Implant exudate leucocyte response to anti-inflammatory drug treatment

Implantation of artificial materials elicits a local inflammatory response. In this study a chamber model technique, allowing sampling of the inflammatory exudate for further analysis in vitro, was used. Male Sprague-Dawley rats were injected daily with two different anti-inflammatory drugs, betamethasone and indomethacin, and the local cellular response was compared with a control group. The retrieved exudate was evaluated with respect to the number of leucocytes, cell viability, differential counts and serum-opsonized zymosan stimulated chemiluminescence (CL). In all groups the majority of cells were polymorphonuclear granulocytes (PMNGs). Betamethasone and high-dose indomethacin (1.92 mg kg^-1 body weight day^-1) treatment caused a marked reduction in the number of accumulated leucocytes 6 days after implantation. A substantial inhibition of the CL response was observed 6 days after treatment with betamethasone (4.23 mg kg^-1 body weight day^-1). An increased CL responsiveness was observed after 24 h with low-dose indomethacin (0.03 mg kg^-1 body weight day^-1) and after 6 days with high-dose indomethacin (1.92 mg kg^-1 body weight day^-1) treatment. In summary, depending on the anti-inflammatory drug treatment, dose and time after implant surgery, either an inhibition or stimulation of leucocyte accumulation and activation was observed. This study shows the possibilities of sampling the inflammatory exudate adjacent to a biomaterial implanted in vivo. This chamber model may be useful for the analysis of the inflammatory reaction around an implanted biomaterial during pharmacological treatment.     

Incidence of neoplasms in ages 0-19 y in parts of Sweden with high 137Cs fallout after the Chernobyl accident

An experiment was conducted to elucidate the origin of tetraploids (2n = 4x = 44) of Paragonimus westermani that occur together with diploid (2n = 2x = 22) and triploid (2n = 3x = 33) types in Liaoning Province, the People's Republic of China. Metacercariae of the diploid type, obtained from Hyogo Prefecture, Japan, and those of the triploid type from Tsushima, Nagasaki Prefecture, Japan, were mixed and inoculated into dogs and cats. The following results were obtained. The flukes were found in pairs within cysts in random combinations of 2x + 2x, 2x + 3x, and 3x + 3x (7:15:7). Oocytes in the oviduct were at stages from diplotene to metaphase. In a triploid fluke encysted with a diploid fluke, the primary oocytes were intruded by sperms from the diploid fluke. In the primary oocytes of diploid as well as triploid flukes, from diplotene to diakinesis, the homologues of the nucleolar chromosomes were heteromorphic as far as the size of the short arm was concerned. This implies that the triploid is an autotriploid generated in an ancestral diploid population that was polymorphic for the nucleolar chromosome.     

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.     

Structural features of an arabinan fragment isolated from the water-soluble fraction of dehulled rapeseed

A water-soluble polysaccharide fraction was prepared from dehulled rapeseed meal (winter rapeseed variety Casino). Further purification yielded two major fractions having a high content of arabinose and galactose residues, with Ara/Gal ratios of 5.4 (G1) and 1.8 (G2). The Ara/Gal ratio of the high molecular weight fraction G1 was stable over the whole gel filtration peak, indicating that the arabinose and galactose residues are part of the same polysaccharide. The high molecular weight fraction G1 was studied further by methylation analysis and several NMR techniques. Structural studies showed G1 to consist mainly of arabian fragments, which have terminal alpha-L-arabinofuranosyl groups with anomeric carbons bound (1-->5) (A) or (1-->2) (B), and 2,5-substituted arabinosyl residues with anomeric carbons bound (1-->5) (D) or (1-->2) (C) to adjacent arabinosyl residues. The A:B:C:D ratios were 2:1:1:1 according to results from NMR and methylation analysis.     

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.