Regulation of prostaglandin biosynthesis by interleukin-1 in cultured bovine endometrial cells

Interleukin-1 (IL1) has been shown to be a potent stimulator of prostaglandin (PG) production in bovine endometrium. The aim of the present study was to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGE2 and PGF2alpha in response to IL1A, and the intracellular mechanisms of IL1A action. Cultured bovine epithelial and stromal cells were exposed to IL1A or IL1B (0.006-3.0 nM) for 24 h. IL1A and IL1B dose-dependently stimulated PGE2 and PGF2alpha production in the stromal cells, but not in the epithelial cells. The stimulatory effect of IL1A (0.06-3.0 nM) on PG production was greater than that of IL1B. The stimulatory actions of IL1A on PG production was augmented by supplementing arachidonic acid (AA). When the stromal cells were incubated with IL1A and inhibitors of phospholipase (PL) C or PLA2 (1 microM; anthranilic acid), only PLA2 inhibitor completely stopped the stimulatory action of IL1A on PG production. Moreover, a specific cyclooxygenase-2 (COX2) inhibitor blocked the stimulatory effect of IL1A on PG production. IL1A (0.06 nM) promoted COX2 and microsomal PGE synthase-1 (PGES1) gene and its protein expression. The expression of COX1, PGES2, PGES3, and PGF synthase (PGFS) mRNA was not affected by IL1A in the stromal cells. The overall results indicate that 1) the target of IL1A and IL1B for stimulating both PGE2 and PGF2alpha production is the stromal cells, 2) IL1A is a far more potent stimulator than IL1B on PG production in stromal cells, 3) the stimulatory effect of IL1A on PG production is mediated via the activation of PLA2 and COX2, and (4) IL1A induced PG production by increasing expressions of COX2 and PGES1 mRNAs and their proteins in bovine stromal cells.     

R-wave amplitude response to myocardial ischemia in hypertrophic cardiomyopathy

Background and Purpose

R-wave amplitude change during exercise has been reported to enhance diagnostic value for myocardial ischemia in coronary heart disease.

Methods

We summed up R-wave amplitude in all the 12 leads during exercise testing and correlated the results with regional myocardial ischemia or diffuse subendocardial ischemia as detected by scintigraphy in 49 patients with hypertrophic cardiomyopathy (HCM) and 16 controls.

Results

The sum of R-wave amplitude decreased during exercise in patients with HCM (mean, 12.4 mV to 11.7 mV, P < .01) as well as in controls (8.0 mV to 7.7 mV, P < .05). Percent changes in the sum of R-wave amplitude did not differ between 4 subgroups of patients with HCM: one having both regional and subendocardial ischemia, one only the former, one only the latter, and one neither of them (mean, 6.5%, 7.7%, 4.6%, and 5.1%; P = .79).

Conclusions

R-wave amplitude response to exercise failed to demonstrate myocardial ischemia in our patients with HCM.     

Histopathologically-diagnosed splenic metastasis in a hepatocellular carcinoma case with adrenal metastasis

We encountered a patient with hepatocellular carcinoma (HCC), with adrenal gland metastasis, in whom splenic metastasis was diagnosed histopathologically. A 59-year-old man visited our hospital in May 2001 with chief complaints of abdominal distension and pretibial pitting edema. Multiple HCCs associated with HCV-positive liver cirrhosis were detected. Transarterial embolization (TAE) was performed a total of 4 times for HCCs. A left adrenal gland metastatic lesion was detected and it was found to increase in diameter from 3 cm to 6 cm over a four-month period; left adrenalectomy was performed in June 2002. Because of marked splenomegaly and findings of hypersplenism, the spleen was also resected. Although no metastatic lesions were evident on macroscopic examination of the spleen, a small metastatic lesion from moderately differentiated HCC, approximately 0.5 mm in diameter, was detected histopathologically. Splenic metastasis from HCC is rare, usually occurring with metastases involving other organs. Our patient also had adrenal gland metastasis. Therefore, hematogenous metastasis to the congested spleen via the systemic circulation was suspected.     

Acquired deficit of forebrain glucocorticoid receptor produces depression-like changes in adrenal axis regulation and behavior

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of major depressive disorder. A number of studies have shown that this dysregulation is correlated with impaired forebrain glucocorticoid receptor (GR) function. To determine whether a primary, acquired deficit in forebrain GR signaling is an etiologic factor in the pathogenesis of depression, we generated a line of mice with time-dependent, forebrain-specific disruption of GR (FBGRKO). These mice develop a number of both physiological and behavioral abnormalities that mimic major depressive disorder in humans, including hyperactivity of the HPA axis, impaired negative feedback regulation of the HPA axis and, increased depression-like behavior. Importantly, a number of these abnormalities are normalized by chronic treatment with the tricyclic antidepressant, imipramine. Our findings suggest that imipramine's proposed activities on forebrain GR function are not essential for its antidepressant effects, and that alteration in GR expression may play a causative role in disease onset of major depressive disorder.     

