Mitogen-activated protein kinases in the acute diabetic myocardium

Diabetes mellitus (DM) causes myocardial remodeling on the subcellular level and alterations in the function of the cell membranes ion transport systems resulting in contractile dysfunction. The present study was aimed to investigate the expression and activation of mitogen-activated protein kinases (MAPKs) and their possible role in the acute diabetic rat hearts. Rats were injected with single dose of streptozotocin (45 mg/kg, i.v.), and after 1 week the disease was manifested by hyperglycemia and cardiac dysfunction. The Langendorff-perfused hearts were subjected to ischemia (5 or 30 min occlusion of LAD coronary artery). The protein pattern in cytosolic fraction of the heart tissue was determined after electrophoretic separation. The levels and activation of MAPKs were determined by Western blot analysis using specific antibodies. No differences between the diabetics and controls in the level of ERKs were found at baseline. However, in DM samples ERKs phosphorylation was markedly increased, and further changes occurred during ischemia. Also content of phoshorylated c-Raf kinase (an upstream activator of ERKs) was slightly increased at baseline conditions in the diabetic samples. In contrast, no significant changes in the contents and phosphorylation of p38-MAPK were observed at baseline. But some differences in the p38-MAPK phosphorylation were found during ischemia.The results show that differential pattern of protein kinase cascades activation in the diabetic hearts might be account for the modulation of their response to ischemia.     

HASTY,the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis

Loss-of-function mutations of HASTY (HST) affect many different processes in Arabidopsis development. In addition to reducing the size of both roots and lateral organs of the shoot, hst mutations affect the size of the shoot apical meristem, accelerate vegetative phase change, delay floral induction under short days, adaxialize leaves and carpels, disrupt the phyllotaxis of the inflorescence, and reduce fertility. Double mutant analysis suggests that HST acts in parallel to SQUINT in the regulation of phase change and in parallel to KANADI in the regulation of leaf polarity. Positional cloning demonstrated that HST is the Arabidopsis ortholog of the importin beta-like nucleocytoplasmic transport receptors exportin 5 in mammals and MSN5 in yeast. Consistent with a potential role in nucleocytoplasmic transport, we found that HST interacts with RAN1 in a yeast two-hybrid assay and that a HST-GUS fusion protein is located at the periphery of the nucleus. HST is one of at least 17 members of the importin-beta family in Arabidopsis and is the first member of this family shown to have an essential function in plants. The hst loss-of-function phenotype suggests that this protein regulates the nucleocytoplasmic transport of molecules involved in several different morphogenetic pathways, as well as molecules generally required for root and shoot growth.     

Dental Ontogeny in Pliocene and Early Pleistocene Hominins

Until recently, our understanding of the evolution of human growth and development derived from studies of fossil juveniles that employed extant populations for both age determination and comparison. This circular approach has led to considerable debate about the human-like and ape-like affinities of fossil hominins. Teeth are invaluable for understanding maturation as age at death can be directly assessed from dental microstructure, and dental development has been shown to correlate with life history across primates broadly. We employ non-destructive synchrotron imaging to characterize incremental development, molar emergence, and age at death in more than 20 Australopithecus anamensis, Australopithecus africanus, Paranthropus robustus and South African early Homo juveniles. Long-period line periodicities range from at least 6–12 days (possibly 5–13 days), and do not support the hypothesis that australopiths have lower mean values than extant or fossil Homo. Crown formation times of australopith and early Homo postcanine teeth fall below or at the low end of extant human values; Paranthropus robustus dentitions have the shortest formation times. Pliocene and early Pleistocene hominins show remarkable variation, and previous reports of age at death that employ a narrow range of estimated long-period line periodicities, cuspal enamel thicknesses, or initiation ages are likely to be in error. New chronological ages for SK 62 and StW 151 are several months younger than previous histological estimates, while Sts 24 is more than one year older. Extant human standards overestimate age at death in hominins predating Homo sapiens, and should not be applied to other fossil taxa. We urge caution when inferring life history as aspects of dental development in Pliocene and early Pleistocene fossils are distinct from modern humans and African apes, and recent work has challenged the predictive power of primate-wide associations between hominoid first molar emergence and certain life history variables.     

Similar but Different: Dynamic Social Network Analysis Highlights Fundamental Differences between the Fission-Fusion Societies of Two Equid Species,the Onager and Grevy’s Zebra

Understanding why animal societies take on the form that they do has benefited from insights gained by applying social network analysis to patterns of individual associations. Such analyses typically aggregate data over long time periods even though most selective forces that shape sociality have strong temporal elements. By explicitly incorporating the temporal signal in social interaction data we re-examine the network dynamics of the social systems of the evolutionarily closely-related Grevy’s zebras and wild asses that show broadly similar social organizations. By identifying dynamic communities, previously hidden differences emerge: Grevy’s zebras show more modularity than wild asses and in wild asses most communities consist of solitary individuals; and in Grevy’s zebras, lactating females show a greater propensity to switch communities than non-lactating females and males. Both patterns were missed by static network analyses and in general, adding a temporal dimension provides insights into differences associated with the size and persistence of communities as well as the frequency and synchrony of their formation. Dynamic network analysis provides insights into the functional significance of these social differences and highlights the way dynamic community analysis can be applied to other species.     

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.Abbreviations: FR3, Filariasis Research Reagent Resource CenterLymphatic filariasis a major threat to human health worldwide. More than one billion people in more than 90 countries around the globe are at risk from lymphatic filariasis, and between 120 and 150 million people are infected.^9,11,25 Infection with the filarioid parasitic worms Brugia malayi and Wuchereria bancrofti can result in severe sequelae, including elephantiasis and hydrocoele formation.^3,11,15,25 In addition to the clinical manifestations of filariasis are the potential associated psychologic, social, and cultural effects in persons exhibiting visible signs of infection.^9,23,34The life cycle of filarioid nematodes requires an arthropod intermediate host and a definitive vertebrate host. Within the definitive host, dioecious adult filarial nematodes reproduce sexually. Inseminated adult female worms then release live, sheathed microfilariae into the lymph that circulate in the peripheral blood.²¹ In the case of B. malayi and W. bancrofti, the intermediate host is the mosquito.²¹ When an uninfected mosquito ingests a blood meal from an infected human, ingested microfilariae unsheathe to penetrate the midgut of the mosquito to reach the thoracic muscles and molt twice, to become the infectious third-stage larvae. The third-stage larvae then migrate to the mosquito''s proboscis and can infect another human when the mosquito takes a blood meal.^10,11 The third-stage larvae enter the new host''s lymphatic system which is their final location, where they undergo 2 molts into adults.Because of the complexity of filarioid life cycles, research involving these parasites can be logistically challenging. Although mice can be infected with W. bancrofti, they do not maintain the infection.³⁵ Furthermore, there is no suitable nonhuman host that can maintain a patent infection, with the exception of the silvered leaf monkey (Trachypithecus cristatus).⁹ Because the closely related parasites B. malayi and B. pahangi have more extensive host ranges than does W. bancrofti, they are easier to maintain in a research setting. Domestic cats (Felis catus) can be experimentally infected with B. malayi and develop a patent infection, and both domestic cats and dogs (Canis familiaris) can be experimentally infected with B. pahangi^13,29,37 and are suitable for obtaining microfilaremic blood for experimental feeding of mosquitoes. The Mongolian gerbil can be infected with B. pahangi. Because replacing a phylogenetically higher species with a lower species is preferable³⁶ and because performing experiments involving dogs and cats can be logistically difficult and cost-prohibitive, many researchers prefer a rodent model, specifically gerbils.The Filariasis Research Reagent Resource Center (FR3) is an NIH center whose mandate is to support filariasis research worldwide. The FR3 provides parasitic and molecular resources, as well as training in animal procedures, to researchers from many nations. The FR3 maintains both B. malayi and B. pahangi, and researchers occasionally require gerbils with patent infections. Because the required level of microfilaria counts varies among investigators, an accurate microfilaria count must be obtained prior to the shipment of gerbils. For example, some experiments require that live mosquitoes feed directly on infected gerbils, and when the microfilaria level is too low, the mosquitoes do not become infected. Conversely when the level is too high, the migration of microfilariae and the later larval stages can kill the mosquitoes. Historically, the FR3 has used retroorbital sampling under general anesthesia to obtain the blood for microfilaria counts.²⁸ Although this method has been fairly successful, the FR3 has encountered occasional complications secondary to the procedure, including exophthalmia and, rarely, death under anesthesia. The NIH guidelines for survival bleeding of mice and rats notes that compared with other blood collection methods, retroorbital sampling has a greater potential for complications. The guidelines recommend a 10- to 14-d period between retroorbital blood collections and state that the procedure is “…best conducted under general anesthesia.”³¹ By comparison, collecting blood from the lateral saphenous vein is considered to have a low potential for complications or tissue damage, can be performed without general anesthesia,^12,18,31 and can be performed repeatedly, even daily.³¹In the current study, we proposed to refine the blood collection method being used by FR3 by developing sampling from the lateral saphenous vein as the new standard blood-collection method for monitoring microfilaremia. Our goal was to assess blood collection from the lateral saphenous vein as a feasible refinement technique to potentially replace retroorbital sampling by determining whether the microfilaria counts in blood collected from the lateral saphenous vein without anesthesia were sufficiently similar to those from retroorbital blood sampling with anesthesia to provide adequate information about the microfilaremia level.     

Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Sumoylation Influences DNA Break Repair Partly by Increasing the Solubility of a Conserved End Resection Protein

Protein modifications regulate both DNA repair levels and pathway choice. How each modification achieves regulatory effects and how different modifications collaborate with each other are important questions to be answered. Here, we show that sumoylation regulates double-strand break repair partly by modifying the end resection factor Sae2. This modification is conserved from yeast to humans, and is induced by DNA damage. We mapped the sumoylation site of Sae2 to a single lysine in its self-association domain. Abolishing Sae2 sumoylation by mutating this lysine to arginine impaired Sae2 function in the processing and repair of multiple types of DNA breaks. We found that Sae2 sumoylation occurs independently of its phosphorylation, and the two modifications act in synergy to increase soluble forms of Sae2. We also provide evidence that sumoylation of the Sae2-binding nuclease, the Mre11-Rad50-Xrs2 complex, further increases end resection. These findings reveal a novel role for sumoylation in DNA repair by regulating the solubility of an end resection factor. They also show that collaboration between different modifications and among multiple substrates leads to a stronger biological effect.     

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells

Tumor-suppressive miR-34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin-induced oxidative DNA damage culminates in p53 activation to up-regulate expression of miR-34a in non-small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR-34a inhibits B cell lymphoma-2 (Bcl-2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down-regulated, p53-transactivated Bcl-2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR-34a regulatory axis might be critical in sensitizing drug-resistant NSCLC cells.     

The Conserved PFT1 Tandem Repeat Is Crucial for Proper Flowering in Arabidopsis thaliana

It is widely appreciated that short tandem repeat (STR) variation underlies substantial phenotypic variation in organisms. Some propose that the high mutation rates of STRs in functional genomic regions facilitate evolutionary adaptation. Despite their high mutation rate, some STRs show little to no variation in populations. One such STR occurs in the Arabidopsis thaliana gene PFT1 (MED25), where it encodes an interrupted polyglutamine tract. Although the PFT1 STR is large (∼270 bp), and thus expected to be extremely variable, it shows only minuscule variation across A. thaliana strains. We hypothesized that the PFT1 STR is under selective constraint, due to previously undescribed roles in PFT1 function. We investigated this hypothesis using plants expressing transgenic PFT1 constructs with either an endogenous STR or synthetic STRs of varying length. Transgenic plants carrying the endogenous PFT1 STR generally performed best in complementing a pft1 null mutant across adult PFT1-dependent traits. In stark contrast, transgenic plants carrying a PFT1 transgene lacking the STR phenocopied a pft1 loss-of-function mutant for flowering time phenotypes and were generally hypomorphic for other traits, establishing the functional importance of this domain. Transgenic plants carrying various synthetic constructs occupied the phenotypic space between wild-type and pft1 loss-of-function mutants. By varying PFT1 STR length, we discovered that PFT1 can act as either an activator or repressor of flowering in a photoperiod-dependent manner. We conclude that the PFT1 STR is constrained to its approximate wild-type length by its various functional requirements. Our study implies that there is strong selection on STRs not only to generate allelic diversity, but also to maintain certain lengths pursuant to optimal molecular function.