Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.     

Expression and characterization of recombinant beta-secretase from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Gal beta1,4-GlcNAc alpha 2,6-sialytransferase

Beta-Secretase (betaSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Gal beta1,4-GlcNAc alpha 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 57 to 59 k Da. A lectin blot analysis indicated that recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant beta-secretase from Tn5B1-4 beta SEC/GalT-ST cells had a lower isoelectric point than beta-secretase from control Tn5B1-4 betaSEC cells (Tn5B1-4 cells transformed only with beta-secretase cDNA). The enzyme activity of recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4 betaSEC cells. The concentrations at half-maximum inhibition (IC(50)) values estimated from inhibition analyses using purified beta-secretases from Tn5B1-4/betaSEC and Tn5B1-4/betaSEC/GalT-ST cells were 32 and 290 nM, respectively.     

Activation of defense responses in Chinese cabbage by a nonhost pathogen, Pseudomonas syringae pv. tomato

Pseudomonas syringae pv. tomato (Pst) causes a bacterial speck disease in tomato and Arabidopsis. In Chinese cabbage, in which host-pathogen interactions are not well understood, Pst does not cause disease but rather elicits a hypersensitive response. Pst induces localized cell death and H2O2 accumulation, a typical hypersensitive response, in infiltrated cabbage leaves. Pre-inoculation with Pst was found to induce resistance to Erwinia carotovora subsp. carotovora, a pathogen that causes soft rot disease in Chinese cabbage. An examination of the expression profiles of 12 previously identified Pst-inducible genes revealed that the majority of these genes were activated by salicylic acid or BTH; however, expressions of the genes encoding PR4 and a class IV chitinase were induced by ethephon, an ethylene-releasing compound, but not by salicylic acid, BTH, or methyl jasmonate. This implies that Pst activates both salicylate-dependent and salicylate-independent defense responses in Chinese cabbage.     

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea)

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.     

Solution structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein

The structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein, was determined by NMR spectroscopy. The structure is dominated by a beta-barrel sandwich. A two-stranded anti-parallel beta-sheet, which seals off one end of the beta-barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the beta-barrel. The three-dimensional structure of At3g04780.1-des15 provides an entry point for understanding its functional role and those of its mammalian homologs.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.     

c-Jun N-terminal kinase is involved in the suppression of adiponectin expression by TNF-alpha in 3T3-L1 adipocytes

Adiponectin, one of adipokines that is secreted from adipocytes, plays an important role in the regulation of glucose and lipid metabolism. Paradoxically, serum concentrations of adiponectin are decreased in obese and type 2 diabetic patients, although it is produced in adipose tissue. On the other hand, plasma TNF-alpha levels are increased in such subjects. In the present study, the mechanism by which adiponectin is regulated by TNF-alpha was investigated. The decreased adiponectin mRNA levels by TNF-alpha were partially recovered by treatment with a c-Jun N-terminal kinase (JNK) inhibitor or the PPAR-gamma agonist rosiglitazone in 3T3-L1 adipocytes. Interestingly, however, cotreatment with the JNK inhibitor and rosiglitazone led to a recovery of TNF-alpha-mediated adiponectin suppression to the control level. The JNK inhibitor regulated the expression of adiponectin by the increase of PPAR-gamma DNA binding activity and the recovery of its mRNA expression while rosiglitazone acted via a PPAR-gamma independent pathway which remains to be elucidated. These findings suggest that the JNK signaling pathway, activated by TNF-alpha, is involved in the regulation of adiponectin expression.     

The dynamic stress responses to area change in planar lipid bilayer membranes

The viscoelastic properties of planar phospholipid (dimyristoylphosphatidylcholine) bilayer membranes at 308 K are studied, many of them for the first time, using the nonequilibrium molecular dynamics simulation (NEMD) method for membrane area change. First, we present a unified formulation of the intrinsic three-dimensional (3D) and apparent in-plane viscoelastic moduli associated with area change based on the constitutive relations for a uniaxial system. The NEMD simulations of oscillatory area change process are then used to obtain the frequency-domain moduli. In the 4-250 GHz range, the intrinsic 3D elastic moduli of 20-27 kbar and viscous moduli of 0.2-9 kbar are found with anisotropy and monotonic frequency dispersion. In contrast, the apparent in-plane elastic moduli (1-9 kbar) are much smaller than, and the viscous moduli (2-6 kbar) comparable to, their 3D counterparts, due to the interplay between the lateral and normal relaxations. The time-domain relaxation functions, separately obtained by applying stepwise strains, can be fit by 4-6 exponential decay modes spanning subpicosecond to nanosecond timescale and are consistent with the frequency-domain results. From NEMD with varying strain amplitude, the linear constitutive model is shown to be valid up to 6 and 20% area change for the intrinsic 3D elastic and viscous responses, respectively, and up to 20% area change for the apparent in-plane viscoelasticity. Inclusion of a gramicidin A dimer (approximately 1 mol %) yields similar response properties with possibly smaller (<10%) viscous moduli. Our results agree well with available data from ultrasonic experiments, and demonstrate that the third dimension (thickness) of the planar lipid bilayer is integral to the in-plane viscoelasticity.     

Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5

We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.