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB''s poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB’s ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein–DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.DNA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes (1). Although typically transposons exhibit no strong sequence selectivity for the target DNA site, certain transposons avoid self-destructive insertion (reviewed in ref. 2), a phenomenon called “target immunity” because the presence of a copy of the transposon renders nearby DNA sites “immune” to additional insertion by the same transposon (3–8). MuB plays critical roles in this self-immunity in the bacteriophage Mu transposition process.Phage Mu is one of the most complex and efficient transposable elements (reviewed in refs. 3 and 4). Two phage-coded proteins, MuA and MuB, are essential for efficient Mu transposition. MuA is the transposase responsible for synapsing the two Mu end sequences and for all of the DNA cutting and joining steps in the initial stages of transposition. However, transposition is inefficient in the absence of MuB, and the residual Mu insertion that takes place uses only DNA target sites near or within the transposing element, often leading to self-destruction (9–11). MuB is a small (35-kDa) ATP-dependent nonspecific DNA-binding protein with relatively low ATPase activity (10, 12, 13). Upon ATP binding, MuB polymerizes preferentially on DNA, but in the absence of DNA it still can form polymers of variable sizes (14, 15). When observed by total internal reflection fluorescence microscopy, GFP-MuB-ATP binds along the DNA molecule forming many short separate segments of polymers, and, as more GFP-MuB is added, the protein-covered segments elongate to form an apparently continuous polymer that fully coats the DNA. Hydrolysis of ATP reverses this process, triggering disassembly of the MuB polymer (16–18).The MuB–DNA complex controls MuA activity at several steps, including stimulation of the strand-transfer reaction, which joins Mu DNA ends to the target DNA site (19, 20), leading to utilization of MuB-bound DNA as the preferred target for transposition (11). MuB-stimulated transposition displays target immunity, avoiding target sites near preexisting Mu-end sequences where MuA binds. This is because MuA stimulates the ATPase activity of MuB, and because this interaction takes place more frequently when the two proteins are bound on the same DNA molecule relatively close to each other, MuA triggers the dissociation of MuB from DNA sites near the Mu ends more efficiently than from those sites at a distance (21). Thus, by the time MuA assembles the active complex with synapsed Mu DNA ends ready to capture target DNA, MuB has been depleted from the vicinity of the active complex and accumulates on DNA sites away from the Mu-end sequence. This explains the strong preference of the transposing Mu to target DNA sites at substantial distances away (5–25 kb) from its original site (22).Beyond the mechanistic framework outlined above, study of MuB function has been hampered by the lack of structural information. The 312-amino-acid MuB polypeptide can be divided into two fragments by limited proteolysis (23). The N-terminal 25-kDa fragment contains Walker A and Walker B motifs (24) and also a putative helix-turn-helix motif (12), but it lacks ATPase activity and is prone to aggregate. The C-terminal 10-kDa fragment consists of a four-helix bundle, whose structure was determined by NMR spectroscopy and reported to resemble the N-terminal domain of Escherichia coli replicative helicase DnaB (25) (Fig. S1). Beyond this, no sequence similarity between MuB and other proteins has been reported. Studies of mutant proteins have been limited mostly to those involving the Walker motifs (26). MuB’s tendency to aggregate in the presence of ATP hampered efforts at structural characterization. Without such information, it has not been clear how ATP controls MuB polymerization, what kind of polymer MuB forms, how MuB polymer interacts with DNA, or how MuA interacts with MuB polymer and triggers ATP hydrolysis and DNA dissociation.In this study we combine bioinformatic, mutagenic, biochemical, and electron microscopy (EM) techniques to characterize the structure of MuB and its role in DNA binding and transposition. First, we present evidence that MuB is a member of the AAA+ (ATPases Associated with diverse cellular Activities) ATPase superfamily. A large number of these proteins have been studied structurally and functionally (27, 28). Guided by this finding, we identified the critical residues that control MuB ATPase, DNA substrate binding, and protein polymerization, yielding a clear picture of the communication between MuB and its interaction partners, MuA and DNA. Furthermore, we studied the structure of MuB polymers by cryo-EM and image reconstruction. We demonstrate that MuB-ATP forms a solenoid-like filament (150 Å in diameter with a pitch of 48 Å) and that DNA is bound in the axial channel. The helical parameters of the MuB filament (5.4 subunits and 48-Å per turn) do not match those of the B-form DNA (10 bp and 36 Å per turn). However, despite this mismatched symmetry between the protein and DNA, MuB does not significantly distort the bound DNA duplex. Therefore, individual MuB subunits in the filament must interact with DNA nonuniformly.     

