Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase

The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose-response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05-0.1 nmol/l insulin stimulated, 1-100 nmol/l had no effect, whereas concentrations >/=250 nmol/l ("high insulin"), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3'-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt(2)cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3'-kinase and are counteracted by raising islet cyclic AMP levels.     

Nuclear Localization of the Mitochondrial Factor HIGD1A during Metabolic Stress

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.     

Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov

Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99-S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. However, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa. The three newly isolated strains 99-1, 99-2, and 99-S differed by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa.     

Distinct modes of vertebrate hypaxial muscle formation contribute to the teleost body wall musculature

The formation of the body wall musculature in vertebrates is assumed to be initiated by direct ventral extension of the somites/myotomes. This contrasts to the formation of limb muscles and muscles involved in feeding or respiration/ventilation, which are founded by migratory muscle precursors (MMPs) distant to the somites. Here, we present evidence from morphology and expression of molecular markers proposing that the formation of the two muscle layers of the teleost body wall involves both of the above mechanisms: (1) MMPs from somites 5 and 6 found an independent muscle primordium–the so-called posterior hypaxial muscle (PHM)–which subsequently gives rise to the most anterior two segments of the medial obliquus inferioris (OI) muscle. (2) Direct epithelial extension of the hypaxial myotomes generates the OI segments from somite 7 caudalward and the entire lateral obliquus superioris (OS) muscle. The findings are discussed in relation to the evolution of hypaxial myogenic patterning including functional considerations. We hypothesise that the potential of the most anterior somites to generate migratory muscle precursors is a general vertebrate feature that has been differently utilised in the evolution in vertebrate groups.     

Designing an implementation strategy to improve interprofessional shared decision making in sciatica: study protocol of the DISC study

ABSTRACT: BACKGROUND: Sciatica is a common condition worldwide that is characterized by radiating leg pain and regularly caused by a herniated disc with nerve root compression. Sciatica patients with persisting leg pain after six to eight weeks were found to have similar clinical outcomes and associated costs after prolonged conservative treatment or surgery at one year follow-up. Guidelines recommend that the team of professionals involved in sciatica care and patients jointly decide about treatment options, so-called interprofessional shared decision making (SDM). However, there are strong indications that SDM for sciatica patients is not integrated in daily practice. We designed a study aiming to explore the barriers and facilitators associated with the everyday embedding of SDM for sciatica patients. All related relevant professionals and patients are involved to develop a tailored strategy to implement SDM for sciatica patients. METHODS: The study consists of two phases: identification of barriers and facilitators and development of an implementation strategy. First, barriers and facilitators are explored using semi-structured interviews among eight professionals of each (para)medical discipline involved in sciatica care (general practitioners, physical therapists, neurologists, neurosurgeons, and orthopedic surgeons). In addition, three focus groups will be conducted among patients. Second, the identified barriers and facilitators will be ranked using a questionnaire among a representative Dutch sample of 200 GPs, 200 physical therapists, 200 neurologists, all 124 neurosurgeons, 200 orthopedic surgeons, and 100 patients. A tailored team-based implementation strategy will be developed based on the results of the first phase using the principles of intervention mapping and an expert panel. DISCUSSION: Little is known about effective strategies to increase the uptake of SDM. Most implementation strategies only target a single discipline, whereas multiple disciplines are involved in SDM among sciatica patients. The results of this study can be used as an example for implementing SDM in other patient groups receiving multidisciplinary complex care (e.g., elderly) and can be generalized to other countries with similar context, thereby contributing to a worldwide increase SDM in preference sensitive choices.     

Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading

The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1.     

The receptor for advanced glycation endproducts and its ligands in patients with myasthenia gravis

ObjectiveMyasthenia gravis (MG) is a T- and B-cell mediated autoimmune disorder affecting the neuromuscular junction. The receptor for advanced glycation endproducts (RAGE) plays a role in the amplification of chronic inflammatory disorders and autoimmune diseases. We sought to investigate the role of RAGE and its ligands in the pathophysiology of MG.MethodsIn this cross-sectional study we enrolled 42 patients with MG and 36 volunteers. We employed enzyme-linked immunosorbent assays to determine the concentration of soluble RAGE (sRAGE) and high mobility group box 1 (HMGB1) in serum of patients and volunteers. In a subpopulation of patients we measured the serum levels of endogenous secretory (es) RAGE and various RAGE ligands, such as S100B, S100A8 and advanced glycation endproducts (AGE-CML). Reported are means and standard error mean.ResultsWe found significantly reduced levels of the soluble receptors sRAGE and esRAGE in patients with MG compared to volunteers without MG (sRAGE [pg/ml] 927.2 ± 80.8 vs. 1400.1 ± 92.4; p < 0.001; esRAGE [pg/ml] 273.5 ± 24.6 vs. 449.0 ± 22.4; p < 0.001). Further categorization of patients with MG according to the distribution of muscle involvement revealed the following sRAGE concentrations: generalized MG 999.4 ± 90.8 and ocular MG 696.1 ± 161.8 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: generalized vs. ocular MG: p = 0.264, generalized MG vs. control: p = 0.008, ocular MG vs. control: p = 0.001). In patients with detectable antibodies specific for acetylcholine receptors (Anti-AChR positive) the sRAGE concentration was 970.0 ± 90.2 compared to those without (seronegative) 670.6 ± 133.1 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: Pos vs. Neg.: p = 0.418, Pos vs. control: p = 0.003, Neg. vs. control: p = 0.008). We next investigated the role of RAGE ligands in MG. The concentrations of RAGE ligands in patients with MG and controls were as follows: (HMGB1 [ng/ml] 1.7 ± 0.1 vs. 2.1 ± 0.2; p = 0.058; S100B [pg/ml] 22.5 ± 22.5 vs. 14.4 ± 9.2; p = 0.698; S100A8 [pg/ml] 107.0 ± 59.3 vs. 242.5 ± 103.6; p = 0.347; and AGE-CML [ng/ml] 1100.8 ± 175.1 vs. 1399.8 ± 132.8; p = 0.179).ConclusionsOur data suggest a role for the RAGE pathway in the pathophysiology of MG. Further studies are warranted to elucidate more about this immunological axis in patients with MG.