Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Role of EP(2) and EP(3) PGE(2) receptors in control of murine renal hemodynamics

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.     

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.     

Solution behavior of hematin under acidic conditions and implications for its interactions with chloroquine

During the intraerythrocytic stage of its lifecycle, the malaria parasite digests host erythrocyte hemoglobin, producing free ferriprotoporhyrin IX (FP). Crystallization of FP into hemozoin is essential for its detoxification and is the target of quinoline antimalarials. To gain further insight into the mechanism of hemozoin formation and quinoline action we have studied the behavior of FP and related derivatives in 40% methanol in water at different concentrations across a broad pH range (2–12). The complex behavior of FP can be modeled by incorporating a pH-dependent dimerization constant that reflects the influence of the ionization state of the propionate groups on the level of self-association. The analysis reveals that aqua-ligated FP has a low propensity to self-associate and that the predominant self-associated species are homodimeric hydroxide-ligated FP and heterodimeric aqua/hydroxide-ligated FP. The latter is predicted to be the main self-associated species at the pH of the parasite digestive vacuole. The state of FP also affects its interaction with chloroquine, with maximum affinity under neutral conditions and a more than 1,000-fold decrease in affinity under acidic (pH 2) and basic (pH 12) conditions. First-derivative absorption spectra of the chloroquine–FP complex indicate that the high-affinity interaction requires the chloroquine ring in its neutral aminoquinoline form and this in turn requires at least one of the FP species in the complex to be aqua-ligated.     

Fenoldopam use in a burn intensive care unit: a retrospective study

Background

We sought to evaluate agreement between a new and widely implemented method of temperature measurement in critical care, temporal artery thermometry and an established method of core temperature measurement, bladder thermometry as performed in clinical practice.

Methods

Temperatures were simultaneously recorded hourly (n = 736 observations) using both devices as part of routine clinical monitoring in 14 critically ill adult patients with temperatures ranging ≥1°C prior to consent.

Results

The mean difference between temporal artery and bladder temperatures measured was -0.44°C (95% confidence interval, -0.47°C to -0.41°C), with temporal artery readings lower than bladder temperatures. Agreement between the two devices was greatest for normothermia (36.0°C to < 38.3°C) (mean difference -0.35°C [95% confidence interval, -0.37°C to -0.33°C]). The temporal artery thermometer recorded higher temperatures during hypothermia (< 36°C) (mean difference 0.66°C [95% confidence interval, 0.53°C to 0.79°C]) and lower temperatures during hyperthermia (≥38.3°C) (mean difference -0.90°C [95% confidence interval, -0.99°C to -0.81°C]). The sensitivity for detecting fever (core temperature ≥38.3°C) using the temporal artery thermometer was 0.26 (95% confidence interval, 0.20 to 0.33), and the specificity was 0.99 (95% confidence interval, 0.98 to 0.99). The positive likelihood ratio for fever was 24.6 (95% confidence interval, 10.7 to 56.8); the negative likelihood ratio was 0.75 (95% confidence interval, 0.68 to 0.82).

Conclusions

Temporal artery thermometry produces somewhat surprising disagreement with an established method of core temperature measurement and should not to be used in situations where body temperature needs to be measured with accuracy.     

A longitudinal study of medicaid coverage for tobacco dependence treatments in Massachusetts and associated decreases in hospitalizations for cardiovascular disease

Background

Insurance coverage of tobacco cessation medications increases their use and reduces smoking prevalence in a population. However, uncertainty about the impact of this coverage on health care utilization and costs is a barrier to the broader adoption of this policy, especially by publicly funded state Medicaid insurance programs. Whether a publicly funded tobacco cessation benefit leads to decreased medical claims for tobacco-related diseases has not been studied. We examined the experience of Massachusetts, whose Medicaid program adopted comprehensive coverage of tobacco cessation medications in July 2006. Over 75,000 Medicaid subscribers used the benefit in the first 2.5 years. On the basis of earlier secondary survey work, it was estimated that smoking prevalence declined among subscribers by 10% during this period.

Methods and Findings

Using claims data, we compared the probability of hospitalization prior to use of the tobacco cessation pharmacotherapy benefit with the probability of hospitalization after benefit use among Massachusetts Medicaid beneficiaries, adjusting for demographics, comorbidities, seasonality, influenza cases, and the implementation of the statewide smoke-free air law using generalized estimating equations. Statistically significant annualized declines of 46% (95% confidence interval 2%–70%) and 49% (95% confidence interval 6%–72%) were observed in hospital admissions for acute myocardial infarction and other acute coronary heart disease diagnoses, respectively. There were no significant decreases in hospitalizations rates for respiratory diagnoses or seven other diagnostic groups evaluated.

Conclusions

Among Massachusetts Medicaid subscribers, use of a comprehensive tobacco cessation pharmacotherapy benefit was associated with a significant decrease in claims for hospitalizations for acute myocardial infarction and acute coronary heart disease, but no significant change in hospital claims for other diagnoses. For low-income smokers, removing the barriers to the use of smoking cessation pharmacotherapy has the potential to decrease short-term utilization of hospital services. Please see later in the article for the Editors'' Summary     

Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production

Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation.     

Engineering primary metabolic pathways of industrial micro-organisms

Metabolic engineering is a powerful tool for the optimisation and the introduction of new cellular processes. This is mostly done by genetic engineering. Since the introduction of this multidisciplinary approach, the success stories keep accumulating. The primary metabolism of industrial micro-organisms has been studied for long time and most biochemical pathways and reaction networks have been elucidated. This large pool of biochemical information, together with data from proteomics, metabolomics and genomics underpins the strategies for design of experiments and choice of targets for manipulation by metabolic engineers. These targets are often located in the primary metabolic pathways, such as glycolysis, pentose phosphate pathway, the TCA cycle and amino acid biosynthesis and mostly at major branch points within these pathways. This paper describes approaches taken for metabolic engineering of these pathways in bacteria, yeast and filamentous fungi.     

Roles of phosphatidylinositol 3-kinase and NF-kappaB in human cytomegalovirus-mediated monocyte diapedesis and adhesion: strategy for viral persistence

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-kappaB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the beta(1) integrins, the beta(2) integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-kappaB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-kappaB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.