To test or not: A registry-based observational study of an online decision support for prostate-specific antigen tests

BackgroundWatchful waiting is an essential part in the handling of patients with prostate cancer (PC). More effective disease management may be achieved by this strategy. Correct use of prostate-specific antigen (PSA) test is crucial and the general practitioner (GP) may therefore benefit from access to a clinical decision support system (CDSS) that focuses on this challenge. There are many barriers to the use of CDSSs. The aim of the present paper is to study if such barriers may be overcome by granting GPs easier access to a web-based CDSS via a hyperlink in the GPs’ electronic medical record system (EMR).MethodsIn the present population-based observational registry study with an intervention and control group, we created a web-based CDSS that was made accessible to GPs via hyperlink inserted into the EMR medical chart contents. The intervention was introduced 1 January 2011. Our outcome measure was the number of age-standardised PSA test rates per 1000 men per practice during three equally sized periods of 6 months within the study period from 1 January 2010 to 30 June 2011.ResultsWe found that none of the differences between intervention and control groups were statistically significant.ConclusionsProviding GPs with access to a CDSS to aid their decision to use the PSA, had no measurable effect on the GPs’ PSA testing behaviour.     

Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8⁺ T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8⁺ T cells and perforin-producing effector CD8⁺ T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.     

Estrogen synthesis in the brain—Role in synaptic plasticity and memory

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200–1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus.Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1–10 nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2 h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.     

CSN5/Jab1 inhibits cardiac L-type Ca2+ channel activity through protein-protein interactions

L-type Ca(2+) channels have a wide tissue distribution and play essential roles in physiological responses. Recent studies have indicated that regulation of L-type Ca(2+) channels involves the assembly of macromolecular signaling complexes such as the beta(2)-adrenergic receptor signaling complex, the small G-protein kir/Gem and the BK channel. Here, we report the previously unidentified role of another protein in binding to the II-III linker of the alpha(1C) subunit of the L-type Ca(2+) channel. This protein is COP9 signalosome subunit 5 (CSN5)/Jun activation domain-binding protein 1 (Jab1). We have demonstrated that CSN5 interacts specifically with the II-III linker of the alpha(1C) subunit in a yeast two-hybrid system. The alpha(1C) subunit and CSN5 were coimmunoprecipitated in rat heart and both proteins were colocalized in sarcolemmal membranes and transverse tubules of cardiac myocytes. Silencing of CSN5 mRNA using siRNA decreased the endogenous protein level of CSN5 and activated L-type Ca(2+) channels expressed in COS7 cells. These data indicate that CSN5 is a protein that plays a newly defined functional role in association with the cardiac L-type Ca(2+) channel.     

Hepatosplenic αβ T-Cell Lymphoma with Myelodysplastic Syndrome

We describe a patient with hepatosplenic 33 T-cell lymphoma who showed pancytopenia and myelodysplasia. A 35-year-old man was admitted with fever, pancytopenia, and hepatosplenomegaly but with no lymphadenopathy. We also found trilineage myelodysplasia in the bone marrow on his first admission.The patient had high fever and anemia but no evidence of infection and was tentatively treated with prednisolone. This treatment resulted in a transient improvement of the cytopenia and a reduction of spleen size. However, 10 months after the first manifestation, progression of the splenomegaly and fever became apparent, and a splenectomy was performed. The pathologic findings for the spleen showed diffuse and disseminated infiltration of medium- to large-sized T-lymphocytes in the splenic red pulp. These cells were immunohistochemically positive for CD3, CD5, CD7, CD8, CD16, CD56,T-cell receptor 33 (TCR33),T-cell intracellular antigen 1, and granzyme B but were negative for CD4, CD30, CD57, and TCR33. These data suggested a diagnosis of hepatosplenic 33 T-cell lymphoma. A Southern blot analysis revealed gene rearrangement of the TCR 3-chain gene but not the immunoglobulin heavy chain gene in the spleen cells. An in situ hybridization analysis for the Epstein-Barr virus revealed negative results. The patient received 8 courses of combination chemotherapy and achieved a partial remission; however, the dysplastic features of the marrow cells persisted after the partial remission was obtained. Additional treatment with allogeneic bone marrow transplantation resulted in a transient complete remission; however, the patient relapsed 11 months later. Because he had experienced no lymphadenopathy and showed dysplastic features in the bone marrow, the diagnosis was highly dependent on the pathologic findings for the resected spleen.     

Simvastatin antagonizes tumor necrosis factor-alpha inhibition of bone morphogenetic proteins-2-induced osteoblast differentiation by regulating Smad signaling and Ras/Rho-mitogen-activated protein kinase pathway

Recent studies have shown that the mevalonate pathway plays an important role in skeletal metabolism. Statins stimulate bone morphogenetic proteins-2 (BMP-2) production in osteoblasts, implicating a possible beneficial role for statins in promoting anabolic effects on bone. Here, we investigated the effects of a lipophilic simvastatin on osteoblast differentiation using mouse myoblast C2C12 cells, in the presence of tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine that inhibits osteogenesis. The addition of TNF-alpha to C2C12 cells suppressed the BMP-2-induced expression of key osteoblastic markers including Runx2 and alkaline phosphatase (ALP) activity. Simvastatin had no independent effects on Runx2 and alkaline phosphatase activity; however, it reversed the suppressive effects of TNF-alpha. The ability of simvastatin to reverse TNF-alpha inhibition of BMP-induced Smad1,5,8 phosphorylation and Id-1 promoter activity suggests the involvement of Smad signaling pathway in simvastatin action. In addition, cDNA array analysis revealed that simvastatin increased expression levels of Smads in C2C12 cells exposed to TNF-alpha that also activated mitogen-activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase 1/2 (ERK1/2), P38, and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Simvastatin potently suppressed TNF-alpha-induced phosphorylation of ERK1/2 and SAPK/JNK by inhibiting TNF-alpha-induced membrane localization of Ras and RhoA. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) reversed the simvastatin effects on TNF-alpha-induced activation of Ras/Rho/MAPK pathways. FPP and GGPP also restored the simvastatin effects on TNF-alpha-induced suppression of Runx2 and ALP activity. In addition, simvastatin decreased the expression levels of TNF type-1 and -2 receptor mRNAs. Collectively, simvastatin supports BMP-induced osteoblast differentiation through antagonizing TNF-alpha-to-Ras/Rho/MAPK pathway and augmenting BMP-Smad signaling, suggesting a potential usage of statins to ameliorate inflammatory bone damage.     

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

AIMS/HYPOTHESIS: Na(+)/K(+)-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na(+)/K(+)-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined. METHODS: Isolated islets from Wistar rats and diabetic Goto-Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2',7'-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed. RESULTS: Ouabain caused a rapid Tyr(418) phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets. CONCLUSIONS/INTERPRETATION: These results indicate that Src plays a role in the signal-transducing function of Na(+)/K(+)-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.     

Isolation of Circular DNA Molecules from Whole Cellular DNA by Use of ATP-Dependent Deoxyribonuclease

A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (lambdadv1 and lambdadv21), a colicinogenic factor (Col E2), a sex factor (F(8)' gal), and "minicircles" in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique.