CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Identification of FLRT1, FLRT2, and FLRT3: a novel family of transmembrane leucine-rich repeat proteins

FLRT1, FLRT2, and FLRT3 comprise a novel gene family isolated in a screen for extracellular matrix proteins expressed in muscle. The three genes encode putative type I transmembrane proteins, each containing 10 leucine-rich repeats flanked by N-terminal and C-terminal cysteine-rich regions, a fibronectin/collagen-like domain, and an intracellular tail. FLRT1 is expressed in kidney and brain, FLRT2 is expressed in pancreas, skeletal muscle, brain, and heart, and FLRT3 is expressed in kidney, brain, pancreas, skeletal muscle, lung, liver, placenta, and heart. FLRT1 localized to 11q12-q13, FLRT2 to 14q24-q32, and FLRT3 to 20p11. When expressed in SF9 and COS-1 cells, FLRT1 and FLRT2 migrate as 90- and 85-kDa proteins, respectively, and both are glycosylated. Given the overall structure of the three proteins, a function in cell adhesion and/or receptor signaling is predicted.     

Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

Mutations in the Pseudomonas syringae avrRpt2 gene that dissociate its virulence and avirulence activities lead to decreased efficiency in AvrRpt2-induced disappearance of RIN4

The avrRpt2 gene from Pseudomonas syringae pv. tomato exhibits avirulence activity on Arabidopsis expressing the resistance gene RPS2 but promotes bacterial virulence on susceptible rps2 Arabidopsis. To understand the functional relationship between the avirulence and virulence activities of avrRpt2, we analyzed a series of six avrRpt2 mutants deficient in eliciting the RPS2-dependent hypersensitive response. We show that the mutants are also severely impaired in triggering RSP2-dependent resistance. Four of these mutants are severely impaired in their virulence activity, whereas two alleles, encoding C-terminal deletions of AvrRpt2, retain significant but slightly reduced virulence activity. Thus, the avirulence and virulence activities of avrRpt2 can be genetically uncoupled. We tested the ability of the two C-terminal deletion mutants to trigger AvrRpt2-induced elimination of the Arabidopsis RIN4 protein and show that they retain this activity but are less efficient than wild-type AvrRpt2. Thus, reduced AvrRpt2 virulence activity is correlated with reduced efficiency in the induction of RIN4 disappearance. This suggests that an alteration in kinetics of RIN4 disappearance triggered by the C-terminal deletion mutants may provide the mechanistic basis for the uncoupling of the avirulence and virulence activities of avrRpt2.     

The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana

AvrRpt2, an effector protein from Pseudomonas syringae pv. tomato (Pst), behaves as an avirulence factor that activates resistance in Arabidopsis thaliana lines expressing the resistance gene RPS2. AvrRpt2 can also enhance pathogen fitness by promoting the ability of the bacteria to grow and to cause disease on susceptible lines of A. thaliana that lack functional RPS2. The activation of RPS2 is coupled to the AvrRpt2-induced disappearance of the A. thaliana RIN4 protein. However, the significance of this RIN4 elimination to AvrRpt2 virulence function is unresolved. To clarify our understanding of the contribution of RIN4 disappearance to AvrRpt2 virulence function, we generated new avrRpt2 alleles by random mutagenesis. We show that the ability of six novel AvrRpt2 mutants to induce RIN4 disappearance correlated well with their avirulence activities but not with their virulence activities. Moreover, the virulence activity of wild-type AvrRpt2 was detectable in an A. thaliana line lacking RIN4. Collectively, these results indicate that the virulence activity of AvrRpt2 in A. thaliana is likely to rely on the modification of host susceptibility factors other than, or in addition to, RIN4.     

The Pseudomonas syringae type III effector AvrRpt2 functions downstream or independently of SA to promote virulence on Arabidopsis thaliana

AvrRpt2, a Pseudomonas syringae type III effector protein, functions from inside plant cells to promote the virulence of P. syringae pv. tomato strain DC3000 (PstDC3000) on Arabidopsis thaliana plants lacking a functional copy of the corresponding RPS2 resistance gene. In this study, we extended our understanding of AvrRpt2 virulence activity by exploring the hypothesis that AvrRpt2 promotes PstDC3000 virulence by suppressing plant defenses. When delivered by PstDC3000, AvrRpt2 suppresses pathogen-related (PR) gene expression during infection, suggesting that AvrRpt2 suppresses defenses mediated by salicylic acid (SA). However, AvrRpt2 promotes PstDC3000 growth on transgenic plants expressing the SA-degrading enzyme NahG, indicating that AvrRpt2 does not promote bacterial virulence by modulating SA levels during infection. AvrRpt2 general virulence activity does not depend on the RPM1 resistance gene, as mutations in RPM1 had no effect on AvrRpt2-induced phenotypes. Transgenic plants expressing AvrRpt2 displayed enhanced susceptibility to PstDC3000 strains defective in type III secretion, indicating that enhanced susceptibility of these plants is not because of suppression of defense responses elicited by other type III effectors. Additionally, avrRpt2 transgenic plants did not exhibit increased susceptibility to Peronospora parasitica and Erysiphe cichoracearum, suggesting that AvrRpt2 virulence activity is specific to P. syringae.     

Reinnervation of arterial grafts by adrenergic nerves occurs in rats as indicated by increased levels of noradrenaline

To investigate the changes in noradrenaline concentrations in transplanted arterial grafts in rats, 31 female rats 4 to 6 weeks old of the AO/Ks:OC strain were operated on. Femoral arterial grafts were anastomosed to carotid arteries and compared with control femoral segments. Six rats were included in each follow-up group at 0, 1, 4, and 12 weeks, and there were seven rats in the 20-week follow-up group. High-performance liquid chromatography was used to determine the concentrations of noradrenaline. The operation itself decreased noradrenaline concentrations in the grafts to 76 percent of that in the control segments. One week after the operation, the noradrenaline concentration had fallen to 1.7 percent of the control values and started to recover thereafter. One month after the operation, it was 23 percent; at 3 months, it was 31 percent; and at 5 months, it was 43 percent of control values. The decrease from time 0 to 1 week was significant (p = 0.001), as was the increase from 1 week to 20 weeks (p = 0.004). Noradrenaline concentrations had fallen significantly 1 week after the operation and thereafter they increased to levels comparable to those seen in the immediate postoperative period.     

Daily and annual variations of free fatty acid, glycerol and leptin plasma concentrations in goats (Capra hircus) under different photoperiods

The aim of the study was to differentiate the impact of lighting conditions and feeding times on the regulation of lipid metabolism of goats under different photoperiods throughout the year. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60 degrees N (the longest light period 18 h, the shortest 6 h). Ambient temperature and feeding regime were kept constant. Blood samples were collected six times a year at 2-h intervals for 2 days, first in light/dark (LD) conditions and then after 3 days in constant darkness (DD). Significant daily variations were detected in the concentrations of plasma free fatty acids (FFA) and glycerol throughout the year. The nocturnal decrease and morning rise of FFA levels were related to the photoperiod, while the trough levels of glycerol were associated with the concentrate meal times. In DD conditions, FFA and glycerol rhythms were unstable. A significant seasonal variation was detected in the overall FFA and glycerol levels suggesting decreased lipogenesis in winter, increased lipolysis in spring and high lipogenesis in summer and fall. There was no significant daily rhythm in serum leptin levels, nor did the profiles in LD and DD conditions differ. The leptin level was slightly lower in early fall than in the other seasons, paralleling a small decrease of body mass in the goats after the grazing season. The daily or annual variations of FFA and glycerol levels were not clearly related to leptin concentrations. The results suggest that lipid metabolism of goats is regulated by light even in constant temperature and feeding conditions; however, no significant contribution of leptin levels could be shown.     

Efficiency, fidelity and enzymatic switching during translesion DNA synthesis

More than half of the 16 human DNA polymerases may have some role in DNA replication and potentially modulate the biological effects of DNA template lesions that impede replication fork progression. As one approach to understand how multiple polymerases are coordinated at the fork, we recently quantified the efficiency and fidelity with which one particular translesion synthesis enzyme, human DNA polymerase eta, copies templates containing cis-syn thymine dimers. Several observations from that study were unanticipated. Here we discuss the structural and biological implications of those results in light of earlier studies of translesion synthesis.