Gel-free and gel-based proteomics in Bacillus subtilis: a comparative study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.     

Dietary ratio of protein to carbohydrate induces plastic responses in the gastrointestinal tract of mice

Some vertebrates change the size of their digestive system in response to quantity and fibre content of ingested food, but the effects of dietary nutrients on gut structure remain poorly understood. Here we investigate how the protein to carbohydrate ratio of diets affects the mass of the gastrointestinal tract in mice. We fed 6-week-old male mice one of five isocaloric diets differing only in protein to carbohydrate ratio (the “no-choice” treatments), while a further four treatment groups received nutritionally complementary food pairings from which they could self-select a diet (the “choice” treatments). After 32 days, we measured the resulting dry mass of stomachs, intestines, caeca and colons. In the no-choice treatments, the stomachs were heavier in the mice fed diets containing more protein and less carbohydrate, indicating that larger stomachs may be needed for efficient digestion of the protein-rich food. In contrast, intestines, caeca and colons were heavier when diets contained more carbohydrates and less protein. This response may function to increase the digestive rate of carbohydrates when the dietary content of this macronutrient increases, but it may also indicate a compensatory response to increase amino acid uptake from a protein-deficient food. Mice in the choice treatments self-selected a diet with a protein to carbohydrate ratio of 0.46, and had gut dimensions similar to the expectation derived from no-choice treatments for this diet composition. Our results provide an example of plasticity in the differential allocation of resources to organ function, which is triggered by variation in resource quality.     

Ethanol-induced sleeping time in mice: age and sex differences

Ethanol-induced sleeping time was determined in mice of both sexes at ages ranging from 3 weeks to just over 1 year. A progressive increase in sleeping time was seen in both sexes up to 26 weeks of age; no subsequent changes were observed, except for a modest decrease in the oldest female group. In the majority of age groups, sleeping time values were higher in females, but few statistically significant sex differences were found.     

Altered protein expression pattern in colon tissue of mice upon supplementation with distinct selenium compounds

The essential trace element selenium (Se) is controversially discussed concerning its role in health and disease. Its various physiological functions are largely mediated by Se incorporation in the catalytic center of selenoproteins. In order to gain insights into the impact of Se deficiency and of supplementation with different Se compounds (selenite, selenate, selenomethionine) at defined concentrations (recommended, 150 μg/kg diet; excessive, 750 μg/kg diet) in murine colon tissues, a 20‐week feeding experiment was performed followed by analysis of the protein expression pattern of colon tissue specimens by 2D‐DIGE and MALDI‐TOF MS. Using this approach, 24 protein spots were identified to be significantly regulated by the different Se compounds. These included the antioxidant enzyme peroxiredoxin‐5 (PRDX5), proteins with binding capabilities, such as cofilin‐1 (COF1), calmodulin, and annexin A2 (ANXA2), and proteins involved in catalytic processes, such as 6‐phosphogluconate dehydrogenase (6PGD). Furthermore, the Se compounds demonstrated a differential impact on the expression of the identified proteins. Selected target structures were validated by qPCR and Western blot which mainly confirmed the proteomic profiling data. Thus, novel Se‐regulated proteins in colon tissues have been identified, which expand our understanding of the physiologic role of Se in colon tissue.     

Dietary supplementation of N-acetylcysteine enhances early inflammatory responses during cutaneous wound healing in protein malnourished mice

Prolonged wound healing is a complication that contributes to the morbidity and mortality of protein malnutrition (PM). The molecular mechanisms that underlie impaired wound healing in PM may begin in the early inflammatory stage of the process. We hypothesized that the impaired wound healing observed in PM occurs as a consequence of excessive reactive oxygen species (ROS) production that impairs the wound healing process by depressing nuclear factor kappa B (NFkappaB) activation and the subsequent synthesis and release of proinflammatory cytokines that are critical mediators of the inflammatory response. In this study, we showed that the time to wound closure was significantly prolonged in PM mice. During the early wound healing in PM, inhibitory kappa B alpha (IkappaBalpha), interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) expression and neutrophil infiltration were significantly decreased in PM mice. The role of excess ROS in PM was demonstrated by using transgenic mice with overexpression of copper zinc superoxide dismutase and with dietary supplementation of N-acetylcysteine (NAC). Both interventions improved the extent of wound closure in PM mice. Moreover, NAC supplementation in PM mice restored the expression of IkappaBalpha, IL-1beta and TNF-alpha and infiltration of neutrophils to levels observed in control animals. These findings support the notion that wound healing defects in PM may result from dysregulation of ROS-mediated and NFkappaB-regulated signaling pathways.     

Global protein expression pattern of Bradyrhizobium japonicum bacteroids: a prelude to functional proteomics

As a prelude to using functional proteomics towards understanding the process of symbiotic nitrogen fixation between the legume soybean and the soil bacteria Bradyrhizobium japonicum, we examined the total protein expression pattern of the nodule bacteria, often referred to as bacteroids. A partial proteome map was constructed by separating the total bacteroid proteins using high-resolution 2-DE. Of the several hundred protein spots analyzed using PMF, 180 spots were tentatively identified by searching the available database for B. japonicum, (http://www.kazusa.or.jp/index.html). The data showed that the bacteroid expressed a dominant and elaborate protein network for nitrogen and carbon metabolism, which is closely dependent on the plant supplied metabolites, and seems aptly supported by a selective group of bacteroid transporter proteins. However, they seem to lack a defined fatty acid and nucleic acid metabolism. Interestingly, the proteins related to protein synthesis, scaffolding and degradation were among the most predominant spots of the bacteroid proteome. In addition, several proteins, which showed fairly good expression, were identified to be involved with cellular detoxification, stress regulation and signaling communication components. This preliminary proteomic data matches very well with several biochemical and genetic reports, and clearly shows the inter-connection between several metabolic pathways that meet the needs of the bacteroid. It is expected that in the future this will allow us to develop testable hypotheses about the roles of several of these proteins in context to the metabolic pathway connections and metabolite fluxes.     

Dietary supplementation with acetyl-l-carnitine in seizure treatment of pentylenetetrazole kindled mice

In spite of the availability of new antiepileptic drugs a considerable number of epilepsy patients still have pharmacoresistant seizures, and thus there is a need for novel approaches. Acetyl-l-carnitine (ALCAR), which delivers acetyl units to mitochondria for acetyl-CoA production, has been shown to improve brain energy homeostasis and protects against various neurotoxic insults. To our knowledge, this is the first study of ALCAR's effect on metabolism in pentylenetetrazole (PTZ) kindled mice. ALCAR or the commonly used antiepileptic drug valproate, was added to the drinking water of mice for 25days, and animals were injected with PTZ or saline three times a week during the last 21days. In order to investigate ALCAR's effects on glucose metabolism, mice were injected with [1-(13)C]glucose 15min prior to microwave fixation. Brain extracts from cortex and the hippocampal formation (HF) were studied using (1)H and (13)C NMR spectroscopy and HPLC. PTZ kindling caused glucose hypometabolism, evidenced by a reduction in both glycolysis and TCA cycle turnover in both brain regions investigated. Glutamatergic and GABAergic neurons were affected in cortex and HF, but the amount of glutamate was only reduced in HF. Slight astrocytic involvement could be detected in the cortex. Interestingly, the dopamine content was increased in the HF. ALCAR attenuated the PTZ induced reduction in [3-(13)C]alanine and the increase in dopamine in the HF. However, TCA cycle metabolism was not different from that seen in PTZ kindled animals. In conclusion, even though ALCAR did not delay the kindling process, it did show some promising ameliorative effects, worthy of further investigation.     

Cell transplantation for myocardial injury: a preliminary comparative study

Background aimsCell transplantation may restore viable muscle after myocardial infarction. Because many studies have focused on one cell type, we compared the characteristics of skeletal myoblasts (SKM), bone marrow stromal/stem cells (BMSC) and smooth muscle cells (SMC) and their effects on cardiac function after myocardial injury.MethodsIn vitro cell characteristics, including proliferation, hypoxic survival and vascular endothelial cell growth factor (VEGF) expression, of SKM, BMSC and SMC were compared. An in vivo left anterior descending artery ligation rat model was used, and cells were implanted into the infarct (n = 16 per cell type). Cell survival was determined by PKH26 staining and real-time polymerase chain reaction (PCR). Cardiac function, tissue VEGF and stem cell factor (SCF) expression and vasculogenesis were evaluated.ResultsAlthough cell morphologies were distinct, in vitro proliferation was similar. In vitro studies showed that SKM had the highest hypoxic survival, whereas BMSC had the lowest hypoxic survival but the highest VEGF expression. After implantation, SKM showed the highest overall survival and in vivo SCF expression, and both SMC and SKM expressed the highest VEGF levels. Vasculogenesis was significantly (P < 0.001) improved after transplantation of each cell type. Overall, BMSC and SKM promoted the greatest improvement in cardiac function.ConclusionsSKM, BMSC and SMC expressed VEGF and SCF and promoted vasculogenesis. Although BMSC showed the greatest regenerative potential relative to cell survival and growth factor expression, the greatest improvement in cardiac function was observed with BMSC and SKM.     

氯胺酮诱导不同年龄和性别小鼠的过度活动

虽然氯胺酮(Ketamine)在历史上作为麻醉剂用于人类和家畜,但由于其分解特性, 作为一种娱乐药物似乎更具知名度.以前的研究表明相对于成年人, 孩子使用氯胺酮不易显示出不利影响,但是对其发生的机理几乎没有研究.本文研究了氯胺酮对小鼠的活动程度和固有行为的作用.结果表明:使用氯胺酮可增加22、35和50日龄小鼠的运动器官的敏捷性,并证明了氯胺酮的作用随年龄的增长而降低;使用氯胺酮所导致的旋转与年龄的变化有关,但站立的减少与年龄无关,这种减少不依赖于小鼠的年龄.     

Dietary nitrate supplementation reduces the O2 cost of walking and running: a placebo-controlled study

Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O(2) cost of submaximal exercise and to increase tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high NO(3)(-) content per se, and not the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q(max)), and physiological responses to walking and moderate- and severe-intensity running following dietary supplementation with BR and NO(3)(-)-depleted BR [placebo (PL)]. After control (nonsupplemented) tests, nine healthy, physically active male subjects were assigned in a randomized, double-blind, crossover design to receive BR (0.5 l/day, containing ～6.2 mmol of NO(3)(-)) and PL (0.5 l/day, containing ～0.003 mmol of NO(3)(-)) for 6 days. Subjects completed treadmill exercise tests on days 4 and 5 and knee-extension exercise tests for estimation of Q(max) (using (31)P-magnetic resonance spectroscopy) on day 6 of the supplementation periods. Relative to PL, BR elevated plasma NO(2)(-) concentration (183 ± 119 vs. 373 ± 211 nM, P < 0.05) and reduced systolic blood pressure (129 ± 9 vs. 124 ± 10 mmHg, P < 0.01). Q(max) was not different between PL and BR (0.93 ± 0.05 and 1.05 ± 0.22 mM/s, respectively). The O(2) cost of walking (0.87 ± 0.12 and 0.70 ± 0.10 l/min in PL and BR, respectively, P < 0.01), moderate-intensity running (2.26 ± 0.27 and 2.10 ± 0.28 l/min in PL and BR, respectively, P < 0.01), and severe-intensity running (end-exercise O(2) uptake = 3.77 ± 0.57 and 3.50 ± 0.62 l/min in PL and BL, respectively, P < 0.01) was reduced by BR, and time to exhaustion during severe-intensity running was increased by 15% (7.6 ± 1.5 and 8.7 ± 1.8 min in PL and BR, respectively, P < 0.01). In contrast, relative to control, PL supplementation did not alter plasma NO(2)(-) concentration, blood pressure, or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high NO(3)(-) content per se.     

Pressures in archaeal protein coding genes: a comparative study

Our studies on the bases of codons from 11 completely sequenced archaeal genomes show that, as we move from GC-rich to AT-rich protein-coding gene-containing species, the differences between G and C and between A and T, the purine load (AG content), and also the overall persistence (i.e. the tendency of a base to be followed by the same base) within codons, all increase almost simultaneously, although the extent of increase is different over the three positions within codons. These findings suggest that the deviations from the second parity rule (through the increasing differences between complementary base contents) and the increasing purine load hinder the chance of formation of the intra-strand Watson-Crick base-paired secondary structures in mRNAs (synonymous with the protein-coding genes we dealt with), thereby increasing the translational efficiency. We hypothesize that the ATrich protein-coding gene-containing archaeal species might have better translational efficiency than their GC-rich counterparts.     

Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: a mechanism for organizational sex differences

The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the rat as a model of sexual differentiation is that treatment of neonatal females with estradiol will permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a nongenomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases, thereby inducing dendritic spine formation. These results reveal a transneuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.     

Dietary L-arginine supplementation increases the hepatic expression of AMP-activated protein kinase in rats

Amino Acids - The goal of this study was to elucidate the molecular mechanisms responsible for the anti-obesity effect of L-arginine supplementation in diet-induced obese rats. Male...     

The evolution of sex differences in mate searching when females benefit: new theory and a comparative test

Sexual selection is thought to have led to searching as a profitable, but risky way of males obtaining mates. While there is great variation in which sex searches, previous theory has not considered search evolution when both males and females benefit from multiple mating. We present new theory and link it with data to bridge this gap. Two different search protocols exist between species in the bush-cricket genus Poecilimon (Orthoptera): females search for calling males, or males search for calling females. Poecilimon males also transfer a costly nuptial food gift to their mates during mating. We relate variations in searching protocols to variation in nuptial gift size among 32 Poecilimon taxa. As predicted, taxa where females search produce significantly larger nuptial gifts than those where males search. Our model and results show that search roles can reverse when multiple mating brings about sufficiently strong material benefits to females.     

Molecular patterns of sex determination in the animal kingdom: a comparative study of the biology of reproduction

Determining sexual fate is an integral part of reproduction, used as a means to enrich the genome. A variety of such regulatory mechanisms have been described so far and some of the more extensively studied ones are being discussed.     

Clinical proteomics: study of a cryogel

Cryoproteins are proteins precipitating at low temperature. Usually, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Very rarely, Igs do not precipitate, but, upon cooling, form a gel. Here, we report a case of cryogel observed in a patient presenting with Waldenström's disease. Using proteomic tools, a monoclonal IgM was identified as being the cause of the gel formation. Furthermore, addition of H₂O before incubation at 4°C demonstrated that the monoclonal IgM was precipitable as a type I cryoglobulin (hypocryoglobulin).     

Differential protein expression in the corpus callosum (splenium) of human alcoholics: a proteomics study

It is widely accepted that the chronic use of alcohol induces metabolic abnormalities and neuronal damage in the brain, which can lead to cognitive dysfunction. Neuroimaging studies reveal that alcohol-induced brain damage is region specific and prominent damage has been observed in both gray and white matter of the prefrontal cortex, and a wide range of white matter structures including the corpus callosum. Molecular mechanisms underlying these structural changes are largely unknown. Using proteomics we have analysed the changes in protein expression in the splenium of the corpus callosum in two different alcoholic groups. Protein extracts from splenium of 22 human brains (nine controls, seven uncomplicated alcoholics and six complicated alcoholics with hepatic cirrhosis-designated complicated) were separated using two-dimensional gel electrophorosis. Image analysis revealed that there were significant alterations in protein expression for 25 protein spots in the uncomplicated alcoholic group and 45 in the complicated group compared to control (P<0.05; ANOVA). In a total of 72 spots (identified as 36 proteins), 15 (identified as 14 proteins) spots overlapped between two alcoholic groups. Another 32 protein spots (26 different proteins) were identified only in the complicated alcoholics. It is therefore possible that these 26 proteins in the complicated group are likely to be the results of hepatic compromise. When compared with our previous data of white matter from the prefrontal cortex in alcoholics, large numbers of identified proteins in the splenium are different. This suggests that there may be different mechanisms causing alcohol-induced brain damage in different regions of the white matter. Our data also indicate the importance of other pathways including oxidative stress, lipid peroxidation and apoptosis as potential causes of alcohol-induced brain damage.     

Sexual jealousy as a facultative trait: Evidence from the pattern of sex differences in adults from China and the United States

Across two studies, 716 and 308 undergraduate students from the United States and mainland China, respectively, were administered a series of measures on jealousy, emotional responses to partner infidelity, family background, and personality. Across both studies for the U.S. and Chinese samples, a higher proportion of males than females reported more distress to a partner's imagined sexual infidelity than to emotional infidelity, whereas a higher proportion of females than males reported more distress to a partner's emotional infidelity than to sexual infidelity, consistent with theoretical expectations and previous empirical research. However, a much higher proportion of U.S. males and females reported more distress to sexual infidelity than their same-sex Chinese peers, suggesting that the tendency toward sexual jealousy might be facultatively influenced by sexual permissiveness in the general culture. The overall pattern of results is considered in terms of individual and contextual differences in the expression of jealousy, as well as in terms of the emotional and behavioral responses associated with jealousy reactions.