Coordinated activation of AMP‐activated protein kinase,extracellular signal‐regulated kinase,and autophagy regulates phorbol myristate acetate‐induced differentiation of SH‐SY5Y neuroblastoma cells

We explored the interplay between the intracellular energy sensor AMP‐activated protein kinase (AMPK), extracellular signal‐regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)‐induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells. PMA‐triggered expression of neuronal markers (dopamine transporter, microtubule‐associated protein 2, β‐tubulin) was associated with an autophagic response, measured by the conversion of microtubule‐associated protein light chain 3 (LC3)‐I to autophagosome‐bound LC3‐II, increase in autophagic flux, and expression of autophagy‐related (Atg) proteins Atg7 and beclin‐1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference‐mediated silencing of AMPK suppressed PMA‐induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA‐induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA‐induced differentiation of SH‐SY5Y cells. Therefore, PMA‐induced neuronal differentiation of SH‐SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response.

     

Conclusion: applying South East Asia Rainforest Research Programme science to land-use management policy and practice in a changing landscape and climate

The context and challenges relating to the remaining tropical rainforest are briefly reviewed and the roles which science can play in addressing questions are outlined. Key messages which articles in the special issue, mainly based on projects of the Royal Society South East Asia Rainforest Research Programme (SEARRP), have raised of relevance to policies on land use, land management and REDD+ are then considered. Results from the atmospheric science and hydrology papers, and some of the ecological ones, demonstrate the very high ecosystem service values of rainforest (compared with oil palm) in maintaining high biodiversity, good local air quality, reducing greenhouse emissions, and reducing landslide, flooding and sedimentation consequences of climate change-and hence provide science to underpin the protection of remaining forest, even if degraded and fragmented. Another group of articles test ways of restoring forest quality (in terms of biodiversity and carbon value) or maintaining as high biodiversity and ecological functioning levels as possible via intelligent design of forest zones and fragments within oil palm landscapes. Finally, factors that have helped to enhance the policy relevance of SEARRP projects and dissemination of their results to decision-makers are outlined.     

Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.     

Human plasma glycome in attention-deficit hyperactivity disorder and autism spectrum disorders

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.     

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats

The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 μl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.     

Gender-related differences in the oxidant state of cells in Fanconi anemia heterozygotes

Abstract Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.     

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

Background  

Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.     

Identification of multiple HPV types on spermatozoa from human sperm donors

Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.     

Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.     

Establishment of a transgenic mouse model specifically expressing human serum amyloid A in adipose tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA.