We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans-Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.

Substrates of the metalloprotease ADAM17 (also known as TNF-a converting enzyme or TACE) undergo ectodomain shedding and include various inflammatory modulators. Though polymorphonuclear leukocytes contribute significantly to inflammation, direct analyses of ADAM17 on human neutrophils are very limited. In addition, the current understanding of the processes regulating ADAM17 activity primarily relate to its rapid activation. Therefore, to extend insights into the mechanisms of ADAM17 activity, we examined its surface expression and the shedding of its substrates during extended periods of neutrophil activation and apoptosis. Contrary to studies with immortalized hematopoietic cell lines, we report that surface expression of ADAM17 is maintained by human neutrophils activated with formyl peptides or by FcR/complement receptormediated phagocytosis. Interestingly, bacterial phagocytosis resulted in a significant increase in ADAM17 expression several hours after pathogen engulfment. We provide novel evidence that ADAM17 surface expression is also maintained during spontaneous and anti-Fas-induced neutrophil apoptosis. The well-validated ADAM17 substrates Lselectin and proTNF-a were shed efficiently by neutrophils under each of the conditions tested. Our data thus indicate prolonged ADAM17 expression during neutrophil effector functions. The implications of this may be a role by ADAM17 in both the induction and down-regulation of neutrophil activity. IntroductionParticular members of the a disintegrin and metalloprotease (ADAM) family function as sheddases, and the cell surface proteins that undergo ectodomain proteolysis participate in most cellular processes and various pathologies, such as Alzheimer's disease, cancer, and damaging inflammation [1,2]. At this time, ADAM17[also referred to as TNF-a converting enzyme (TACE)] is the best characterized of the sheddases, and its putative substrates are widespread, including various inflammatory modulators (e.g. TNF-a, IL-1RII, TNFR55, TNFR75, IL-6R, MCSF-R, fractalkine, VCAM-1, and L-selectin) [3][4][5][6][7][8][9][10][11][12]. L-selectin and proTNF-a in particular are wellcharacterized substrates of ADAM17. Gene-targeting of ADAM17 is lethal [5], yet studies have revealed that immature and mature leukocytes as well as cell lines deficient in functional ADAM17 are greatly impaired in their shedding of L-selectin and proTNF-a [4,5,7,12,13], indicating that the primary means of their shedding is not functionally redundant.Ectodomain shedding can be rapid and robust. For instance, shedding of the *1 Â 10 5 L-selectin molecules on the surface of a leukocyte occurs in the first few To that end, we examined the surface expression and activity of ADAM17 on human neutrophils during periods of stimulation and apoptosis. We demonstrate prolonged ADAM17 expression on the surface of human neutrophils under both conditions. We also show that ADAM17 substrates undergo efficient turnover during extended neutrophil activation and apoptosis. Our study thus provides new insights into th...

Invasive pulmonary aspergillosis (IPA) has significantly increased over the last decade. Here, a fusion protein consisting of the Dectin-1 extracellular domain linked to the Fc portion of murine immunoglobulin G1 augmented alveolar macrophage killing of Aspergillus fumigatus and shifted mortality associated with IPA via attenuation of A. fumigatus growth in the lung.

Background: MUC1 apical delivery in polarized MDCK cells employs a path used by proteins with glycan-dependent targeting signals. Results: Core O-glycans on mucin-like repeats of MUC1 act as an apical targeting signal. Conclusion: MUC1 apical targeting is O-glycan-dependent.Significance: We have identified a specific sequence with a post-translational modification that can direct apical delivery of MUC1 or a reporter protein.