Macrophages (Mphi) comprise a heterogeneous population of cells with various immune and homeostatic functions. Recently, we have described type-1 and type-2 human monocyte-derived Mphi subsets. Although both support outgrowth of intracellular mycobacteria, Mphi-1 secretes interleukin (IL)-23/IL-12 and supports T helper cell type 1 (Th1) responses, whereas Mphi-2 fails to produce IL-23/IL-12, predominantly secretes IL-10, and inhibits Th1 function. Here, we further describe the phenotypic and functional profiles of Mphi-1 and Mphi-2 in response to microbial antigens and interferon-gamma (IFN-gamma) and CD40L as costimulatory T cell back-talk signals. Activated IL-23(+)/IL-12(+) Mphi-1 secreted IL-1beta, IL-18, IL-6, and tumor necrosis factor-alpha (TNF-alpha), as well as IL-8, monocyte chemoattractant protein-1 (MCP-1), IFN-inducible protein 10 (IP-10), Mphi inflammatory protein-1beta (MIP-1beta), regulated on activation, normal T expressed and secreted (RANTES), Mphi-derived chemokine (MDC), and (low levels of) pulmonary and activation-regulated chemokine and thymus and activation-regulated chemokine (TARC), corroborating their proinflammatory function. Regardless of the stimulus, Mphi-2 maintained their IL-10(+) signature cytokine profile and produced no or relatively low levels of IL-12p40, IL-1beta, IL-6, TNF-alpha, MDC, or TARC. It is remarkable that Mphi-2 secreted high levels of IL-8, MCP-1, IP-10, MIP-1beta, and RANTES, suggesting an active role for these cells in regulating cellular immunity and homeostasis. Mphi-1 and Mphi-2 expressed similar levels of Toll-like receptor and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin as microbial pattern recognition receptors. Mphi-2, unlike Mphi-1 but like other nonclassical Mphi described previously, expressed CD163 and down-modulated human leukocyte antigen and costimulatory molecules specifically upon activation. These findings demonstrate how Mphi-1/Mphi-2 polarization can differentially skew the host response toward pro- or anti-inflammatory immune responses, respectively. This is likely to be relevant for host-pathogen interactions in chronic bacterial infections and provides a model for dissecting pro- and anti-inflammatory cascades.

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In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild‐type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate‐decarboxylase‐negative (Pdc−) mutant lacking all three PDC genes exhibited a three‐fold lower growth rate in complex medium with glucose than the isogenic wild‐type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc− strains. Furthermore, in ethanol‐limited chemostat cultures, the biomass yield of Pdc− and wild‐type S. cerevisiae were identical. However, Pdc− S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol‐limited chemostat cultures (D = 0·10 h−1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed‐substrate cultures were switched to a feed containing glucose as the sole carbon source, wash‐out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl‐CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose‐limited chemostat cultures.

All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3 end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.Central in the life cycle of plus-strand RNA viruses is the process of RNA-templated RNA synthesis, which is required to replicate and transcribe the viral genome and which occurs in association with cytoplasmic membranes in the infected host cell. However, the enzymes required for this activity are not present in the eukaryotic host cell. Consequently, all plusstrand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit for viral RNA synthesis, and it operates in concert with other viral and/or host proteins. Correct initiation of RNA synthesis is essential for the integrity of the viral genome. Although diverse RNA viruses use an amazing variety of replication scenarios, there are only two principally different mechanisms by which RNA synthesis can be initiated: initiation can occur either de novo or in a primer-dependent fashion (reviewed in references 27 and 61). Upon de novo initiation, the starting nucleotide provides the 3Ј hydroxyl group for the addition of the next nucleotide. Primer-dependent initiation requires the use of either an oligonucleotide or a protein primer to provide the hydroxyl nucleophile. For primer-dependent initiation in RNA viruses, the most commonly used primer is either (i) an oligonucleotide that is covalently linked to a protein, as used, for example, by picornaviruses like poliovirus (41), or (ii) a capped primer that is cleaved from the 5Ј end of a host mRNA by a so-called cap-snatching mechanism, which is, e.g., used for transcription by some of...

Employability orientation of college students Employability orientation of college students K. van Dam, L. van der Zanden & Valeria Piras, Gedrag & Organisatie, volume 20, June 2007, nr. 2, pp. Based on Fugate, Kinicki and Ashforth's (2004) employability model, we investigated students' employability-oriëntation: what is their attitude toward their flexibility and availability in their study and future work situation, and how does this employability-orientation relate to intended job search behavior? Data from 109 students showed that the aspects of personal adaptability (learning orientation, core-self evaluations, and extraversion) had positive relationships with employability-orientation. And whereas career-planning showed a positive relationship, career commitment showed a negative relationship with employability-orientation. In turn, employability-orientation predicted intended job search behavior. In addition, education and work practice made a difference. The findings have theoretical and practical relevance.