Dissecting the structural determinants of the interaction between the human cytomegalovirus UL18 protein and the CD85j immune receptor

UL18 is a glycoprotein encoded by the human cytomegalovirus genome and is thought to play a pivotal role during human cytomegalovirus infection, although its exact function is still a matter of debate. UL18 shares structural similarity with MHC class I and binds the receptor CD85j on immune cells. Besides UL18, CD85j binds MHC class I molecules. The binding properties of CD85j to MHC class I molecules have been thoroughly studied. Conversely, very little information is available on the CD85j/UL18 complex, namely that UL18 binds CD85j through its alpha3 domain with an affinity that is approximately 1000-fold higher than the MHC class I affinity for CD85j. Deeper knowledge of features of the UL18/CD85j complex would help to disclose the function of UL18 when it binds to CD85j. In this study we first demonstrated that the UL18alpha3 domain is not sufficient per se for binding and that beta2-microglobulin is necessary for UL18-CD85j interaction. We then dissected structural determinants of binding UL18 to CD85j. To this end, we constructed a three-dimensional model of the complex. The model was used to design mutants in selected regions of the putative interaction interface, the effects of which were measured on binding. Six regions in both the alpha2 and alpha3 domains and specific amino acids within them were identified that are potentially involved in the UL18-CD85j interaction. The higher affinity of UL18 to CD85j, compared with MHC class I, seems to be due not to additional interaction regions but to an overall better fit of the two molecules.     

Specific recognition of the viral protein UL18 by CD85j/LIR-1/ILT2 on CD8+ T cells mediates the non-MHC-restricted lysis of human cytomegalovirus-infected cells

Immune evasion mechanisms of human CMV are known; however, the immune control of infection remains poorly elucidated. We show that interaction between the viral protein UL18 on infected cells and the invariant receptor CD85j/LIR-1/ILT2 expressed on CTL is relevant for the control of infection. Resting and activated CD8(+) T cells lysed UL18 expressing cells, whereas cells infected with CMV defective for UL18 were not killed. Lysis was not dependent on CD8(+) T cell Ag specificity, MHC-unrestricted and specifically blocked by anti-CD85j and anti-UL18 mAb. Moreover, soluble recombinant UL18Fc immunoprecipitated CD85j from T cells. Activation is mediated by CD85j and its pathway is unrelated to CD3/TCR engagement. UL18 is detected in immunocompromised patients with productive infection and the mechanism used in vivo by human CMV to ensure survival of the immunocompetent host may be mediated by activation signals delivered by infected cells to T lymphocytes via UL18/CD85j interactions.     

The sema domain

The sema domain was first defined from sequence by Kolodkin and colleagues in the early 1990s, and constitutes the distinctive structural and functional element of semaphorins, their plexin receptors and the receptor tyrosine kinases MET and RON, three protein families with major roles in development, tissue regeneration and cancer. Recently determined crystal structures of two semaphorins (SEMA3A and SEMA4D) and the MET receptor have shown that the sema domain consists of a highly conserved variant form of the seven-blade beta-propeller fold. The structures, however, also suggest differences between these families with respect to the mode of dimerisation and the regions of the domain involved in ligand-receptor interactions. This reflects the considerable plasticity and adaptation of the sema domain in order to meet different binding requirements, properties that may underlie the vast array of ligand-receptor specificities and functions of the semaphorin superfamily.     

Cytochrome P450-dependent metabolism of l-deprenyl in monkey (Cercopithecus aethiops) and C57BL/6 mouse brain microsomal preparations

