Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.     

Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

Dynamics of proteins predicted by molecular dynamics simulations and analytical approaches: application to alpha-amylase inhibitor

The dynamics of alpha-amylase inhibitors has been investigated using molecular dynamics (MD) simulations and two analytical approaches, the Gaussian network model (GNM) and anisotropic network model (ANM). MD simulations use a full atomic approach with empirical force fields, while the analytical approaches are based on a coarse-grained single-site-per-residue model with a single-parameter harmonic potential between sufficiently close (r 相似文献   

Evaluation of antral follicle growth in the macaque ovary during the menstrual cycle and controlled ovarian stimulation by high‐resolution ultrasonography

To date, ultrasonography of monkey ovaries is rare and typically of low resolution. The objectives of this study were to use state‐of‐the‐art, high‐resolution, transabdominal ultrasonography with real‐time Doppler capabilities to: (1) determine whether one can reliably detect in real time the large dominant follicle, the corpus luteum (CL), and small (<2 mm) antral follicles on the ovaries of rhesus monkeys during the natural menstrual cycle; and (2) predict the follicular response of rhesus ovaries to controlled ovarian stimulation (COS) protocols. Rhesus monkeys were selected for transabdominal ultrasonography using a GE Voluson 730 Expert Doppler System at discrete stages of the menstrual cycle. Subsequently, serial ultrasound scanning was employed to observe growth of antral follicles and the CL. Finally, females were scanned to assess follicular growth during COS. The dominant structure and small antral follicles (<2 mm) were reliably visualized in real time. The follicle destined to ovulate could be identified by size differential by day 3 of the follicular phase. The number of small antral follicles present before onset of COS protocol correlated positively with the number of metaphase II‐stage oocytes collected after treatment. The results of this study demonstrate that the population dynamics of antral follicle pools can be noninvasively evaluated in monkeys during natural and pharmacologic ovarian cycles. Am. J. Primatol. 71:384–392, 2009. © 2009 Wiley‐Liss, Inc.     

A comparative analysis of the equilibrium dynamics of a designed protein inferred from NMR,X‐ray,and computations

A detailed analysis of high‐resolution structural data and computationally predicted dynamics was carried out for a designed sugar‐binding protein. The mean‐square deviations in the positions of residues derived from nuclear magnetic resonance (NMR) models and those inferred from X‐ray crystallographic B‐factors for two different crystal forms were compared with the predictions based on the Gaussian Network Model (GNM) and the results from molecular dynamics (MD) simulations. GNM systematically yielded a higher correlation than MD, with experimental data, suggesting that the lack of atomistic details in the coarse‐grained GNM is more than compensated for by the mathematically exact evaluation of fluctuations using the native contacts topology. Evidence is provided that particular loop motions are curtailed by intermolecular contacts in the crystal environment causing a discrepancy between theory and experiments. Interestingly, the information conveyed by X‐ray crystallography becomes more consistent with NMR models and computational predictions when ensembles of X‐ray models are considered. Less precise (broadly distributed) ensembles indeed appear to describe the accessible conformational space under native state conditions better than B‐factors. Our results highlight the importance of using multiple conformations obtained by alternative experimental methods, and analyzing results from both coarse‐grained models and atomic simulations, for accurate assessment of motions accessible to proteins under native state conditions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

Oxygenation measurement by multi‐wavelength oxygen‐dependent phosphorescence and delayed fluorescence: catchment depth and application in intact heart

Oxygen delivery and metabolism represent key factors for organ function in health and disease. We describe the optical key characteristics of a technique to comprehensively measure oxygen tension (PO₂) in myocardium, using oxygen‐dependent quenching of phosphorescence and delayed fluorescence of porphyrins, by means of Monte Carlo simulations and ex vivo experiments. Oxyphor G2 (microvascular PO₂) was excited at 442 nm and 632 nm and protoporphyrin IX (mitochondrial PO₂) at 510 nm. This resulted in catchment depths of 161 (86) µm, 350 (307) µm and 262 (255) µm respectively, as estimated by Monte Carlo simulations and ex vivo experiments (brackets). The feasibility to detect changes in oxygenation within separate anatomical compartments is demonstrated in rat heart in vivo.

Schematic of ex vivo measurements.     

Effect of genetic variants of chemokine receptors on the development of myocardial infarction in Turkish population

Inflammation is a crucial component of coronary atherosclerosis and myocardial infarction (MI). Chemokine receptors are important modulators of inflammation. Polymorphisms in genes coding for chemokine receptors, CCR2 and CCR5, have been studied as genetic markers of coronary artery disease. In the present study, we investigated whether genetic variants of CCR2-V64I and CCR5-delta32 chemokine receptors have any effect on the development of myocardial infarction. A total of 146 MI patients and 202 control subjects were genotyped for CCR2 and CCR5. CCR2-V64I genotypes were not significantly different between patients with MI and controls (P > 0.05). CCR5-delta32 genotype distribution in cases was significantly different from that of controls (P = 0.042). The CCR5-delta32 wt/deletion genotype frequencies for controls and cases were 0.10 and 0.19, respectively and individuals with CCR5-delta32 wt/deletion genotype had a 2.13-fold increased risk of myocardial infarction (P = 0.0013). Individuals carrying the CCR5-delta32 heterozygote or homozygous variant genotype (deletion/deletion + wt/deletion) had a 1.96-fold increased risk of myocardial infarction compared with the wild-type genotype (wt/wt) (p: 0.016). In conclusion, our data have suggested that genetic variant of CCR5 might be associated with the development of MI. Further larger sample size studies are required to confirm our findings.     

The effects of dietary boric acid and borax supplementation on lipid peroxidation,antioxidant activity,and DNA damage in rats

The aims of this study were to clarify the effects of high dietary supplementation with boric acid and borax, called boron (B) compounds, on lipid peroxidation (LPO), antioxidant activity, some vitamin levels, and DNA damage in rats. Thirty Sprague Dawley male rats were divided into three equal groups: the animals in the first group (control) were fed with a standard rodent diet containing 6.4 mg B/kg, and the animals in the experimental group were fed with a standard rodent diet added with a supra-nutritional amount of boric acid and borax (100 mg B/kg) throughout the experimental period of 28 days. The B compounds decreased malondialdehyde (MDA), DNA damage, the protein carbonyl content (PCO) level in blood, and glutathione (GSH) concentration in the liver, Cu–Zn superoxide dismutase (SOD), and catalase (CAT) activity in the kidney. The B compounds increased GSH concentration in blood and the vitamin C level in plasma. Consequently, our results demonstrate that B supplementation (100 mg/kg) in diet decreases LPO, and enhances the antioxidant defense mechanism and vitamin status. There are no differences in oxidant/antioxidant balance and biochemical parameters except for serum vitamin A and liver GSH concentration, between the boron compounds used in this study.