Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.     

An Auto-Catalytic Surface for Conformational Replication of Amyloid Fibrils—Genesis of an Amyloid World?

Amyloid fibrils are composed of self assembled stacked peptide or protein molecules folded and trapped in a stable cross-beta-sheet conformation. The amyloid fibrillation mechanism represents an intriguing self-catalyzed process rendering replication of a molecular conformational memory of interest for prebiotic chemistry. Herein we describe how a solid surface can be rendered auto-catalytic for fibrillation of a protein solution. We have discovered that a hydrophobic silicon or glass surface can be made to continuously fibrillate solutions of insulin monomers under stressed conditions (pH 1.6, 65°C). It was found that the surface acts as a platform for the formation of nascent seeds that induce fibril replication on and at the surface. This autocatalytic effect stems from a layer a few insulin molecules thick representing an oligomeric layer of misfolded, conformationally trapped, insulin molecules that rapidly through epitaxial growth catalyze the rate determining step (nucleation) during fibril replication. This autocatalytic layer is generated by the protein-solid surface interaction and conformational changes of the adsorbed protein during exposure at the air-water interface. The resulting autocatalytic surface thus both initiates local conformational molecular self-replication and acts as a reservoir for fibril seeds budding off into solution spreading fibril replication entities to the surrounding medium. The possibility of catalysis of the conformational replication process by minute amounts of nucleation sites located on a recruiting surface can evade the issue of dramatic concentration dependence of amyloidogenesis.     

Mass spectrometric analysis reveals changes in phospholipid, neutral sphingolipid and sulfatide molecular species in progressive epilepsy with mental retardation, EPMR, brain: a case study

Progressive epilepsy with mental retardation, EPMR, belongs to a group of inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The CLN8 gene that underlies EPMR encodes a novel transmembrane protein that localizes to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment. Recently, CLN8 was linked to a large eukaryotic protein family of TLC (TRAM, Lag1, CLN8) domain homologues with postulated functions in lipid synthesis, transport or sensing. By using liquid chromatography/mass spectrometry we analysed molecular species of major phosholipid and simple sphingolipid classes from cerebral samples of two EPMR patients representing a progressive and advanced state of the disease. The progressive state brain showed reduced levels of ceramide, galactosyl- and lactosylceramide and sulfatide as well as a decrease in long fatty acyl chain containing molecular species within these classes. Among glycerophospholipid classes, an increase in species containing polyunsaturated acyl chains was detected especially in phosphatidylserines and phosphatidylethanolamines. By contrast, saturated and monounsaturated species were overrepresented among phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol classes in the advanced state sample. The observed changes in brain sphingo- and phospholipid molecular profiles may result in altered membrane stability, lipid peroxidation, vesicular trafficking or neurotransmission and thus may contribute to the progression of the molecular pathogenesis of EPMR.     

Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry

Phospholipids (PL) in cerebral cortex from patients with infantile (INCL or CLN1) and juvenile (JNCL or CLN3) forms of neuronal ceroid-lipofuscinosis (NCL) and controls were analysed by normal phase HPLC and on-line electrospray ionization ion-trap mass spectrometric detection (LC-ESI-MS). The method provided quantitative data on numerous molecular species of different PL classes, which are not achieved by using the conventional chromatographic methods. Compared with the controls, the INCL brains contained proportionally more phosphatidylcholine (PC), and less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Different molecular species of PC, PE, PS, phosphatidylinositol and sphingomyelin were quantified using multiple internal PL standards that differed in fatty acyl chain length and thus allowed correction for chain length dependency of instrument response. In INCL cortex, which had lost 65% of the normal PL content, the proportions of polyunsaturated molecular species, especially the PS and PE that contained docosahexaenoic acid (22:6n-3), were dramatically decreased. The membranes may have adapted to this alteration by increasing the proportions of PL molecules substituted with monounsaturated and short-chain fatty acids. Lysobisphosphatidic acid was highly elevated in the INCL brain and consisted mostly of polyunsaturated species. It is possible that changes in the composition of PL membranes accelerate progression of INCL by altering signalling and membrane trafficking in neurons.     

Reproductive performance of Common Goldeneye Bucephala clangula females in relation to age and lifespan

We studied the effects of female age and length of lifespan on reproductive performance in the Common Goldeneye Bucephala clangula , a precocial species with self-feeding chicks. Dependent variables studied were the occurrence of failed nesting attempts and non-breeding years, clutch size, nesting and hatching success, female condition and recovery rate of offspring. To avoid misinterpretations resulting from age-dependent quality changes in the cohorts, individuals with different lifespans were separated in the analyses. There was no evidence that females of different lifespan differed in reproductive strategy. The data revealed no significant differences in nest losses, occurrence of non-breeding years, timing of nesting or clutch size, but the physiological condition of short-lived females was poorer than that of longer living females. This accords with the prediction that poor-quality females invest proportionally more in reproduction than high-quality females and consequently die earlier. The most productive individuals were those that lived longest. The only indication of senescence was the low recovery rate of offspring produced by old females. The last breeding attempt did not differ in any observable respect from the penultimate attempt, implying that the last investment in reproduction was no heavier than the previous one.     

Gene Expression Profiling of Peri-Implant Healing of PLGA-Li+ Implants Suggests an Activated Wnt Signaling Pathway In Vivo

Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li⁺). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li⁺, while PLGA without Li⁺ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li⁺ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li⁺, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li⁺ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li⁺. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li⁺ compared with Ctrl. The presence of β-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors'' knowledge the first report evaluating the effect of a local release of Li⁺ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li⁺ in PLGA implants, Li⁺ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.