Perinatal Environmental Tobacco Smoke Exposure in Rhesus Monkeys: Critical Periods and Regional Selectivity for Effects on Brain Cell Development and Lipid Peroxidation

Perinatal environmental tobacco smoke (ETS) exposure in humans elicits neurobehavioral deficits. We exposed rhesus monkeys to ETS during gestation and through 13 months postnatally, or postnatally only (6–13 months). At the conclusion of exposure, we examined cerebrocortical regions and the midbrain for cell damage markers and lipid peroxidation. For perinatal ETS, two archetypal patterns were seen in the various regions, one characterized by cell loss (reduced DNA concentration) and corresponding increases in cell size (increased protein/DNA ratio), and a second pattern suggesting replacement of larger neuronal cells with smaller and more numerous glia (increased DNA concentration, decreased protein/DNA ratio). The membrane/total protein ratio, a biomarker of neurite formation, also indicated potential damage to neuronal projections, accompanied by reactive sprouting. When ETS exposure was restricted to the postnatal period, the effects were similar in regional selectivity, direction, and magnitude. These patterns resemble the effects of prenatal nicotine exposure in rodent and primate models. Surprisingly, perinatal ETS exposure reduced the level of lipid peroxidation as assessed by the concentration of thiobarbituric acid reactive species, whereas postnatal ETS did not. The heart, a tissue that, like the brain, has high oxygen demand, displayed a similar but earlier decrease (2–3 months) in lipid peroxidation in the perinatal exposure model, whereas values were reduced at 13 months with the postnatal exposure paradigm. Our results provide a mechanistic connection between perinatal ETS exposure and neurobehavioral anomalies, reinforce the role of nicotine in these effects, and buttress the importance of restricting or eliminating ETS exposure in young children

A redox state-dictated signalling pathway deciphers the malignant cell specificity of CD40-mediated apoptosis

CD40, a member of the tumour necrosis factor receptor (TNFR) superfamily, has the capacity to cause extensive apoptosis in carcinoma cells, while sparing normal epithelial cells. Yet, apoptosis is only achieved by membrane-presented CD40 ligand (mCD40L), as soluble receptor agonists are but weakly pro-apoptotic. Here, for the first time we have identified the precise signalling cascade underpinning mCD40L-mediated death as involving sequential TRAF3 stabilisation, ASK1 phosphorylation, MKK4 (but not MKK7) activation and JNK/AP-1 induction, leading to a Bak- and Bax-dependent mitochondrial apoptosis pathway. TRAF3 is central in the activation of the NADPH oxidase (Nox)-2 component p40phox and the elevation of reactive oxygen species (ROS) is essential in apoptosis. Strikingly, CD40 activation resulted in down-regulation of Thioredoxin (Trx)-1 to permit ASK1 activation and apoptosis. Although soluble receptor agonist alone could not induce death, combinatorial treatment incorporating soluble CD40 agonist and pharmacological inhibition of Trx-1 was functionally equivalent to the signal triggered by mCD40L. Finally, we demonstrate using normal, ‘para-malignant’ and tumour-derived cells that progression to malignant transformation is associated with increase in oxidative stress in epithelial cells, which coincides with increased susceptibility to CD40 killing, while in normal cells CD40 signalling is cytoprotective. Our studies have revealed the molecular nature of the tumour specificity of CD40 signalling and explained the differences in pro-apoptotic potential between soluble and membrane-bound CD40 agonists. Equally importantly, by exploiting a unique epithelial culture system that allowed us to monitor alterations in the redox-state of epithelial cells at different stages of malignant transformation, our study reveals how pro-apoptotic signals can elevate ROS past a previously hypothesised ‘lethal pro-apoptotic threshold’ to induce death; an observation that is both of fundamental importance and carries implications for cancer therap

Proteome Analyses of Cellular Proteins in Methicillin-Resistant Staphylococcus aureus Treated with Rhodomyrtone, a Novel Antibiotic Candidate

The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC) values ranged from 31.25–62.5 µg/ml, and the minimal bactericidal concentration (MBC) was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5–125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml) affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Patients with chronic idiopathic neutropenia of adults have increased serum concentrations of inflammatory cytokines and chemokines

Serum levels of inflammatory cytokines and chemokines were measured in 132 patients with chronic idiopathic neutropenia of adults (CINA) and 34 healthy Volunteers (controls) using commercially available micro-ELISA determination kits, We found that serum interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (1) (TGF-beta (1)), and soluble tumor necrosis factor receptor p55 (sTNF-RI) were all significantly increased in CINA patients compared to controls. Individual cytokine values inversely correlated with the number of circulating neutrophils. Serum levels of interleukin-8 (IL-8) and RANTES, two potent chemokines for neutrophils and lymphocytes, respectively, were also significantly increased in the group of patients and they inversely correlated with the number of circulating neutrophils, Contrarily, serum levels of interleukin-l (IL-4), interferon-gamma (IFN gamma), soluble CD23 (sCD23), and soluble interleukin-a receptor (sIL-2R) did not show any significant change in the patients studied, We assume that CINA patients have increased serum concentrations of inflammatory cytokines and chemokines mainly produced by activated macrophages, while they disclose normal levels of inflammatory molecules mainly released from activated lymphocytes. These findings provide further evidence for an underlying low-grade chronic inflammatory process in CINA patients, as we previously have suggested, If this chronic inflammation is really the cause of the disorder or it simply represents the result of neutropenia remains to be elucidated. (C) 2000 Wiley-Liss, Inc