Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway.

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.

[Enantioselective Synthesis of (R)- and (S)-3-Methylspermidines].

Earlier unknown enantiomerically pure (R)- and (S)-1,8-diamino-3-methyl-4-azaoctane's (3-MeSpd's) were synthesized with high overall yields and optical purity starting from commercially available R- and S-isomers of N-Boc-2-aminopropanol-1. Application of R- and S-isomers of 3-MeSpd for the investigation of the stereospecificity of spermidine transporter and peculiarities of deoxyhypusine synthase reaction are discussed.

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism.

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells "proinhibitor" of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.

INPPL1 is associated with the metabolic syndrome in men with Type 1 diabetes, but not with diabetic nephropathy.

AIMS: The metabolic syndrome is a frequent phenomenon in people with Type 1 diabetes and is associated with diabetic nephropathy. The aim of this study was to investigate if the INPPL1 (inositol polyphosphate phosphatase-like 1) gene encoding lipid phosphatase SHIP2 is associated with the metabolic syndrome and diabetic nephropathy in Finnish people with Type 1 diabetes. METHODS: Participants were selected from the FinnDiane study for this cross-sectional study. The individuals were divided into controls without the metabolic syndrome (n = 1074) and cases with the metabolic syndrome (n = 1328), or into groups based upon their albumin excretion rate. Nine single-nucleotide polymorphisms covering the INPPL1 gene +/- 20 kb were genotyped. The associations between the single-nucleotide polymorphisms and outcome variables were analysed with the χ(2) test and logistic regression. RESULTS: Two INPPL1 single-nucleotide polymorphisms, rs2276048 (silent mutation) and rs2276047 (intronic), were associated with the metabolic syndrome in men with odds ratios of 0.23 (95% CI 0.11-0.45, P = 2.1 × 10(-5) ), and 0.37 (0.21-0.65, P = 0.001), adjusted for age, duration of diabetes and history of smoking. When both sexes were included, these associations were less significant. No association between the genotyped single-nucleotide polymorphisms and diabetic nephropathy was observed. CONCLUSIONS: INPPL1 gene variants may contribute to susceptibility to the metabolic syndrome in men with Type 1 diabetes, but not to diabetic nephropathy.

Osteoinduction by combining bone morphogenetic protein (BMP)-2 with a bioactive novel nanocomposite.

OBJECTIVES: There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite. METHODS: BMP-2 was synthesised using an Escherichia coli expression system and purified. In vitro bioactivity was confirmed using C2C12 cells and an alkaline phosphatase assay. The modified solution-evaporation method was used to fabricate α-TCP/PLGA nanocomposite and this was characterised using X-ray diffraction and scanning electron microscopy. Functionalisation of α-TCP/PLGA nanocomposite by adsorption of BMP-2 was performed and release of BMP-2 was characterised using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Alkaline phosphatase activity of C2C12 cells was increased by the presence of all BMP-2/nanocomposite discs compared with the presence of a blank disc (p = 0.0022), and increased with increasing incubation concentrations of BMP-2, showing successful adsorption and bioactivity of BMP-2. A burst release profile was observed for BMP-2 from the nanocomposite. CONCLUSIONS: Functionalisation of α-TCP/PLGA with BMP-2 produced osteoinduction and was dose-dependent. This material therefore has potential application as an osteoinductive agent in regenerative medicine.

A molecular dynamics approach for the association of apolipoproteinB-100 and chondroitin-6-sulfate.

A force field has been previously designed for a dodecasaccharide chain of chondroitin-6-sulfate (C6S) and has proved to yield valuable data going from basic conformational properties to a more detailed H-bonding network. This force field is further used here to unravel the interaction of C6S with its pathological counterpart in low density lipoprotein (LDL) particles. In particular, well-selected peptide fragment p2 (residues 3359-3377) also identified as the principal proteoglycan binding site (PPBS) of the major protein in LDL, apolipoproteinB-100 (apoB-100), was chosen. We study here the interaction between C6S and p2. The role of arginine and lysine, positively charged amino acids of p2, in the crucial interaction of C6S with LDL is highlighted. The secondary structure of p2 is shown to affect the efficiency of the interaction, as the α-helical structure of p2 allows optimal interaction with C6S also in dynamic conditions. One point mutation in p2 appeared to affect consequently p2-C6S interaction.

Acute pancreatitis induced by activated polyamine catabolism is associated with coagulopathy: effects of alpha-methylated polyamine analogs on hemostasis.

BACKGROUND/AIMS: Polyamines are ubiquitous organic cations essential for cellular proliferation and tissue integrity. We have previously shown that pancreatic polyamine depletion in rats overexpressing the catabolic enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT), results in the development of severe acute pancreatitis, and that therapeutic administration of metabolically stable alpha-methylated polyamine analogs protects the animals from pancreatitis-associated mortality. Our aim was to elucidate the therapeutic mechanism(s) of alpha-methylspermidine (MeSpd). METHODS: The effect of MeSpd on hemostasis and the extent of organ failure were studied in SSAT transgenic rats with either induced pancreatitis or lipopolysaccharide (LPS)-induced coagulopathy. The effect of polyamines on fibrinolysis and coagulation was also studied in vitro. RESULTS: Pancreatitis caused a rapid development of intravascular coagulopathy, as assessed by prolonged coagulation times, decreased plasma fibrinogen level and antithrombin activity, enhanced fibrinolysis, reduced platelet count and presence of schistocytes. Therapeutic administration of MeSpd restored these parameters to almost control levels within 24 h. In vitro, polyamines dose-dependently inhibited fibrinolysis and intrinsic coagulation pathway. In LPS-induced coagulopathy, SSAT transgenic rats were more sensitive to the drug than their syngeneic littermates, and MeSpd-ameliorated LPS-induced coagulation disorders. CONCLUSION: Pancreatitis-associated mortality in SSAT rats is due to coagulopathy that is alleviated by treatment with MeSpd.

Atomistic insight into chondroitin-6-sulfate glycosaminoglycan chain through quantum mechanics calculations and molecular dynamics simulation.

Chondroitin-6-sulfate (C6S) is a glycosaminoglycan (GAG) constituent in the extracellular matrix, which participates actively in crucial biological processes, as well as in various pathological conditions, such as atherosclerosis and cancer. Molecular interactions involving the C6S chain are therefore of considerable interest. A computational model for atomistic simulation was built. This work describes the design and validation of a force field for a C6S dodecasaccharide chain. The results of an extensive molecular dynamics simulation performed with the new force field provide a novel insight into the structure and dynamics of the C6S chain. The intramolecular H-bonds in the disaccharide linkage region are suggested to play a major role in determining the chain structural dynamics. Moreover, the unravelling of an additional H-bond involving the sulfate groups in C6S is interesting as changes in sulfation have been claimed to be an important factor in several diseases. The force field will prove useful for future studies of crucial interactions between C6S and various nanoassemblies. It can also be used as a basis for modeling of other GAGs.

Does the frequency of Prevotella intermedia increase during pregnancy?

INTRODUCTION: The former Bacteroides intermedius, currently including Prevotella intermedia and Prevotella nigrescens, has been associated with hormone-induced pregnancy gingivitis. The aim of the present longitudinal study was to determine whether only P. intermedia or P. nigrescens, or both species, are involved in the demonstrated microbial shift during pregnancy. METHODS: Subgingival plaque and saliva samples, collected from 30 healthy pregnant women and 24 healthy non-pregnant women as their controls, were examined for the presence of pigmented gram-negative anaerobes. Altogether 2628 isolates were preliminarily identified as P. intermedia sensu lato, based on phenotypic testing. Their further identification was performed by using a 16S ribosomal DNA-based polymerase chain reaction (PCR). RESULTS: A mean of 8.3 P. intermedia sensu lato isolates from each subject/sampling was examined. During the second trimester, the mean number of P. intermedia sensu lato in plaque increased along with increasing signs of pregnancy gingivitis, and then both decreased. After delivery, gingival inflammation still decreased while the number of P. intermedia sensu lato transiently increased both in plaque and saliva. In the present study, the vast majority of isolates (95.3%) proved to be P. nigrescens and 2.5% were P. intermedia. The remaining 2.2% of the isolates could not be identified with PCR as P. intermedia or P. nigrescens. The corresponding percentages in the control population were 94.2%, 5.5%, and 0.3%. CONCLUSION: In the oral cavity of relatively young women without periodontitis, P. nigrescens, unlike P. intermedia, is a frequent finding. Conceivably, pregnant women harbor increasing numbers of P. nigrescens associated with pregnancy gingivitis.

[Methylated analogues of spermine and spermidine as tools to investigate cellular functions of polyamines and the enzymes of their metabolism].

Biogenic amines spermine and spermidine are essential factors of cellular growth. Polyamine analogues are widely used to investigate and to regulate the enzymes of polyamine metabolism and functions of spermine and spermidine in vitro and in vivo. Recently, it was demonstrated that alpha-methylated derivatives of spermine and spermidine are capable to fulfill key cellular functions of polyamines, moreover in some cases of (R)- and (S)-isomers are actually different. Using these alpha-methylated spermine and spermidine analogues it turned possible to prevent the development of acute pancreatitis of SSAT-transgenic rats and to demostrate for the first time that polyamine oxidase, spermine oxidase and deoxyhypusine synthase have dormant stereospecificity. An original approach to regulate the stereospecificity of polyamine oxidase was suggested. It was also demonstrated that the depletion of the intracellular polyamine pool has both hypusine-related consequences and also the consequences unrelated to the posttranslational modification of eukaryotic initiation translation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogues for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.

Mechanisms of polyamine catabolism-induced acute pancreatitis.

Acute pancreatitis is an autodigestive disease, in which the pancreatic tissue is damaged by the digestive enzymes produced by the acinar cells. Among the tissues in the mammalian body, pancreas has the highest concentration of the natural polyamine, spermidine. We have found that pancreas is very sensitive to acute decreases in the concentrations of the higher polyamines, spermidine and spermine. Activation of polyamine catabolism in transgenic rats overexpressing SSAT (spermidine/spermine-N(1)-acetyltransferase) in the pancreas leads to rapid depletion of these polyamines and to acute necrotizing pancreatitis. Replacement of the natural polyamines with methylated polyamine analogues before the induction of acute pancreatitis prevents the development of the disease. As premature trypsinogen activation is a common, early event leading to tissue injury in acute pancreatitis in human and in experimental animal models, we studied its role in polyamine catabolism-induced pancreatitis. Cathepsin B, a lysosomal hydrolase mediating trypsinogen activation, was activated just 2 h after induction of SSAT. Pre-treatment of the rats with bismethylspermine prevented pancreatic cathepsin B activation. Analysis of tissue ultrastructure by transmission electron microscopy revealed early dilatation of rough endoplasmic reticulum, probable disturbance of zymogen packaging, appearance of autophagosomes and later disruption of intracellular membranes and organelles. Based on these results, we suggest that rapid eradication of polyamines from cellular structures leads to premature zymogen activation and autodigestion of acinar cells.

Molecular dynamics simulations of lipid bilayers: major artifacts due to truncating electrostatic interactions.

We study the influence of truncating the electrostatic interactions in a fully hydrated pure dipalmitoylphosphatidylcholine (DPPC) bilayer through 20 ns molecular dynamics simulations. The computations in which the electrostatic interactions were truncated are compared to similar simulations using the particle-mesh Ewald (PME) technique. All examined truncation distances (1.8-2.5 nm) lead to major effects on the bilayer properties, such as enhanced order of acyl chains together with decreased areas per lipid. The results obtained using PME, on the other hand, are consistent with experiments. These artifacts are interpreted in terms of radial distribution functions g(r) of molecules and molecular groups in the bilayer plane. Pronounced maxima or minima in g(r) appear exactly at the cutoff distance indicating that the truncation gives rise to artificial ordering between the polar phosphatidyl and choline groups of the DPPC molecules. In systems described using PME, such artificial ordering is not present.

Application of self-organizing maps in conformational analysis of lipids.

The characteristics of lipid assemblies are important for the functions of biological membranes. This has led to an increasing utilization of molecular dynamics simulations for the elucidation of the structural features of biomembranes. We have applied the self-organizing map (SOM) to the analysis of the complex conformational data from a 1-ns molecular dynamics simulation of PLPC phospholipids in a membrane assembly. Mapping of 1.44 million molecular conformations to a two-dimensional array of neurons revealed, without human intervention, the main conformational features in hours. Both the whole molecule and the characteristics of the unsaturated fatty acid chains were analyzed. All major structural features were easily distinguished, such as the orientational variability of the headgroup, the mainly trans state dihedral angles of the sn-1 chain, and both straight and bent conformations of the unsaturated sn-2 chain. Furthermore, presentation of the trajectory of an individual lipid molecule on the map provides information on conformational dynamics. The present results suggest that the SOM method provides a powerful tool for routinely gaining rapid insight to the main molecular conformations as well as to the conformational dynamics of any simulated molecular assembly without the requirement of a priori knowledge.

Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles.

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH(3))(3) resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.

Changes in a phospholipid bilayer induced by the hydrolysis of a phospholipase A2 enzyme: a molecular dynamics simulation study.

Phospholipase A2 (PLA2) enzymes are important in numerous physiological processes. Their function at lipid-water interfaces is also used as a biophysical model for protein-membrane interactions. These enzymes catalyze the hydrolysis of the sn-2 bonds of various phospholipids and the hydrolysis products are known to increase the activity of the enzymes. Here, we have applied molecular dynamics (MD) simulations to study the membrane properties in three compositionally different systems that relate to PLA2 enzyme action. One-nanosecond simulations were performed for a 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and for two of its PLA2-hydrolyzed versions, i.e., bilayers consisting of lysophospholipids and of either free charged linoleate or free uncharged linoleic acid molecules. The results revealed loosening of the structure in the hydrolyzed bilayer due to increased mobility of the molecules in the direction normal to the bilayer. This loss of integrity due to the hydrolysis products is in accord with observations that not only the presence of hydrolysis products, but also a variety of other perturbations of the membrane may activate PLA2. Additionally, changes were observed in other structural parameters and in the electrostatic potential across the membrane-water interface. These changes are discussed in relation to the simulation methodology and the experimental observations of PLA2-hydrolyzed membranes.

Protein-protein interactions in receptor activation and intracellular signalling.

We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin GPry, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 A2, such as Cdk6INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the betagamma subunits (Gtbetagamma) of the heterotrimeric G protein transducin.

Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL.

Low density lipoprotein (LDL) particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. In the process of atherogenesis these particles are modified and they accumulate in the arterial wall. Although the composition and overall structure of the LDL particles is well known, the fundamental molecular interactions and their impact on the structure of LDL particles are not well understood. Here, the existing pieces of structural information on LDL particles are combined with computer models of the individual molecular components to give a detailed structural model and visualization of the particles. Strong evidence is presented in favor of interactions between LDL lipid constituents that lead to specific domain formation in the particles. A new three-layer model, which divides the LDL particle into outer surface, interfacial layer, and core, and which is capable of explaining some seemingly contradictory interpretations of molecular interactions in LDL particles, is also presented. A new molecular interaction model for the beta-sheet structure and phosphatidylcholine headgroups is introduced and an overall view of the tertiary structure of apolipoprotein B-100 in the LDL particles is presented. This structural information is also utilized to understand and explain the molecular characteristics and interactions of modified, atherogenic LDL particles.

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate.

BACKGROUND: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. RESULTS: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D-myo-inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P4). The inositol Ins (1,3,4,5)P4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C-delta. A second Ins (1,3,4,5)P4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P4 to the wild type (Kd = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. CONCLUSIONS: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a Galpha protein.

Structure and dynamic properties of diunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine lipid bilayer from molecular dynamics simulation.

Unsaturated fatty acid chains are known to be an essential structural part of biomembranes, but only monounsaturated chains have been included in the molecular dynamics (MD) simulations of membrane systems. Here we present a 1-ns MD simulation for a diunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC; 16:0/18:2[delta9,12]) bilayer. The structural behavior of the phosphatidylcholine headgroup, the glycerol backbone, and the hydrating water were assessed and found to be consistent with the existing information about similar systems from both experimental and computational studies. Further analysis was focused on the structure of the double bond region and the effects of the diunsaturation on the bilayer interior. The behavior of the diunsaturated sn-2 chains is affected by the tilted beginning of the chain and the four main conformations of the double bond region. The double bonds of the sn-2 chains also influenced the characteristics of the saturated chains in the sn-1 position. Furthermore, extreme conformations of the sn-2 chains existed that are likely to be related to the functional role of the double bonds. The results here point out the importance of polyunsaturation for the biological interpretations deduced from the membrane MD simulations.