1.アクラシノマイシンAのマウスL細胞に対する細胞致死効果および細胞周期へ及ぼす影響(第576回千葉医学会例会・第1回千葉大学小児外科教室例会)

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Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated Δycf54 Strain of the Cyanobacterium Synechocystis PCC 6803

In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use 35S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b6f and the HliD– Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed

The Heart Is an Early Target of Anthrax Lethal Toxin in Mice: A Protective Role for Neuronal Nitric Oxide Synthase (nNOS)

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS−/− mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS−/− mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS−/− and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS−/− mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS−/− and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012

OBJECTIVE: To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008. DESIGN: A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (1) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (1) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations. RESULTS: Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within 1 h of the recognition of septic shock (1B) and severe sepsis without septic shock (1C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (1B); infection source control with attention to the balance of risks and benefits of the chosen method within 12 h of diagnosis (1C); initial fluid resuscitation with crystalloid (1B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (1B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (1C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (1B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (1C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (1B); low tidal volume (1A) and limitation of inspiratory plateau pressure (1B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (1B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤100 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (1B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (1C); protocols for weaning and sedation (1A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (1B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (1C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) 180 mg/dL, targeting an upper blood glucose ≤180 mg/dL (1A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (1B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (1B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-10 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C). CONCLUSIONS: Strong agreement existed among a large cohort of international experts regarding many level 1 recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients

Medical Guidelines and Performance Measures: The Need to Keep Them Free of Industry Influence

To be unbiased and represent best practice, medical guidelines should be free of industry influence, carefully vetted among experts in the field and encompass treatments widely accepted based on difficult to refute evidence ( Shaneyfelt, Mayo-Smith and Rothwang, 2006; Grilli, et al. , 2000; Grissom, 2000). These requirements are even more important if individual guideline recommendations are to be grouped into "bundles" to serve as performance measures and provide a possible basis for reimbursement. In a fall 2006 issue of the New England Journal of Medicine , we presented our concerns when these requirements are not met ( Eichacker, Natanson and Danner, 2006 ). As an example, which directly affected us as physicians in the field of critical care medicine, we described the close relationship between the development and implementation of the Surviving Sepsis Campaign guidelines and the marketing efforts of Eli Lilly and Company, the primary financial sponsor for this campaign. Considering solutions to such a problem in the context of our original concerns is worthwhile. [No abstract available.

Lethal and Edema Toxins in the Pathogenesis of Bacillus anthracis Septic Shock: Implications for Therapy

Recent research regarding the structure and function of Bacillus anthracis lethal (LeTx) and edema (ETx) toxins provides growing insights into the pathophysiology and treatment of shock with this lethal bacteria. These are both binary-type toxins composed of protective antigen necessary for their cellular uptake and either lethal or edema factors, the toxigenic moieties. The primary cellular receptors for protective antigen have been identified and constructed and key steps in the extracellular processing and internalization of the toxins clarified. Consistent with the lethal factor's primary action as an intracellular endopeptidase targeting mitogen-activated protein kinase kinases, growing evidence indicates that shock with this toxin does not result from an excessive inflammatory response. In fact, the potent immunosuppressive effects of LeTx may actually contribute to the establishment and persistence of infection. Instead, shock with LeTx may be related to the direct injurious effects of lethal factor on endothelial cell function. Despite the importance of LeTx, very recent studies show that edema factor, a potent adenyl cyclase, has the ability to make a substantial contribution to shock caused by B. anthracis and works additively with LeTx. Furthermore, ETx may contribute to the immunosuppressive effects of LeTx. Therapies under development that target several different steps in the cellular uptake and function of these two toxins have been effective in in vitro and in vivo systems. Understanding how best to apply these agents clinically and how they interact with conventional treatments should be goals for future research