The aim of the present investigation was to characterize the cytochrome P450 (CYP)-dependent metabolism of l-deprenyl by brain microsomal preparations obtained from two different animal models that have been extensively used in Parkinson's disease studies, namely monkey (Cercopithecus aethiops) and C57BL/6 mouse. In monkey brain microsomal fractions, the apparent Km values for methamphetamine formation from l-deprenyl were 67.8 +/- 1.0 and 72.0 +/- 1.6 microm, in the cortex and striatum, respectively. Similarly, for nordeprenyl formation from l-deprenyl, Km values in cortex and striatum were 21.3 +/- 3.2 and 27.3 +/- 4.0 microm, respectively. Both metabolic pathways appear to be more efficient in the cortex than in the striatum as the Vmax for microsomal preparation was lower in the striatum for the formation of both metabolites. The formation rate of l-methamphetamine was up to one order of magnitude greater than that of nordeprenyl. Inhibition analysis of both pathways in monkey brain suggested that l-methamphetamine formation is catalysed by CYP2A and CYP3A, whereas only CYP3A appears to be involved in nordeprenyl formation. With microsomal preparations from whole brain of C57BL/6 mice, the only l-deprenyl metabolite that could be detected was methamphetamine and the Km and Vmax values were similar to those determined in monkey cortex (53.6 +/- 2.9 microm and 33.9 +/- 0.4 pmol/min/mg protein, respectively). 4-Methylpyrazole selectively inhibited methamphetamine formation, suggesting the involvement of CYP2E1. In conclusion, the present study indicates that l-deprenyl is effectively metabolised by CYP-dependent oxidases in the brain, giving rise mainly to the formation of methamphetamine, which has been suggested to play a role in the pharmacological effects of the parent drug. The results also demonstrate that there are differences between species in CYP-dependent metabolism of l-deprenyl.     

CD85j (leukocyte Ig-like receptor-1/Ig-like transcript 2) inhibits human osteoclast-associated receptor-mediated activation of human dendritic cells

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated gamma-chain, induced Ca(2+) flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.     

Upper miocene carbonate ramp deposits from the southernmost part of Maiella Nountain (Abruzzo, Central Italy)

Summary The development of carbonate ramp depositional systems in the Neogene of the Mediterranean Region represents a widespread feature so far analysed in several papers. It is striking to note that the evolution of upper Miocene carbonate ramps, characterized by the presence of coralgal bioherms, highlights the events leading to the Messinian salinity crisis. The coralgal bioherms of preevaporite Messinian age exhibit fossil assemblages indicating marine waters with normal salinity, whereas stromatolitic and microbial encrustations underline the deterioration of the environment during the Messinian salinity crisis. Maiella Mountain is a broad carbonate massif located in Abruzzo (Central Italy). The late lower Oligocene-Messinian part of its stratigraphic succession consists of stacked non-tropical carbonate ramp deposits related to third and higher order sequences. The investigations performed in the southernmost portion of the massif allowed to recognize a complete fourth order carbonate depositional sequence on a homoclinal ramp of pre-evaporite Messinian age. The presence of small coralgal patch reefs and overlaying microbial encrustations is significant. A transect exhibits the stratigraphic framework of the area. The data show how local parameters play a notable role in the development of these deposits.     

Functional and morphologic characterization of human T lymphocytes expressing the TCR gamma /delta

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta+ cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta+ cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC); in addition, MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta+ cells. Both of these mABs, termed BB3 and delta TCS-1 respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively unfrequent in peripheral blood but represent the majority of TCR gamma/delta+ cells in tissues.     

How do reaction conditions affect the enantiopure synthesis of 2‐substituted‐1,4‐benzodioxane derivatives?

Several biologically active compounds structurally include the enantiopure 2‐substituted‐1,4‐benzodioxane scaffold. The straightforward racemization that affects reactions involving most of the common chemical reactives is thus a crucial issue. The developing of a completely stereo‐controlled synthetic route that does not affect the enantiomeric excess is consequently mandatory. It is also important to set up a reliable chiral HPLC method, able to follow the reaction, and to improve the synthetic performances. Here, we report the chiral investigation of two different synthons, we specifically evaluated the synthetic pathways that could be run in order to afford them, avoiding the racemization processes, which could normally occur in basic conditions. In addition, we developed peculiar chiral HPLC methods in order to resolve the enantiomers, define the enantiomeric excess, and fully characterize these compounds.