Chlorophyllide a Oxygenase mRNA and Protein Levels Correlate with the Chlorophyll a/b Ratio in Arabidopsis thaliana

Plants can change the size of their light harvesting complexes in response to growth at different light intensities. Although these changes are small compared to those observed in algae, their conservation in many plant species suggest they play an important role in photoacclimation. A polyclonal antibody to the C-terminus of the Arabidopsis thaliana chlorophyllide a oxygenase (CAO) protein was used to determine if CAO protein levels change under three conditions which perturb chlorophyll levels. These conditions were: (1) transfer to shaded light intensity; (2) limited chlorophyll synthesis, and (3) during photoinhibition. Transfer of wild-type plants from moderate to shaded light intensity resulted in a slight reduction in the Chl a/b ratio, and increases in both CAO and Lhcb1 mRNA levels as well as CAO protein levels. CAO protein levels were also measured in the cch1 mutant, a P642L missense mutation in the H subunit of Mg-chelatase. This mutant has reduced total Chl levels and an increased Chl a/b ratio when transferred to moderate light intensity. After transfer to moderate light intensity, CAO mRNA levels decreased in the cch1 mutant, and a concomitant decrease in CAO protein levels was also observed. Measurements of tetrapyrrole intermediates suggested that decreased Chl synthesis in the cch1 mutant was not a result of increased feedback inhibition at higher light intensity. When wild-type plants were exposed to photoinhibitory light intensity for 3 h, total Chl levels decreased and both CAO mRNA and CAO protein levels were also reduced. These results indicate that CAO protein levels correlate with CAO mRNA levels, and suggest that changes in Chl b levels in vascular plants, are regulated, in part, at the CAO mRNA level.     

Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.     

Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L

Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction.     

Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).     

Physiological mechanisms of regulating alloimmunity: cytokines,CTLA-4, CD25+ cells,and the alloreactive T cell clone size

The mechanisms underlying physiological regulation of alloimmune responses remain poorly defined. We investigated the roles of cytokines, CTLA-4, CD25(+) T cells, and apoptosis in regulating alloimmune responses in vivo. Two murine cardiac transplant models were used, B10.D2 (minor mismatch) and C57BL/6 (major mismatch), into BALB/c recipients. Recipients were wild type, STAT4(-/-) (Th1 deficient), or STAT6(-/-) (Th2 deficient) mice. Minor mismatched allografts were accepted spontaneously in approximately 70% of wild type and STAT4(-/-) mice. By contrast, there was significantly shorter graft survival in minor mismatched STAT6(-/-) mice. Either the adoptive transfer of STAT4(-/-) splenocytes or the administration of IL-4Fc fusion protein into STAT6(-/-) mice resulted in long term graft survival. Blocking CTLA-4 signaling accelerated the rejection in all recipients, but was more pronounced in the minor combination. This was accompanied by an increased frequency of alloreactive T cells. Furthermore, CTLA-4 blockade regulated CD4(+) or CD8(+) as well as Th1 or Th2 alloreactive T cells. Finally, while anti-CD25 treatment prolonged graft survival in the major mismatched combination, the same treatment accelerated graft rejection in the minor mismatched group. The latter was associated with an increased frequency of alloreactive T cells and inhibition of T cell apoptosis. These data demonstrate that cytokine regulation, CTLA-4 negative signaling, and T cell apoptosis play critical roles in regulating alloimmunity, especially under conditions where the alloreactive T cell clone size is relatively small.     

Early events of TCR signaling are distinct in human Th1 and Th2 cells

To study the requirements for activation of human Th1 and Th2 cells, soluble peptide/DR1 complexes were prepared from naturally expressed DR1 protein. When immobilized, this material induced T cell activation, as revealed by CD25 up-regulation. Unexpectedly, Th2 cells required a higher density of peptide/DR1 complexes than Th1 cells to initiate CD25 up-regulation. Similar findings were obtained with immobilized or soluble and cross-linked anti-CD3 mAb. In contrast, peptide/DR1 complexes displayed on the surface of nonprofessional APC similarly induced CD25 up-regulation in Th1 and Th2 cells. Signaling events distinguishing human Th1 and Th2 cells following TCR engagement by anti-CD3 mAb were then studied. It was observed that upon TCR triggering, the overall tyrosine phosphorylation profiles were fainter in Th2 than in Th1 clones. Similar results were obtained with Th1- and Th2-polarized polyclonal lines. Varying the dose of anti-CD3 mAb, the kinetics of activation, and coengagement of CD3 and CD28 failed to increase tyrosine phosphorylation in Th2 cells to levels reached in Th1 cells. In contrast, treatment with the tyrosine phosphatase inhibitor phenylarsine oxide resulted in similar tyrosine phosphorylation levels in Th2 and Th1 cells. These findings indicated that Th2 cells had an intrinsically lower TCR-induced tyrosine phosphorylation capacity than Th1 cells, which might be controlled by Th1- and Th2-specific phosphatase profiles. Finally, a weaker association was found between ZAP-70 and CD3zeta in Th2 than in Th1 cells after TCR engagement. Taken together, these results constituted evidence that early events in the TCR signaling cascades are distinct in human Th1 and Th2 cells.     

Putative roles of the CNF2 and CDTIII toxins in experimental infections with necrotoxigenic Escherichia coli type 2 (NTEC2) strains in calves

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.     

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

Structural,dynamic and mechanical properties of POPC at low cholesterol concentration studied in pressure/temperature space

We have studied the structural, dynamic and mechanical properties of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine (POPC)/cholesterol binary mixtures by small-angle X-ray scattering. Our investigations were concentrated on the biologically most relevant pressure-temperature-cholesterol regime, i.e. the liquid crystalline phase and its phase boundary to the lamellar gel phase within a cholesterol concentration up to 25 mol%. From the dependence of the transition pressure we derived a value of 19 kJ/mol for the transition enthalpy Delta H(m) of POPC in excess water. With increasing cholesterol concentration, Delta H(m) drops to about 7 kJ/mol at 20 mol% cholesterol. Time-resolved pressure-scan (p-scan) and temperature-jump (T-jump) experiments reveal that at low cholesterol content (<5-8 mol%) the fluidity and also the bilayer compressibility increase remarkably. In contrast, at concentrations between 5 and 25 mol% cholesterol the bilayer becomes again more rigid and the lipid bilayer spacing increases about 2 A. Theses changes are attributed to the onset of phase separation between liquid disordered and liquid ordered phases. The fluid-fluid miscibility gap for this mono-unsaturated lecithin species is strongly enlarged compared with saturated lecithins.