Internalization and thermal susceptibility of Shiga toxin-producing Escherichia coli (STEC) in marinated beef products.

This study evaluated the internalization and cooking susceptibility of seven individual Escherichia coli (STEC) serogroups in surface-inoculated (10(5)log CFU/cm(2)) and vacuum tumbled marinated (30 or 60 min) bottom sirloin steaks. After storage for 14 days (0 to 2°C), flaps were cooked to various endpoint temperatures (55, 60, 65, and 71°C) for evaluation of pathogen survival by direct plating or rapid PCR based detection (BAX®). Direct plating of cooked samples yielded no enumerable plates. The data indicate varied internalization, translocation, and heat susceptibility patterns among serogroups. Using the rapid PCR based detection method O26, O103, and O111 were detected in flaps after cooking to 55 and 60°C, while O157:H7 survived in flaps cooked to 60 and 65°C. However, STEC O145 was the only serogroup that survived in all cooking temperatures. Serogroup O121 was not detected by plating or PCR in any cooked products. Intriguingly, STEC serogroups can be internalized during marination and the internalized pathogens vary in thermal susceptibility.

The influence of beef quality characteristics on the internalization and thermal susceptibility of Shiga toxin-producing Escherichia coli (STEC) in blade-tenderized beef steaks.

The risk of Shiga toxin-producing Escherichia coli (STEC) survival in blade-tenderized beef is a concern for beef processors. This study evaluated the internalization and post-cooking survival of individual STEC serogroups (O157:H7, O26, O45, O103, O111, O121, and O145) in blade-tenderized beef steaks with different quality traits. Strip loins representing four combinations of USDA Quality Grade (Choice or Select) and pH category (High pH or Normal pH) were inoculated (10(6)logCFU/cm(2) attachment) with individual STEC serogroups before storage (14 days), blade tenderization, and cooking (50, 60, 71, or 85°C). Serogroup populations on raw steak surfaces and internal cores were determined. Rapid-based methods were used to detect the internal presence of STEC in cooked steaks. Internalization and post-cooking survival varied among STECs. All serogroups, except O45 and O121, were detected in the internal cores of steaks cooked to 50°C, while O103, O111, and O145 STEC were detected in steaks cooked to 50, 60, and 71°C.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster.

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of approximately 40 nitr genes are contiguous in the genome and span approximately 0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway.

Destruction of tumor cells is a key function of lymphocytes, but the molecular processes driving it are unclear. Analysis of signal molecules indicated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase 2 critically controlled lytic function in human NK cells. We now have evidence to indicate that target ligation triggers a Ras-independent MAPK pathway that is required for lysis of the ligated tumor cell. Target engagement caused NK cells to rapidly activate MAPK within 5 min, and PD098059 effectively blocked both MAPK activation and tumoricidal function in NK cells. Target engagement also rapidly activated Ras, detected as active Ras-GTP bound to GST-Raf-RBD, a GST fusion protein linked to the Raf protein fragment containing the Ras-GTP binding domain. However, Ras inactivation by pharmacological disruption with the farnesyl transferase inhibitor, FTI-277, had no adverse effect on the ability of NK cells to lyse tumor cells or to express MAPK activation upon target conjugation. Notably, MAPK inactivation with PD098059, but not Ras inactivation with FTI-277, could interfere with perforin and granzyme B polarization within NK cells toward the contacted target cell. Using vaccinia delivery of N17 Ras into NK cells, we demonstrated that IL-2 activated a Ras-dependent MAPK pathway, while target ligation used a Ras-independent MAPK pathway to trigger lysis in NK cells.

Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells.

The mitogen-activated protein kinase-extracellular signal-regulated kinase signaling element (MAPK-ERK) plays a critical role in natural killer (NK) cell lysis of tumor cells, but its upstream effectors were previously unknown. We show that inhibition of phosphoinositide-3 kinase (PI3K) in NK cells blocks p21-activated kinase 1 (PAK1), MAPK kinase (MEK) and ERK activation by target cell ligation, interferes with perforin and granzyme B movement toward target cells and suppresses NK cytotoxicity. Dominant-negative N17Rac1 and PAK1 mimic the suppressive effects of PI3K inhibitors, whereas constitutively active V12Rac1 has the opposite effect. V12Rac1 restores the activity of downstream effectors and lytic function in LY294002- or wortmannin-treated, but not PD98059-treated, NK cells. These results document a specific PI3K-->Rac1-->PAK1-->MEK-->ERK pathway in NK cells that effects lysis.

Association of DAP12 with activating CD94/NKG2C NK cell receptors.

