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.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

The role of Bcl-2 family members in tumorigenesis

The Bcl-2 family consists of about 20 homologues of important pro- and anti-apoptotic regulators of programmed cell death. The established mode of function of the individual members is to either preserve or disturb mitochondrial integrity, thereby inducing or preventing release of apoptogenic factors like Cytochrome c (Cyt c) from mitochondria. Recent findings also indicate further Bcl-2-controlled mitochondria-independent apoptosis pathways. Bcl-2 represents the founding member of the new and growing class of cell death inhibiting oncoproteins. In this review, we try to briefly summarize current models of Bcl-2 family function and to outline the work demonstrating the influence of deregulated Bcl-2 family member expression on tumorigenesis and cancer therapy. Since several Bcl-2 homologues, in addition to influencing apoptotic behaviour, also impinge on cell cycle progression, we discuss possible implications of this additional role for the expression of Bcl-2 family members in tumor cells.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.     

Structure-toxicity relationships for different types of dinuclear platinum complexes

Nine structurally distinct dinuclear platinum complexes have been evaluated in a novel model system for the investigation of renal epithelial toxicity of platinum drugs. The results showed that these compounds are toxic when applied at the basolateral side of renal epithelia, whereas their toxic effects on the apical side are negligible. Such a difference in toxicity of the complexes has been found to result from their poor uptake through the apical membrane, as compared to the basolateral membrane. Toxicity of the compounds on the basolateral side varies depending on their structure. Structure-toxicity relationships for the group of complexes with rigid ligands and for the group of complexes with flexible ligands are discussed. Among the dinuclear complexes with rigid ligands, sterically hindered complexes are less toxic, due to their poor uptake and low reactivity towards glutathione. Within the group of complexes with flexible ligands, cis-configured isomers are more toxic than their trans-counterparts.     

Nitrogen Cycling and Mass Balance for a Forested Catchment in the Italian Alps. Assessment of Nitrogen Status

During 1999–2001 the chemical composition and fluxes were measured in rainfall, throughfall, soil solution and stream water in a remote forested site in the Italian Alps. The analysis of temporal patterns revealed the differential behaviour of nitrogen and sulphur and suggested that different mechanisms controlled their flux. No important changes in sulphate concentration and fluxes emerged as the solution passed through the various components of the forest ecosystem, and temporal variations of SO₄ in the soil solution and stream were likely driven by the physical process of dilution. The availability of nitrate and ammonia, by contrast, was drastically reduced as throughfall water entered the soil and passed through the mineral layers, irrespective of season. The calculated hydrochemical budget based on throughfall and soil solution N fluxes revealed that ~80% N retention in the forest soil, corresponding to 12 kg ha⁻¹ yr⁻¹, despite a relatively high N deposition loading (15 kg ha⁻¹ yr⁻¹). Most of the leached nitrogen (90%) was in the organic form. Indicators of the N status of this ecosystem, such as C/N ratio in solid and solution phase of the soil and N foliage content as well as land use history were examined. Despite the strong N retention in the forested part of the catchment, the stream water N–NO₃ levels were consistently above 10 μg l⁻¹ suggesting that the Val Masino catchment as a whole was less efficient in processing atmospheric N inputs. This contrasting N behaviour illustrates the role of landscape features, such as the soil cover and vegetation type, that is characteristic of an alpine catchment.     

In vitro effects of GSM modulated radiofrequency fields on human immune cells

Despite the important role of the immune system in defending the body against infections and cancer, only few investigations on possible effects of radiofrequency (RF) radiation on function of human immune cells have been undertaken. Aim of the present investigation was therefore to assess whether GSM modulated RF fields have adverse effects on the functional competence of human immune cells. Within the frame of the multidisciplinary project "Biological effects of high frequency electromagnetic fields (EMF)" sponsored by the National Occupation Hazard Insurance Association (AUVA) in vitro investigations were carried out on human blood cells. Exposure was performed at GSM Basic 1950 MHz, an SAR of 1 mW/g in an intermittent mode (5 min "ON", 10 min "OFF") and a maximum Delta T of 0.06 degrees C for the duration of 8 h. The following immune parameters were evaluated: (1) the intracellular production of interleukin-2 (IL-2) and interferon (INF) gamma in lymphocytes, and IL-1 and tumor necrosis factor (TNF)-alpha in monocytes were evaluated with monoclonal antibodies. (2) The activity of immune-relevant genes (IL 1-alpha and beta, IL-2, IL-2-receptor, IL-4, macrophage colony stimulating factor (MCSF)-receptor, TNF-alpha, TNF-alpha-receptor) and housekeeping genes was analyzed with real time PCR. (3) The cytotoxicity of lymphokine activated killer cells (LAK cells) against a tumor cell line was determined in a flow cytometric test. For each parameter, blood samples of at least 15 donors were evaluated. No statistically significant effects of exposure were found and there is no indication that emissions from mobile phones are associated with adverse effects on the human immune system.     

Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .