Lack of effects of some individual polybrominated diphenyl ether (PBDE) and polychlorinated biphenyl (PCB) congeners on human lymphocyte functions in vitro

The structural similarities between polybrominated diphenyl ethers and immunotoxic halogenated aromatic compounds suggest that the polybrominated diphenyl ethers might affect the immune system. The present study was undertaken to investigate the immunological effects of some purified PBDE-congeners on human lymphocyte function in vitro. Polychlorinated biphenyl congeners were also included in the study. Mitogen-induced DNA synthesis and immunoglobulin synthesis by lymphocytes from blood donors were examined following polybrominated diphenyl ether or polychlorinated biphenyl exposure in vitro in order to determine the immunotoxic potential of these substances. No effects on mitogen-induced proliferation or immunoglobulin synthesis were observed after exposure of cells to concentrations up to 10(-5) M. The negative findings in this study indicate that certain functions of human peripheral lymphocytes, i.e. proliferation and immunoglobulin synthesis, are insensitive to the direct action of polybrominated diphenyl ethers and polychlorinated biphenyls. Our results are in accordance with other recent studies in which no effects on immunological parameters were demonstrated by exposure of lymphocytes to polyhalogenated aromatic hydrocarbons in vitro.     

Cell signaling by the type IV pili of pathogenic Neisseria

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.     

13C solid-state NMR of gramicidin A in a lipid membrane

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.     

Annealing and irradiation studies of MoC x thin films

On single-phase B1 molybdenum carbide thin films prepared by the rf sputtering technique at substrate temperatures between 40 and 800°C isochronous annealing and He-irradiation investigations were performed. The transition temperature to superconductivityT _c ,the lattice parametera ₀, and the specific resistivity were measured as a function of annealing temperature and fluence, respectively. Both during annealing and irradiation MoC _x behaves quite differently from typical refractory compounds such as NbC and NbN. During annealingT _c drops pronouncedly between 250 and 400°C. The B1 structure transforms to -Mo₂C between 600 and 900°C. During irradiationT _c rises slightly and then drops by only 0.5 K. The lattice parametera ₀ changes only by 0.2%.On leave of absence from the Institute of Physics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia.