Variation of the immunolabelling of the α1-isotubulin in the mitotic spindle ofPhysarum polycephalum

Summary Immunofluorescent labelling ofPhysarum microtubules with a new antibody specific for the ₁-isotubulin has been compared with the labelling with an antibody specific for -isotubulins and an antibody with recognizes tubulin chains terminated by an aromatic amino-acid. In agreement with the known presence of only one -isotype in amoebae and several -isotypes in plasmodia, the immunofluorescence of the mitotic spindle was qualitatively identical, but lower in plasmodia than in amoebae. In all cases except one, there were no relative variations of immuno-fluorescence staining with the three antibodies, from metaphase to telophase, in spindles sampled. In plasmodia grown at optimal temperature, both during normal or perturbed mitosis, the immunostaining of the ₁isotype decreased sharply after metaphase, while the staining obtained with the two other antibodies did not vary significantly. The immunologic determination of the relative amount of the ₁-isotubulin in the tubulin pool and in isolated mitotic microtubules could not account for this observation.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Is the origin of type 1 diabetes in the gut?

In type 1 diabetes, insulin-producing beta-cells in the pancreas are destroyed by immune-mediated mechanisms. The manifestation of the disease is preceded by the so-called pre-diabetic period that may last several years and is characterized by the appearance of circulating autoantibodies against beta-cell antigens. The role of the gut as a regulator of type 1 diabetes was suggested in animal studies, in which changes affecting the gut immune system modulated the incidence of diabetes. Dietary interventions, alterations in the intestinal microbiota and exposure to enteric pathogens, regulate the development of autoimmune diabetes in animal models. It has been demonstrated that these modulations affect the gut barrier mechanisms and intestinal immunity. Because the pancreas and the gut belong to the same intestinal immune system, the link between autoimmune diabetes and the gut is not unexpected. The gut hypothesis in the development of type 1 diabetes is also supported by the observations made in human type 1 diabetes. Early diet could modulate the development of beta-cell autoimmunity; weaning to hydrolysed casein formula decreased the risk of beta-cell autoimmunity by age 10 in the infants at genetic risk. Increased gut permeability, intestinal inflammation with impaired regulatory mechanisms and dysregulated oral tolerance have been observed in children with type 1 diabetes. The factors that contribute to these intestinal alterations are not known, but interest is focused on the microbial stimuli and function of innate immunity. It is likely that our microbial environment does not support the healthy maturation of the gut and tolerance in the gut, and this leads to the increasing type 1 diabetes as well as other immune-mediated diseases regulated by intestinal immune system. Thus, the interventions, aiming to prevent or treat type 1 diabetes in humans, should be targeting the gut immune system.     

Population genetics of α1-Antitrypsin in the Netherlands

Summary Two groups of 708 healthy blood donors and 563 patients affected with chronic obstructive lung disease (C.O.L.D.) respectively, have been screened for ₁-antitrypsin (₁AT) variants by electrophoresis on agarose-polyacrylamide gels at pH 4.7 and isoelectric focusing (IEF).The frequencies of the Pi (Protease inhibitor) alleles are comparable to those observed in the North European populations. As expected, the frequency of the Z gene is higher in the group of patients with C.O.L.D. Also the frequency of MZ phenotypes is higher among these patients, but in this case the difference is not statistically significant.With the aid of the electrophoretic methods described in the text we were able to detect a new electrophoretic variant (M₃) showing a mobility intermediate between the M₁ and the M₂ phenotypes.     

Structure of the Nidogen Binding LE Module of the Laminin γ1 Chain in Solution

The structure of the single LE module between residues 791 and 848 of the laminin γ1 chain, which contains the high affinity binding site for nidogen, has been probed using NMR methods. The module folds into an autonomous domain which has a stable and unique three-dimensional (3D) structure in solution. The 3D structure was determined on the basis of 362 interproton distance constraints derived from nuclear Overhauser enhancement measurements and 39 π angles, supplemented by 5 ψ and 22 χ₁angles. The main features of the NMR structures are two-stranded antiparallel β-sheets which are separated by loops and cross-connected by four disulfide bridges. The N-terminal segment which contains the first three disulfide bridges is similar to epidermal growth factor. The C-terminal segment has an S-like backbone profile with a crossover at the last disulfide bridge and comprises two three-residue long β-strands that form an antiparallel β-sheet. The LE module possesses an exposed nidogen binding loop that projects away from the main body of the protein. The side-chains of three amino acids which are crucial for binding (Asp, Asn, Val) are all exposed at the domain surface. An inactivating Asn-Ser mutation in this region showed the same 3D structure indicating that these three residues, and possibly an additional Tyr in an adjacent loop, provide direct contacts in the interaction with nidogen.     

Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.The Snf1 protein kinase of Saccharomyces cerevisiae is the yeast ortholog of the AMP-activated protein kinase (AMPK) found in mammals and other eukaryotes. AMPK acts as a nutrient and energy sensor, becoming activated under conditions of nutrient and energy depletion (6). In mammals, AMPK plays a key role in glucose homeostasis and is a target for drugs used to treat metabolic syndrome and type 2 diabetes (34). In yeast, the Snf1 kinase plays an essential role during aerobic growth and fermentative growth on alternative carbon sources. Cells lacking Snf1 kinase activity are viable but display numerous phenotypes including poor or no growth on alternative carbon sources, defects in meiosis and sporulation, defects in response to ion stress, and defects in pseudohyphal growth (7).The Snf1 kinase and all members of the AMPK family function as heterotrimers composed of a catalytic α subunit complexed with regulatory β and γ subunits (2). The γ subunit in mammalian enzymes directly binds three molecules of AMP (26, 33), which stimulates enzyme activity by inhibiting the dephosphorylation of the conserved threonine residue in the kinase activation loop (23). In yeast, there is no evidence that the γ subunit binds AMP; however, similar to mammals, the key glucose-regulated step is the dephosphorylation of the kinase activation loop (22).In this study, we examine the role of the β subunits in the regulation of the Snf1 kinase activity. Yeasts express three isoforms of the Snf1 kinase that differ depending on which of the three distinct β subunits, Sip1, Sip2, and Gal83, is incorporated into the enzyme. Previous studies have shown that the Snf1 isoforms have distinct substrate preferences (24), subcellular localizations (32), and stress response capacities (9). Only the Snf1 isoform containing Gal83 as the β subunit is able to localize to the cell nucleus in a process that requires Sak1, one of the three Snf1-activating protein kinases. Since all three of the Snf1-activating kinases (SAKs) are capable of phosphorylating Snf1 on its activation loop (3), it has remained a mystery as to why the Sak1 kinase is specifically required for Snf1 nuclear localization.The β subunits of Snf1 as well as mammalian AMPK contain a domain that is referred to as either a carbohydrate-binding module (CBM) (11) or a glycogen-binding domain (GBD) (19). The structure of this domain has been solved (20), and it was previously shown that this domain binds most tightly to branched oligosaccharides like glycogen that contain α1→6 branches (12). The binding of glycogen to the β subunit causes an allosteric inhibition of AMPK activity and inhibits phosphorylation by the upstream activating kinase. The β subunits of yeast contain the GBDs, but the importance of binding glycogen is questionable since cells that lack the ability to make glycogen show a normal regulation of Snf1 kinase in response to glucose limitation (15). Nonetheless, the deletion of the GBD from the Gal83 protein caused an increased activity of the Snf1 enzyme and release from glucose repression. Therefore, the GBD acts as a negative regulator of kinase activity in both mammalian and fungal cells.In this study we examine the role of the GBD present in the Sip2 and Sip1 proteins. We also extend the characterization of the Gal83 GBD by determining what connection this domain has with the regulated dephosphorylation of the Snf1 kinase. Finally, we have characterized other N-terminal domains in the β subunits that control accumulation and phosphorylation.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Pathogenic role of HIF-1α in prostate hyperplasia in the presence of chronic inflammation

Benign prostatic hyperplasia (BPH) commonly occurs in older men with chronic prostatitis. Although BPH is frequently accompanied by inflammation, it is unclear whether inflammation underlies prostate enlargement. Recently, we reported that hypoxia‐inducible factor 1α (HIF-1α), which is known to be induced by proinflammatory cytokines, is involved in testosterone-induced prostate hyperplasia. Therefore, we hypothesized that cytokines secreted from infiltrated macrophages under inflammatory conditions stimulate prostate enlargement by up‐regulating HIF-1α. In the present study, we injected lipopolysaccharide (LPS) into rat prostates to mimic prostatitis and evaluated prostate hyperplasia 14 days later. Epithelial cells of LPS-treated prostates were found to be highly proliferative and HIF-1α levels in prostate tissues to be elevated. When prostate epithelial cells were incubated in conditioned medium from macrophages activated with LPS, they robustly expressed HIF-1α, and under these conditions IL-1β, IL-6, and TNF-α cytokines were found to mediate HIF-1α induction. In addition, HIF-1α was found to enhance the expression of Twist, which initiates epithelial–mesenchymal transition (EMT). Furthermore, profound EMT features were observed in LPS-treated rat prostates, and the natural HIF-1α inhibitors ascorbate and curcumin were found to attenuate EMT and prostate hyperplasia both in vivo and in vitro. Based on these results, we propose that HIF-1α mediates prostate enlargement under inflammatory conditions, and we suggest that HIF-1α be viewed as a promising target for blocking the transition from prostatitis to BPH.     

Plasma levels of TGF-β1 in homeostasis of the inflammation in sickle cell disease

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The −509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-β1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-β1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Role of transglutaminase 1 in stabilisation of intercellular junctions of the vascular endothelium

Microvascular endothelial monolayers from mouse myocardium (MyEnd) cultured for up to 5 days postconfluency became increasingly resistant to various barrier-compromising stimuli such as low extracellular Ca²⁺ and treatment with the Ca²⁺ ionophore A23187 and with the actin depolymerising compound cytochalasin D. In contrast, microvascular endothelial monolayers from mouse lung microvessels (PulmEnd) remained sensitive to these conditions during the entire culture period which corresponds to the well-known in vivo sensitivity of the lung microvasculature to Ca²⁺ depletion and cytochalasin D treatment. One molecular difference between pulmonary and myocardial endothelial cells was found to be transglutaminase 1 (TGase1) which is strongly expressed in myocardial endothelial cells but is absent from pulmonary endothelial cells. Resistance of MyEnd cells to barrier-breaking conditions correlated strongly with translocation of TGase1 to intercellular junctions. Simultaneous inhibition of intracellular and extracellular TGase activity by monodansylcadaverine (MDC) strongly weakened barrier properties of MyEnd monolayers, whereas inhibition of extracellular TGases by the membrane-impermeable active site-directed TGase inhibitor R281 did not reduce barrier properties. Weakening of barrier properties could be also induced in MyEnd cells by downregulation of TGase1 expression using RNAi-based gene silencing. These findings suggest that crosslinking activity of intracellular TGase1 at intercellular junctions may play a role in controlling barrier properties of endothelial monolayers.     

Properties of the permeability transition in VDAC1−/− mitochondria

Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca²⁺ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a ∼32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812–49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1^−/− mice. The basic properties of the PTP in VDAC1^−/− mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1^−/− mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.     

Defining the role of Emi1 in the DNA replication–segregation cycle

Ordered progression through the cell cycle is essential to maintain genomic stability, and fundamental to this is ubiquitin-mediated proteolysis. In particular, the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase destabilises specific regulators at defined times in the cycle to ensure that each round of DNA replication is followed by cell division. Thus, the proper regulation of the APC/C is crucial in each cell cycle. There are several APC/C regulators that restrict its activity to specific cell cycle phases, and amongst these the early mitotic inhibitor 1 (Emi1) protein has recently come to prominence. Emi1 has been proposed to control APC/C in early mitosis; however, recent evidence questions this role. In this review we discuss new evidence that indicates that Emi1 is essential to restrict APC/C activity in interphase and, by doing so, ensure the proper coordination between DNA replication and mitosis.