Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf-derived fibroblasts are slow to enter the stage of growth arrest induced by culturing normal cells under standard culture conditions at 20% O(2). Dwarf cells cultured at 20% O(2) resemble control cells cultured at 3% O(2) not only in their delayed growth arrest, but also in their rapid growth rates and resistance to both oxidative and nonoxidative forms of cytotoxic stress. Levels of the heat-shock protein HSP-70 respond to serum withdrawal and stress only in control cells, showing that intracellular signals are blunted in dwarf-derived cells. These data suggest a model in which stable epigenetic changes induced in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice.     

Augmented autophagy pathways and MTOR modulation in fibroblasts from long-lived mutant mice

Fibroblasts from long-lived pituitary dwarf mutants, including Snell dwarf, Ames dwarf and the growth hormone receptor knockout (GHRKO) mice, are resistant in culture to multiple forms of lethal stress. We found that fibroblasts from Snell dwarf and GHRKO mice are more susceptible than control cells to autophagy induced by amino acid withdrawal or by oxidative stress. We also found evidence for lower MTOR function in dwarf cells under conditions that induce autophagy, consistent with the evidence that increased autophagy requires lower TOR activity. Our results provide new hints about the connections between autophagy and aging in long-lived mutants with alterations in GH-IGF1 levels, and suggest a role for hyperactive autophagy in the resistance of cells from these mice to lethal stresses.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

European populations of Diabrotica virgifera virgifera are resistant to aldrin,but not to methyl‐parathion

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of cultivated corn in North America and has recently begun to invade Europe. In addition to crop rotation, chemical control is an important option for D. v. virgifera management. However, resistance to chemical insecticides has evolved repeatedly in the USA. In Europe, chemical control strategies have yet to be harmonized and no surveys of insecticide resistance have been carried out. We investigated the resistance to methyl‐parathion and aldrin of samples from nine D. v. virgifera field populations originating from two European outbreaks thought to have originated from two independent introductions from North America. Diagnostic concentration bioassays revealed that all nine D. v. virgifera field populations were resistant to aldrin but susceptible to methyl‐parathion. Aldrin resistance was probably introduced independently, at least twice, from North America into Europe, as there is no evident selection pressure to account for an increase of frequency of aldrin resistance in each of the invasive outbreaks in Europe. Our results suggest that organophosphates, such as methyl‐parathion, may still provide effective control of both larval and adult D. v. virgifera in the European invasive outbreaks studied.     

Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress

Trehalose is known to protect cells from various environmental assaults; however, its role in the ethanol tolerance of Saccharomyces cerevisiae remains controversial. Many previous studies report correlations between trehalose levels and ethanol tolerance across a variety of strains, yet variations in genetic background make it difficult to separate the impact of trehalose from other stress response factors. In the current study, investigations were conducted on the ethanol tolerance of S. cerevisiae BY4742 and BY4742 deletion strains, tsl1 Δ and nth1 Δ, across a range of ethanol concentrations. It was found that trehalose does play a role in ethanol tolerance at lethal ethanol concentrations, but not at sublethal ethanol concentrations; differences of 20–40% in the intracellular trehalose concentration did not provide any growth advantage for cells incubated in the presence of sublethal ethanol concentrations. It was speculated that the ethanol concentration-dependent nature of the trehalose effect supports a mechanism for trehalose in protecting cellular proteins from the damaging effects of ethanol.     

Altered growth characteristics of skin fibroblasts from wild-derived mice, and genetic loci regulating fibroblast clone size

Mouse fibroblast senescence in vitro is an important model for the study of aging at cellular level. However, common laboratory mouse strains may have lost some important allele variations related to aging processes. In this study, growth in vitro of tail skin fibroblasts (TSFs) derived from a wild-derived stock, Pohnpei (Pohn) mice, differed from growth of control C57BL/6 J (B6) TSFs. Pohn TSFs exhibited higher proliferative ability, fewer apoptotic cells, decreased expression of Cip1, smaller surface areas, fewer cells positive for senescence associated-beta-galactosidase (SA-beta-gal) and greater resistance to H(2)O(2)-induced SA-beta-gal staining and Cip1 expression. These data suggest that TSFs from Pohn mice resist cellular senescence-like changes. Using large clone ratio (LCR) as the phenotype, a quantitative trait locus (QTL) analysis in a Pohn/B6 backcross population found four QTLs for LCR: Fcs1 on Chr 3 at 55 CM; Fcs2 on Chr X at 50 CM; Fcs3 on Chr 4 at 51 CM and Fcs4 on Chr 10 at 25 CM. Together, these four QTLs explain 26.1% of the variations in LCRs in the N2 population. These are the first QTLs reported that regulate fibroblast growth. Glutathione S transferase mu (GST-mu) genes are overrepresented in the 95% confidence interval of Fcs1, and Pohn TSFs have higher H(2)O(2)-induced GST-mu 4, 5 and 7 mRNA levels than B6 TSFs. These enzymes may protect Pohn TSFs from oxidation.     

According to the CPLL proteome sheriffs,not all aperitifs are created equal!

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called “Amaro Branzi”, stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products.     

The head and the foot inhibitor from hydra are not dowex artefacts

Summary In a recent publication in this journal (Berking 1983) it was claimed (1) that the head inhibitor we isolated from hydra is a Dowex artefact, (2) that a separate foot inhibitor does not exist in hydra and (3) that the only inhibitor that has so far been isolated from hydra is one which inhibits head and foot regeneration equally well. These statements are incorrect and require a response. In the following, I would like to summarise our evidence that the inhibitors isolated from hydra, including Berking's inhibitor, have different specificities for head and foot regeneration. In addition, I would like to show that none of our substances are Dowex artefacts.     

Increasing water temperature and disease risks in aquatic systems: Climate change increases the risk of some, but not all, diseases

Global warming may impose severe risks for aquatic animal health if increasing water temperature leads to an increase in the incidence of parasitic diseases. Essentially, this could take place through a temperature-driven effect on the epidemiology of the disease. For example, higher temperature may boost the rate of disease spread through positive effects on parasite fitness in a weakened host. Increased temperature may also lengthen the transmission season leading to higher total prevalence of infection and more widespread epidemics. However, to date, general understanding of these relationships is limited due to scarcity of long-term empirical data. Here, we present one of the first long-term multi-pathogen data sets on the occurrence of pathogenic bacterial and parasitic infections in relation to increasing temperatures in aquatic systems. We analyse a time-series of disease dynamics on two fish farms in northern Finland from 1986 to 2006. We first demonstrate that the annual mean water temperature increased significantly on both farms over the study period and that the increase was most pronounced in the late summer (July-September). Second, we show that the prevalence of infection (i.e. proportion of fish tanks infected each year) increased with temperature. Interestingly, this pattern was observed in some of the diseases (Ichthyophthirius multifiliis, Flavobacterium columnare), whereas in the other diseases, the pattern was the opposite (Ichthyobodo necator) or absent (Chilodonella spp.). These results demonstrate the effect of increasing water temperature on aquatic disease dynamics, but also emphasise the importance of the biology of each disease, as well as the role of local conditions, in determining the direction and magnitude of these effects.     

Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli

To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.     

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy.     

Tyrosine hydroxylase and insulin-like growth factor II but not insulin are adjacent in the teleost species barramundi, Lates calcarifer

In humans, the genes encoding tyrosine hydroxylase (TH), insulin and insulin-like growth factor II (IGF-II) form an extremely tight linkage group on chromosome 11p15. Characterisation of the homologous genomic region of a teleost, the barramundi Lates calcarifer , revealed tight linkage of the TH and IGF-II genes, and the absence of the gene encoding insulin.     

Castelli risk indexes 1 and 2 are higher in major depression but other characteristics of the metabolic syndrome are not specific to mood disorders

Aims

This study examined whether Castelli risk indexes 1 (total/high-density lipoprotein (HDL) cholesterol) and 2 (low density lipoprotein (LDL)/HDL cholesterol) and other shared metabolic disorders might underpin the pathophysiology of the metabolic syndrome, major depression or bipolar disorder.

Main methods

This cross-sectional study examined 92 major depressed, 49 bipolar depressed and 201 normal controls in whom the Castelli risk indexes 1 and 2 and key characteristics of the metabolic syndrome, i.e. waist/hip circumference, body mass index (BMI), systolic/diastolic blood pressure, total cholesterol, low-density lipoprotein (LDL) and HDL cholesterol, triglycerides, insulin, glucose, hemoglobin A1c (HbA1c) and homocysteine were assessed.

Key findings

Castelli risk indexes 1 and 2 were significantly higher in major depressed patients than in bipolar disorder patients and controls. There were no significant differences in waist or hip circumference, total and LDL cholesterol, triglycerides, plasma glucose, insulin, homocysteine and HbA1c between depression and bipolar patients and controls. Bipolar patients had a significantly higher BMI than major depressed patients and normal controls.

Significance

Major depression is accompanied by increased Castelli risk indexes 1 and 2, which may be risk factors for cardiovascular disease. Other key characteristics of the metabolic syndrome, either metabolic biomarkers or central obesity, are not necessarily specific to major depression or bipolar disorder.     

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH₂-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H₂O₂/Cl⁻ system is known to react with the NH₂-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH₂Cl) was identified as a breakdown product of this reaction.     

Arfaptins are localized to the trans-Golgi by interaction with Arl1, but not Arfs

Arfaptins (arfaptin-1 and arfaptin-2/POR1) were originally identified as binding partners of the Arf small GTPases. Both proteins contain a BAR (Bin/Amphiphysin/Rvs) domain, which participates in membrane deformation. Here we show that arfaptins associate with trans-Golgi membranes. Unexpectedly, Arl1 (Arf-like 1), but not Arfs, determines the trans-Golgi association of arfaptins. We also demonstrate that arfaptins interact with Arl1 through their BAR domain-containing region and compete for Arl1 binding with golgin-97 and golgin-245/p230, both of which also bind to Arl1 through their GRIP (golgin-97/RanBP2/Imh1p/p230) domains. However, arfaptins and these golgins show only limited colocalization at the trans-Golgi. Time-lapse imaging of cells overexpressing fluorescent protein-tagged arfaptins and golgin-97 reveals that arfaptins, but not golgin-97, are included in vesicular and tubular structures emanating from the Golgi region. These observations indicate that arfaptins are recruited onto trans-Golgi membranes by interacting with Arl1, and capable of inducing membrane deformation via their BAR domains.     

Neuronal cells but not muscle cells are resistant to oxidative stress mediated protein misfolding and cell death: Role of molecular chaperones

Our recent study in a mouse model of familial-Amyotrophic Lateral Sclerosis (f-ALS) revealed that muscle proteins are equally sensitive to misfolding as spinal cord proteins despite the presence of low mutant CuZn-superoxide dismutase, which is considered to be the key toxic element for initiation and progression of f-ALS. More importantly, we observed differential level of heat shock proteins (Hsp’s) between skeletal muscle and spinal cord tissues prior to the onset and during disease progression; spinal cord maintains significantly higher level of Hsp’s compared to skeletal muscle. In this study, we report two important observations; (i) muscle cells (but not neuronal cells) are extremely vulnerable to protein misfolding and cell death during challenge with oxidative stress and (ii) muscle cells fail to mount Hsp’s during challenge unlike neuronal cells. These two findings can possibly explain why muscle atrophy precedes the death of motor neurons in f-ALS mice.