While the inhibitory NK cell receptors for MHC class I express immunoreceptor tyrosine-based inhibitory motifs that recruit intracellular tyrosine phosphatases and prevent NK cell effector function, the activating NK cell receptors lack intrinsic sequences required for cellular stimulation. CD94/NKG2C, an activating NK cell receptor of the C-type lectin superfamily that binds to HLA-E, noncovalently associates with DAP12, a membrane receptor containing an immunoreceptor tyrosine-based activating motif. Efficient expression of CD94/NKG2C on the cell surface requires the presence of DAP12, and charged residues in the transmembrane domains of DAP12 and NKG2C are necessary for this interaction. These results provide a molecular basis for the assembly of NK cell receptors for MHC class I involved in cellular activation and inhibition.

Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.

Natural killer (NK) cells express cell-surface receptors of the immunoglobulin and C-type lectin superfamilies that recognize major histocompatibility complex (MHC) class I peptides and inhibit NK-cell-mediated cytotoxicity. These inhibitory receptors possess ITIM sequences (for immunoreceptor tyrosine-based inhibitory motifs) in their cytoplasmic domains that recruit SH2-domain-containing protein tyrosine phosphatases, resulting in inactivation of NK cells. Certain isoforms of these NK-cell receptors lack ITIM sequences and it has been proposed that these 'non-inhibitory' receptors may activate, rather than inhibit, NK cells. Here we show that DAP12, a disulphide-bonded homodimer containing an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain, non-covalently associates with membrane glycoproteins of the killer-cell inhibitory receptor (KIR) family without an ITIM in their cytoplasmic domain. Crosslinking of KIR-DAP12 complexes results in cellular activation, as demonstrated by tyrosine phosphorylation of cellular proteins and upregulation of early-activation antigens. Phosphorylated DAP12 peptides bind ZAP-70 and Syk protein tyrosine kinases, suggesting that the activation pathway is similar to that of the T- and B-cell antigen receptors.

CD94/NKG2 is the predominant inhibitory receptor involved in recognition of HLA-G by decidual and peripheral blood NK cells.

Prior studies demonstrated that NK cells isolated from adult peripheral blood kill the HLA-A-, HLA-B-, and HLA-C-deficient B lymphoblastoid cell line 721.221, but many are unable to kill 721.221 cells transfected with HLA-G, a molecule expressed preferentially on fetal cytotrophoblasts. To determine the biologic relevance of this recognition, we established NK cell clones from the placenta and demonstrate that these NK cells also were unable to kill 721.221 cells expressing HLA-G. Recognition of HLA-G by NK cells was prevented in the presence of anti-CD94 mAb, implicating CD94/NKG2 as the predominant inhibitory NK cell receptor for HLA-G used by decidual NK cells. In contrast, mAbs against the killer cell inhibitory receptors recognizing HLA-Cw3-related, HLA-Cw4-related, or HLA-Bw4 ligands did not affect NK cell killing of the HLA-G transfectants.

Arousal and inhibition of human NK cells.

NK cells express receptors for polymorphic MHC class I molecules that inhibit killing of potential target cells bearing appropriate class I allotypes. Here we review the membrane receptors on human NK cells that are known to initiate cell-mediated cytotoxicity and demonstrate regulation of these responses by the killer cell inhibitory (KIR) receptors.

Human diversity in killer cell inhibitory receptor genes.

The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.

The position of the gulf stream during quaternary glaciations.

Ocean general circulation theories predict that the position of the boundary between subtropical and subpolar gyres (and therefore the position of the Gulf Stream-North Atlantic Current system and the subpolar-subtropical front) is set by the line of zero "Ekman pumping," where there is no convergence or divergence of water in the directly wind-forced surface layer of the ocean. In the present-day North Atlantic Ocean this line runs southwest to northeast, from off the Carolinas to off Ireland. However, during the last ice age (18,000 years ago) the subpolar-subtropical boundary ran more zonally, directly toward Gibraltar. A numerical atmospheric general circulation model indicates that the field of Ekman pumping 18,000 years ago was modified by the presence of a continental ice cap more than 3 kilometers thick such that the line of zero Ekman pumping overlaid the paleogyre boundary. These results demonstrate that the presence of a thick continental ice sheet could have caused changes in sea surface temperatures in the North Atlantic during Quaternary glaciations by altering wind patterns.

The Eocene/Oligocene boundary event in the deep sea.

Analysis of middle Eocene to early, Oligocene calcareous and siliceous microfossils shows gradual biotic changes with no massive extinction event across the Eocene/Oligocene boundary. Biotic changes in the late Paleogene appear to reflect changing paleoclimatic and paleoceanographic conditions and do not support suggestions of a catastrophic biotic event caused by a bolide impact at the Eocenel Oligocene boundary.