Functional organization of TRPC-Ca2+ channels and regulation of calcium microdomains

TRP family of proteins are components of unique cation channels that are activated in response to diverse stimuli ranging from growth factor and neurotransmitter stimulation of plasma membrane receptors to a variety of chemical and sensory signals. This review will focus on members of the TRPC sub-family (TRPC1-TRPC7) which currently appear to be the strongest candidates for the enigmatic Ca(2+) influx channels that are activated in response to stimulation of plasma membrane receptors which result in phosphatidyl inositol-(4,5)-bisphosphate (PIP(2)) hydrolysis, generation of IP(3) and DAG, and IP(3)-induced Ca(2+) release from the intracellular Ca(2+) store via inositol trisphosphate receptor (IP(3)R). Homomeric or selective heteromeric interactions between TRPC monomers generate distinct channels that contribute to store-operated as well as store-independent Ca(2+) entry mechanisms. The former is regulated by the emptying/refilling of internal Ca(2+) store(s) while the latter depends on PIP(2) hydrolysis (due to changes in PIP(2) per se or an increase in diacylglycerol, DAG). Although the exact physiological function of TRPC channels and how they are regulated has not yet been conclusively established, it is clear that a variety of cellular functions are controlled by Ca(2+) entry via these channels. Thus, it is critical to understand how cells coordinate the regulation of diverse TRPC channels to elicit specific physiological functions. It is now well established that segregation of TRPC channels mediated by interactions with signaling and scaffolding proteins, determines their localization and regulation in functionally distinct cellular domains. Furthermore, both protein and lipid components of intracellular and plasma membranes contribute to the organization of these microdomains. Such organization serves as a platform for the generation of spatially and temporally dictated [Ca(2+)](i) signals which are critical for precise control of downstream cellular functions.     

Morphometric sex determination of Milky and Painted Storks in captivity

Logistic regression was applied to develop a morphometric sexing method of two closely related stork species that were previously sexed through amplification of the CHD gene. Tarsus length (TL) and bill length (BL) measurements were recorded from captive populations of adult Milky Stork (Mycteria cinerea) (n = 60) and Painted Stork (Mycteria leucocephala) (n = 58) at Zoo Negara Malaysia. Despite having monomorphic plumages, both stork species exhibited normal sexual size dimorphism in which males were significantly larger than females in the tested variables. Based on logistic regression analysis, BL correctly classified the sex of sampled individuals from Painted and Milky stork with an overall predicted accuracy of 94.8 and 90.0%, respectively. However, TL measurements generated a lower predicted accuracy level of 86.2% and a same accuracy level of 90% on the sex classification of individuals from Painted and Milky stork, respectively. By comparing the measurements of both species, only the average BL measurements of the Milky storks were significantly lower than that of Painted storks (t-test, P80.001). The logistic regression equation in this study may serve as a simple and more practical option for sexing Milky and Painted storks for their breeding and conservation programmes.     

Effects of heat acclimatisation on work tolerance and thermoregulation in trained tropical natives

This study aimed to investigate the effects of heat acclimatisation on thermoregulatory responses and work tolerance in trained individuals residing in the tropics. Eighteen male trained soldiers, who are native to a warm and humid climate, performed a total of four heat stress tests donning the Skeletal Battle Order (SBO, 20.5 kg) and Full Battle Order (FBO, 24.7 kg) before (PRE) and after (POST) a 10-day heat acclimatisation programme. The trials were conducted in an environmental chamber (dry bulb temperature: 32 °C, relative humidity: 70%, solar radiation: 400 W/m²). Excluding the data sets of which participants fully completed the heat stress tests (210 min) before and after heat acclimatisation, work tolerance was improved from 173±30 to 201±18 min (∼21%, p<0.05, n=9) following heat acclimatisation. Following heat acclimatisation, chest skin temperature during exercise was lowered in SBO (PRE=36.7±0.3 vs. POST=36.5±0.3 °C, p<0.01) and FBO (PRE=36.8±0.4 vs. POST=36.6±0.3 °C, p<0.01). Ratings of perceived exertion were decreased with SBO and FBO (PRE=11±2; POST=10±2; p<0.05) after heat acclimatisation. Heat acclimatisation had no effects on baseline body core temperature, heart rate and sweat rate across trials (p>0.05). A heat acclimatisation programme improves work tolerance with minimal effects on thermoregulation in trained tropical natives.     

Brain Isoprenoids Farnesyl Pyrophosphate and Geranylgeranyl Pyrophosphate are Increased in Aged Mice

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23?months) as compared to middle-aged mice (12?months) and young mice (3?months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

A new tumour suppressor enters the network of intestinal progenitor cell homeostasis

In this issue of The EMBO Journal, Wilson et al (2012) elegantly discovered an important new axis for intestinal homeostasis and cancer, using an RNAi screen to enhance the RAS-induced multivulva (MUV) phenotype in Caenorhabditis elegans.     

Anti-atherogenic and anti-angiogenic activities of polyphenols from propolis

Propolis is a polyphenol-rich resinous substance extensively used to improve health and prevent diseases. The effects of polyphenols from different sources of propolis on atherosclerotic lesions and inflammatory and angiogenic factors were investigated in LDL receptor gene (LDLr?/?) knockout mice. The animals received a cholesterol-enriched diet to induce the initial atherosclerotic lesions (IALs) or advanced atherosclerotic lesions (AALs). The IAL or AAL animals were divided into three groups, each receiving polyphenols from either the green, red or brown propolis (250 mg/kg per day) by gavage. After 4 weeks of polyphenol treatment, the animals were sacrificed and their blood was collected for lipid profile analysis. The atheromatous lesions at the aortic root were also analyzed for gene expression of inflammatory and angiogenic factors by quantitative real-time polymerase chain reaction and immunohistochemistry. All three polyphenol extracts improved the lipid profile and decreased the atherosclerotic lesion area in IAL animals. However, only polyphenols from the red propolis induced favorable changes in the lipid profiles and reduced the lesion areas in AAL mice. In IAL groups, VCAM, MCP-1, FGF, PDGF, VEGF, PECAM and MMP-9 gene expression was down-regulated, while the metalloproteinase inhibitor TIMP-1 gene was up-regulated by all polyphenol extracts. In contrast, for advanced lesions, only the polyphenols from red propolis induced the down-regulation of CD36 and the up-regulation of HO-1 and TIMP-1 when compared to polyphenols from the other two types of propolis. In conclusion, polyphenols from propolis, particularly red propolis, are able to reduce atherosclerotic lesions through mechanisms including the modulation of inflammatory and angiogenic factors.     

A method for quantifying pulmonary Legionella pneumophila infection in mouse lungs by flow cytometry

ABSTRACT: BACKGROUND: Pulmonary load of Legionella pneumophila in mice is normally determined by counting serial dilutions of bacterial colony forming units (CFU) on agar plates. This process is often tedious and time consuming. We describe a novel, rapid and versatile flow cytometric method that detects bacteria phagocytosed by neutrophils. FINDINGS: Mice were infected with L. pneumophila via intratracheal or intranasal administration. At various times after bacteria inoculation, mouse lungs were harvested and analysed concurrently for bacterial load by colony counting and flow cytometry analysis. The number of L. pneumophila-containing neutrophils correlated strongly with CFU obtained by bacteriological culture. CONCLUSIONS: This technique can be utilised to determine pulmonary bacterial load and may be used in conjunction with other flow cytometric based analyses of the resulting immune response.     

iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.Stable isotope labeling techniques have become very popular in recent years to perform quantitative mass spectrometry experiments with high precision and accuracy. In contrast to label-free approaches, multiplexed isotopically labeled samples can be simultaneously analyzed resulting in increased reproducibility and accuracy for quantification of peptides and proteins from different biological states. Isotopic labeling strategies can be grouped into two major categories: isobaric labels and nonisobaric labels. In the former category are iTRAQ¹ (isobaric tags for relative and absolute quantification (1)) and TMT (tandem mass tags (2)) mass tags. In the nonisobaric labeling category are methods such as mTRAQ (mass differential tags for relative and absolute quantification), stable isotope labeling by amino acids in cell culture (SILAC (3)), and reductive dimethylation (4). Isobaric labeling techniques allow relative quantification of peptides based on ratios of low m/z reporter ions produced by fragmentation of the precursor ion, whereas nonisobaric labeling yields precursors with different masses that can be directly quantified from MS1 intensity. iTRAQ and mTRAQ reagents provide a great opportunity to directly compare capabilities of reporter and precursor ion quantification since both labels have identical chemical structures and differ only in their composition and number of ¹³C, ¹⁵N, and ¹⁸O atoms. In fact, iTRAQ-117 and mTRAQ-Δ4 are identical mass tags with a total mass of 145 Da (Fig. 1A). To achieve 4-plex quantification capabilities for iTRAQ labels, the composition of stable isotopes is arranged in a way to obtain the reporter ion/balancing group pairs 114/31, 115/30, 116/29, and 117/28 (1). Three nonisobaric mTRAQ labels were generated by adding or removing four neutrons to the mTRAQ-Δ4 label resulting in mTRAQ-Δ8 and mTRAQ-Δ0, respectively. Both iTRAQ and mTRAQ reagents are available as N-hydroxy-succinimide esters to facilitate primary amine labeling of peptides.Open in a separate windowFig. 1.A, Labeling strategy for comparative evaluation of iTRAQ and mTRAQ tags. Peptides were labeled with the indicated iTRAQ and mTRAQ reagents for combined phosphoproteome and proteome analysis. B, Selection of optimal instrument methods for analysis of iTRAQ- and mTRAQ-labeled peptides. Unfractionated proteome samples (1 ug) and phosphoproteome samples (enriched from 250 μg peptides) were analyzed for iTRAQ samples with a CID/HCD-Top8 method, whereas for mTRAQ we compared CID-Top16 acquisition to HCD-Top8. Note that duty cycle times were for all instrument methods ∼3.1 s.One potential advantage of an iTRAQ labeling strategy is its additive effect on precursor intensities when samples are multiplexed, resulting in increased sensitivity. However, iTRAQ ratios have been demonstrated to be prone to compression. This occurs when other nonregulated background peptides are co-isolated and cofragmented in the same isolation window of the peptide of interest and contribute fractional intensity to the reporter ions in MS2-scans (5–7). Because most peptides in an experiment are present at 1:1:1:1 ratios between multiplexed samples, all ratios in the experiment tend to be dampened toward unity when cofragmentation occurs. This inaccuracy led to the development of mTRAQ labels to facilitate accurate precursor-based quantification of proteins initially identified in iTRAQ discovery experiments with targeted assays, such as multiple reaction monitoring (MRM) (8). Although iTRAQ has been widely used in discovery-based proteomics studies, mTRAQ has only appeared in a small number of studies thus far (8).In this study we investigated the advantages and disadvantages of iTRAQ and mTRAQ labeling for proteome-wide analysis of protein phosphorylation and expression changes. We selected epidermal growth factor (EGF)-stimulated HeLa cells as a model system for our comparative evaluation of iTRAQ and mTRAQ labeling, as both changes in the phosphoproteome (9) as well as the proteome (10) are well described for EGF stimulation. We show that iTRAQ labeling yields superior results to mTRAQ in terms of numbers of quantified phosphopeptides, proteins and regulated components. By means of spike-in experiments with GluC generated peptides of known ratios we find that iTRAQ quantification is more precise but less accurate than mTRAQ due to ratio compression. We identify a linear relationship of observed versus expected logarithmic GluC generated peptide ratios as well as for logarithmic iTRAQ and mTRAQ ratios of the phosphoproteome and proteome analysis. This indicates a uniform degree of ratio compression over two orders of magnitude throughout iTRAQ data sets and explains why iTRAQ ratio compression does not compromise the ability to detect regulated elements in these experiments.