechanism dissipating excess absorbed energy in the phycobilisome, the extramembrane antenna of cyanobacteria. Here, we demonstrate that this photoprotective mechanism, characterized by blue light-induced fluorescence quenching, is indeed phycobilisome-related and that a soluble carotenoid binding protein, ORANGE CAROTENOID PROTEIN (OCP), encoded by the slr1963 gene in Synechocystis PCC 6803, plays an essential role in this process. Blue light is unable to quench fluorescence in the absence of phycobilisomes or OCP. The fluorescence quenching is not DeltapH-dependent, and it can be induced in the absence of the reaction center II or the chlorophyll antenna, CP43 and CP47. Our data suggest that OCP, which strongly interacts with the thylakoids, acts as both the photoreceptor and the mediator of the reduction of the amount of energy transferred from the phycobilisomes to the photosystems. These are novel roles for a soluble carotenoid protein.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Millimeter wave induced reversible externalization of phosphatidylserine molecules in cells exposed in vitro

In vitro exposure of refrigerated samples (4 degrees C) of anti-coagulated blood with millimeter waves (MMWs) at incident power densities (IPDs) between 0.55 and 1.23 W/cm2 has been found to induce clot formation. We found a small but statistically significant change in clot size with increasing IPD value. MMW exposure of blood samples starting at room temperature (22 degrees C) did not induce blood coagulation; neither did conventional heating at temperatures up to 40 degrees C. Since cell-free plasma did not clot upon MMW exposure, the role of blood cells was particularly analyzed. Experiments on various mixtures of blood cells with plasma revealed an important role of red blood cells (RBC) in the coagulation process. Plasma coagulation also developed within the MMW beam above dense keratinocyte (HaCaT) monolayers suggesting it lacked cell-type specificity. We hypothesized that alteration of the membrane surface in exposed cells might be responsible for the circumscribed coagulation. The thrombogenic role of externalized phosphatidylserine (PS) molecules is well known. Therefore, we carried out experiments for immunolabeling PS molecules with fluorescein isothiocyanate (FITC)-conjugated Annexin V on exposed cells. Fluorescence microscopy of the adherent human keratinocytes (HaCaT) and murine melanoma cells (B16F10) showed that MMW exposure at an IPD of 1.23 W/cm2 is capable of inducing reversible externalization of PS molecules in cells within the beam area without detectable membrane damage. Nonadherent Jurkat cells exposed to MMW at an IPD of 34.5 mW/cm2 also showed reversible PS externalization with flow cytometry, whether the cell temperature was held constant or permitted to rise. These results suggest that certain biological effects induced by MMWs could be initiated by membrane changes in exposed cells.     

Targeted and shielded adenovectors for cancer therapy

Conditionally replicative adenovirus (CRAd) vectors are novel vectors with utility as virotherapy agents for alternative cancer therapies. These vectors have already established a broad safety record in humans and overcome some of the limitations of non-replicative adenovirus (Ad) vectors. In addition, one potential problem with these vectors, attainment of tumor or tissue selectivity has widely been addressed. However, two confounding problems limiting efficacy of these drug candidates remains. The paucity of the native Ad receptor on tumor tissues, and host humoral response due to pre-existing titers of neutralizing antibodies against the vector itself in humans have been highlighted in the clinical context. The well-characterized CRAd, AdΔ24-RGD, is infectivity enhanced, thus overcoming the lack of coxsackievirus and adenovirus receptor (CAR), and this agent is already rapidly progressing towards clinical translation. However, the perceived host humoral response potentially will limit gains seen from the infectivity enhancement and therefore a strategy to blunt immunity against the vector is required. On the basis of this caveat a novel strategy, termed shielding, has been developed in which the genetic modification of a virion capsid protein would provide uniformly shielded Ad vectors. The identification of the pIX capsid protein as an ideal locale for genetic incorporation of shielding ligands to conceal the Ad vector from pre-existing neutralizing antibodies is a major progression in the development of shielded CRAds. Preliminary data utilizing an Ad vector with HSV-TK fused to the pIX protein indicates that a shield against neutralizing antibodies can be achieved. The utility of various proteins as shielding molecules is currently being addressed. The creation of AdΔ24S-RGD, an infectivity enhanced and shielded Ad vector will provide the next step in the development of clinically and commercially feasible CRAds that can be dosed multiple times for maximum effectiveness in the fight against cancers in humans.This article is a symposium paper from the Annual Meeting of the "International Society for Cell and Gene Therapy of Cancer", held in Shenzhen, China, on 9–11 December 2005.