Rapid and efficient detection method of Norwalk virus

A rapid and efficient RT-PCR with fluorogenic probe(TaqMan-PCR) was developed for detection of Norwalk virus(NV) genomes in clinical specimens. We designed NV genogroup specific primers and fluorogenic probes in the junction of open reading frame (ORF)1 and ORF2. Eighty specimens from patients of gastroenteritis, in which NV-like particles were detected by electron microscopy, were examined by TaqMan-PCR and RT-PCR using primer sets previously reported; two sets in RdRp region of NV and one set in capsid region. TaqMan-PCR detected NV genome from 79 of 80 specimens(98.8%); this method showed the highest sensitivity of all other RT-PCR tested. Moreover, TaqMan-PCR was considered to be useful to recognize genogroup of NV correctly.     

Prognostic significance of extracellular volume fraction with equilibrium contrast-enhanced computed tomography for pancreatic neuroendocrine neoplasms

Background/Objectives: Identifying reliable pretreatment imaging biomarkers for pancreatic neuroendocrine neoplasm (PanNEN) is a key imperative. Extracellular volume (ECV) fraction quantified with equilibrium contrast-enhanced CT can be easily integrated into routine examinations. This study aimed to determine whether ECV fraction with equilibrium contrast-enhanced computed tomography (CECT) could predict long-term outcomes in patients with PanNEN.MethodsThis study was a retrospective observational study of 80 patients pathologically diagnosed with PanNEN at a single institution. ECV fraction of the primary lesion was calculated using region-of-interest measurement within PanNEN and the aorta on unenhanced and equilibrium CECT. The impact of clinical factors and tumor ECV fraction on progression-free survival (PFS) and overall survival (OS) was assessed with univariate and multivariate analyses using Cox proportional hazards models. The correlation between WHO classification and tumor ECV fraction was evaluated using Kendall rank correlation coefficients.ResultsPFS and OS rates were estimated as 93.4% and 94.6%, 78.7% and 86.2%, 78.7% and 77.0%, and 78.7% and 66.6% at 1, 3, 5, and 10 years, respectively. Multivariate analysis revealed that Union for International Cancer Control (UICC) stage (hazard ratio [HR] = 3.95, P = 0.003), WHO classification (HR = 12.27, P = 0.003), and tumor ECV fraction (HR = 11.93, P = 0.039) were independent predictors of PFS. Patient age (HR = 1.11, P < 0.001), UICC stage (HR = 3.14, P = 0.001), and tumor ECV fraction (HR = 5.27, P = 0.024) were independent significant variables for predicting OS. Tumor ECV fraction had a weak inverse relationship with WHO classification (P = 0.045, τ = ?0.178).ConclusionsECV fraction determined by equilibrium CECT and UICC stage may predict survival in patients with PanNEN.     

Reduced expression and functional impairment of Toll-like receptor 2 on dendritic cells in chronic hepatitis C virus infection.

BACKGROUND: Dendritic cells (DCs) utilize Toll-like receptors (TLRs) to sense virus and initiate immune responses. We aimed at elucidating the roles of TLRs on DCs in hepatitis C virus (HCV) infection. METHODS: Monocyte-derived DCs were obtained from 32 healthy volunteers (HV) and 30 chronically HCV-infected patients (CH). TLR2, TLR3 and TLR4 expressions on immature DCs were quantified by real-time quantitative RT-PCR. We stimulated DCs with specific TLR ligands and examined DC maturation, cytokine production and ability to stimulate allogeneic CD4(+) T cells. RESULTS: TLR2 expression on immature DCs was lower in the CH group, whereas those of TLR3 or TLR4 were not different between the groups. Each TLR ligand induced DC maturation and stimulated them to release comparable levels of IL-12p70, IL-6, IL-10, TNF-alpha and IFN-beta between the groups. TLR2 and TLR4 ligands enhanced DC ability to stimulate T cell proliferation, with the degree due to the TLR2 ligand being lower in the CH group. CONCLUSIONS: In HCV infection, the TLR2 expression on DCs is reduced and TLR2-stimulated DCs show lesser ability to proliferate T cells than healthy counterparts, suggesting that the TLR2 system is involved in HCV-induced immunopathogenesis.