Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Neural regulation of slow-wave frequency in the murine gastric antrum

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.     

Use of a Modified Pediatric Early Warning Score in a Department of Pediatric and Adolescent Medicine

Background

Several versions of the Pediatric Early Warning Score (PEWS) exist, but there is limited information available on the use of such systems in different contexts. In the present study, we aimed to examine the relationship between a modified version of The Brighton Paediatric Early Warning Score (PEWS) and patient characteristics in a Norwegian department of pediatric and adolescent medicine. In addition, we sought to establish guidelines for escalation in patient care based on the PEWS in our patient population.

Methods

The medical records of patients referred for acute care from March to May 2011 were retrospectively reviewed. Children with a PEWS ≥3 were compared to children with a PEWS 0–2 with regard to age, diagnostic group and indicators of severe disease.

Results

A total of 761 patients (0−18 years of age) were included in the analysis. A younger age and diagnostic groups such as lower airway and cardiovascular disease were associated with PEWS ≥3. Upper airway disease and minor injury were more frequent in patients with PEWS 0−2. Children with PEWS ≥3 received fluid resuscitation, intravenous antibiotics, and oxygen supplementation, and were transferred to a higher level of care more often than children with PEWS 0−2.

Conclusions

A PEWS ≥3 was associated with severe illnesses and surrogate markers of cardio-respiratory compromise. Patients with PEWS ≥3 should be carefully monitored to prevent further deterioration.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Staphylococcus aureus ClpC ATPase is a late growth phase effector of metabolism and persistence

Staphylococcus aureus Clp ATPases (molecular chaperones) alter normal physiological functions including an aconitase‐mediated effect on post‐stationary growth, acetate catabolism, and entry into death phase (Chatterjee et al., J. Bacteriol. 2005, 187, 4488–4496). In the present study, the global function of ClpC in physiology, metabolism, and late‐stationary phase survival was examined using DNA microarrays and 2‐D PAGE followed by MALDI‐TOF MS. The results suggest that ClpC is involved in regulating the expression of genes and/or proteins of gluconeogenesis, the pentose‐phosphate pathway, pyruvate metabolism, the electron transport chain, nucleotide metabolism, oxidative stress, metal ion homeostasis, stringent response, and programmed cell death. Thus, one major function of ClpC is balancing late growth phase carbon metabolism. Furthermore, these changes in carbon metabolism result in alterations of the intracellular concentration of free NADH, the amount of cell‐associated iron, and fatty acid metabolism. This study provides strong evidence for ClpC as a critical factor in staphylococcal energy metabolism, stress regulation, and late‐stationary phase survival; therefore, these data provide important insight into the adaptation of S. aureus toward a persister state in chronic infections.     

Effect of GLG-V-13, a class III antiarrhythmic agent, on potassium currents in rabbit ventricular myocytes

The effects of a new Class III antiarrhythmic drug, GLG-V-13, on the 4-aminopyridine sensitive transient outward current, on the inward rectifier potassium current, on the ATP sensitive potassium current and on the rapid and slow components of the delayed rectifier potassium current were studied in single rabbit ventricular myocytes using the whole-cell voltage-clamp technique. GLG-V-13 blocked the rapid component of the delayed rectifier potassium current in a dose-dependent manner, with an estimated EC50 value of 0.36 microM. At high concentration, the slow component of the delayed rectifier potassium current was also depressed by the drug (40% effect at 10 microM concentration). The transient outward current, the inward rectifier potassium current and the ATP sensitive potassium current were not influenced by GLG-V-13, even at 10 microM concentration. Thus, GLG-V-13 blocks predominantly the rapid component of the delayed rectifier potassium current which may play a significant role in the prolongation of repolarization by the drug in ventricular tissue.     

Ammonia formation and soil pH increase caused by decomposing fruitbodies of macrofungi

The formation of gaseous compounds from decomposing fruibodies of three common macrofungi was qualitatively analysed. It was shown that significant production of ammonia took place. When the fruitbodies were decomposing on the surface of a podzolic forest floor more than 90% of the ammonia produced was absorbed by the soil, causing soil pH to increase.