The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation. L eukotriene B 4 (LTB 4 ) is a lipid mediator that is important for host defense, as it stimulates leukocyte chemotaxis, degranulation, phagocytosis, and superoxide generation (reviewed in ref. 1). The underproduction of leukotrienes (LTs) results in susceptibility to infection (2, 3), whereas the overproduction of LTB 4 contributes to inflammatory disease (4, 5). The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of LTB 4 from arachidonic acid (AA). Thus, the action of 5-LO is a key point of regulation in LTB 4 synthesis.In resting leukocytes, 5-LO is a soluble enzyme. Elevated intracellular calcium, as occurs upon cell stimulation, causes translocation of 5-LO to nuclear and perinuclear membranes (6, 7). Membrane association places the enzyme close to its substrate and initiates LT synthesis. LT synthesis can also be stimulated by activation of p38 mitogen-activated protein kinase (MAPK) (8), which is thought to be due to phosphorylation of 5-LO on Ser-271 by MAPK-activated protein (MAPKAP) kinase 2 (9) and phosphorylation-dependent nuclear association of 5-LO (10).Entirely distinct from its ability to move to membranes upon cell stimulation, 5-LO can redistribute in unstimulated cells; 5-LO can be imported into the nucleus (11, 12), and it can be exported from the nucleus (13). The import of soluble 5-LO protein, through nuclear pores on the nuclear envelope, is mediated by three distinct nuclear localization signals (NLSs) on 5-LO (14, 15). The import process can be triggered by adherence (11,12) or by cytokines (16,17). Movement of 5-LO from the cytoplasm into the interior of the nucleus also occurs during leukocyte recruitment into site...

A partial nucleotide sequence that included 1,693 base pairs of the M12 (emml2) gene of group A streptococci (strain CS24) and adjacent upstream DNA was determined. Type 12 M protein-specific mRNA of strain CS24 is transcribed from two promoters (P, and P3) (35) it has been shown that the switch from high to low levels of M-protein expression occurs at high frequency and is reversible, which are characteristics of phase variation described for other bacterial species (7,26). In addition to phase variation of M-protein expression (35), streptococci also exhibit both antigenic variation (more than 70 strains with antigenically distinct M proteins have been described) and size variation of this protein (11). The mechanisms that control phase and antigenic variation and the relationship between them is not understood. Although the nature of M protein as an antiphagocytic molecule has been demonstrated, a role for the M-state in the survival of the organism has not been identified. Phase variation has been postulated to be a virulence factor that controls the expression of the type 1 pilus of Escherichia coli (6, 7) and the pilus of Neisseria gonorrhoeae (26 report results of a study of the role of upstream neighboring sequences in the regulation of expression of the emmi2 gene of strain CS24 in E. coli, describe the physical relationship between the deletion in strain CS64 and the 5' end of the emml2 gene, and study the transcriptional activity of the emml2 gene by Northern and primer extension analyses. We found that transcription of the emml2 gene in strain CS64 is diminished by more than 100-fold and that the deletion is more than 400 bases upstream from the transcription start sites of the emml2 gene. MATERIALS AND METHODSBacterial strains, plasmids, bacteriophage, and media. M type 12 group A streptococcal strain CS24 and variant CS64 have been described previously (3,39). Liquid cultures of group A streptococci were grown in Todd-Hewitt broth supplemented with 1% Neopeptone (Difco Laboratories, Detroit, Mich.) for 15 to 18 h at 37°C. E. coli JM83, JM103, and JM110, carrying constructs of plasmids pUC19 and pUC18, were propagated in L broth or on L agar containing ampicillin (30 ,ug/ml) and 5-bromo-4-chloro-3-indolyl-P-Dgalactoside (0.03%) at 37°C. All plasmids used and constructed in this study are listed in Fig. 1

No abstract

The Serratia entomophila antifeeding prophage (Afp) is thought to form a virus-like structure that has activity towards the New Zealand grass grub, Costelytra zealandica. Through the trans based expression of AnfA1, an RfaH - like transcriptional antiterminator, the Afp, was able to be induced. The expressed Afp was purified and visualized by electron microscopy. The Afp resembled a phage tail-like bacteriocin, exhibiting two distinct morphologies: an extended and a contracted form. The purified Afp conferred rapid activity towards C. zealandica larvae, causing cessation of feeding and a change to an amber colouration within 48 h postinoculation, with increased dose rates causing larval mortality.

The adequacy of lymph node dissection of colonic resection specimens influences the clinical and pathologic staging, leading to important postsurgical treatment decisions. Although manual lymph node dissection is the current standard at most institutions, recent statistical studies indicate that all lymph nodes, including those measuring 1-2 mm, should be recovered to be assured of lymph node negative status. Thus, we tested the efficacy of gross dissection by submitting the entire residual mesenteric fat. We analyzed 15 randomly chosen colonic resections (2 pT1, 1 pT2, 11 pT3, 1 pT4). After standard gross dissection of lymph nodes and submission of colonic material for diagnosis, the entire remaining mesenteric material was dehydrated over several days by serial washing in alcohol and acetone. All of the mesenteric tissue was submitted for histology. The average number of nodes found by original gross inspection was 20.8, while the average number of additional nodes found after clearing was 68.6. In all, 83% of the additional nodes were 2.0 mm or less in size. There were seven pN0 cases; one was upstaged by additional findings that may have been artifactual. There were four pN1 cases; three were upstaged to pN2 after submission of the mesenteric material. All four pN2 tumors had additional metastases identified. In all, 75% of all positive nodes were under 2.0 mm in size. In this limited sample, standard gross dissection proved sufficient for most pN0 tumors to remain node negative. However, our findings within the pN1 group show that examination of all of the mesenteric material may be necessary to be assured of correct pN status. Keywords: colon; adenocarcinoma; lymph node; clearing; dissection; staging; prognosisThe presence or absence of lymph node metastasis is pivotal for predicting the clinical outcome of patients who have undergone radical surgery for colorectal carcinoma.1 Lymph nodes are an integral component of the TNM classification system, a major determinant of adjunct therapy, and a prognostic marker in colorectal adenocarcinoma.2 Recovering a greater number of lymph nodes guards against missing a lymph node metastasis and allows for more accurate staging of patients. The identification of a single lymph node metastasis is sufficient to offer adjunctive therapy. The most important determinant of survival is the presence or absence of metastases in regional lymph nodes.3 Thus, the detection and examination of the largest possible number of lymph nodes are essential for correct staging, therapeutic decisions, and prognosis. 4,5 Despite claims of a meticulous search for lymph nodes in surgical specimens, wide variations in the number of total nodes and lymph node metastases continue to exist. 2,6,7 This variation may be due to the size of the specimen, the number of regional lymph nodes present in the specimen, and the number of nodes with metastases present.3,8 Pathologists also vary in their diligence, skill, and patience in dissecting the lymph nodes of a surgical specimen. 9In this study, we enr...