A Conserved, Lipid-Mediated Sorting Mechanism of Yeast Ist2 and Mammalian STIM Proteins to the Peripheral ER

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS^Ist2) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS^Ist2 targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P₂ at the PM created ER–PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca²⁺ entry (SOCE). The CSS^Ist2 and the C-terminus of the ER-located Ca²⁺ sensor STIM2 were sufficient to bind PI(4,5)P₂ and PI(3,4,5)P₃ at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P₂, suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.     

Binding of Plasma Membrane Lipids Recruits the Yeast Integral Membrane Protein Ist2 to the Cortical ER

Recruitment of cytosolic proteins to individual membranes is governed by a combination of protein–protein and protein–membrane interactions. Many proteins recognize phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the cytosolic surface of the plasma membrane (PM). Here, we show that a protein–lipid interaction can also serve as a dominant signal for the sorting of integral membrane proteins. Interaction with phosphatidly-inositolphosphates (PIPs) at the PM is involved in the targeting of the polytopic yeast protein Ist2 to PM-associated domains of the cortical endoplasmic reticulum (ER). Moreover, binding of PI(4,5)P₂ at the PM functions as a dominant mechanism that targets other integral membrane proteins to PM-associated domains of the cortical ER. This sorting to a subdomain of the ER abolishes proteasomal degradation and trafficking along the classical secretory (sec) pathway. In combination with the localization of IST2 mRNA to the bud tip and other redundant signals in Ist2, binding of PIPs leads to efficient accumulation of Ist2 at domains of the cortical ER from where the protein may reach the PM independently of the function of the sec-pathway.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.     

Conformation of Bovine Nerve Root P2 Protein: Characteristics of the Molecule from Circular Dichroism Spectra

Abstract: Circular dichroism (CD) was used to study the conformations of bovine nerve root P₂ basic protein, its reduced and carboxymethylated derivative (RCM-P₂), and its large cyanogen bromide fragment (CN1). Data in the far UV show that both the parent protein and RCM-P₂ have conformations dominated by a large amount of β structure. However, the CN1 peptide appears to exist in a largely unordered conformation. Since CN1 lacks short (20 residue) amino- and carboxy-terminal segments of the P₂ protein, the spectral data suggest that these regions are important for determining and/or maintaining folding of the P₂ protein in aqueous solutions. The P₂ protein was found to have a distinctive CD spectrum in the near UV. The characteristics of molar ellipticities indicate that the spectrum contains significant contributions from tyrosine residues, and that these contributions suggest different environments for the two tyrosines in P₂ protein. Both environments depend on protein conformation, since CD side chain absorptions are lost when P₂ protein is denatured with 5 M urea.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

RanBPM, a novel interaction partner of the brain-specific protein p42IP4/centaurin alpha-1

The protein p42^IP4 (aka Centaurin α-1) is highly enriched in the brain and has specific binding sites for the membrane lipid phosphatidylinositol 3,4,5-trisphosphate and the soluble messenger inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄; IP4). p42^IP4 shuttles between plasma membrane, cytosol and cell nucleus. However, the molecular function of p42^IP4 is still largely unclear. Here, we report a novel interaction partner for p42^IP4, Ran binding protein in microtubule-organizing center (RanBPM). RanBPM is ubiquitously expressed and seems to act as scaffolding and modulator protein. In our studies, we established this interaction in vitro and in vivo . The in vivo interaction was demonstrated with endogenous RanBPM from rat brain. Both proteins co-localize in transfected HEK 293 cells. We could show that the interaction does not require additional proteins. D-Ins(1,3,4,5)P₄, a specific ligand for p42^IP4, is a concentration-dependent and stereoselective inhibitor of this interaction; the l -isoform is much less effective. We found that mainly the SPRY domain of RanBPM mediates the p42^IP4-RanBPM association. The ARFGAP domain of p42^IP4 is important for the interaction, without being the only interaction site. Recently, p42^IP4 and RanBPM were shown to be involved in dendritic differentiation. Thus, we hypothesize that RanBPM could act as a modulator together with p42^IP4 in synaptic plasticity.     

Phosphoinositide recognition domains

Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3-phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind PtdIns(3,4,5)P₃ and/or PtdIns(3,4)P₂], but requires an additional membrane-insertion and/or oligomerization component in the case of the PtdIns(3)P-targeting phox homology (PX) and FYVE domains. Domains that target PtdIns(4,5)P₂, which is more abundant by some 25-fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding .     

SUBCELLULAR FRACTIONS OF NORMAL HUMAN SUBSTANTIA NIGRA AND CAUDATE NUCLEUS; A STUDY OF THEIR MORPHOLOGY AND SOME ENZYMES INCLUDING GLUTAMATE DECARBOXYLASE AND CHOLINE ACETYLTRANSFERASE

Abstract— Subcellular fractions have been prepared from normal human caudate nucleus and substantia nigra by a standard fractionation technique and the fractions assayed for the following enzymes, which were studied because of their relevance to neurotransmission and pathological change: glutamate decarboxylase (EC 4.1.1.15), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), acid phosphatase (EC 3.1.3.2) and succinate dehydrogenase (EC 1.3.99.1). The distribution of these enzymes was assessed in relation to the morphology of the fractions as observed by electron microscopy. As with preparations from animal cerebral cortex, acetylcholinesterase and acid phosphatase were found mainly in fractions known to contain plasma membranes, synaptosomal membranes and microsomes. The levels of choline acetyltransferase in fractions from the substantia nigra were too low to measure but, in the caudate nucleus, the enzyme was concentrated in the crude mitochondrial fraction (P₂), especially in the P₂B and P₂C subfractions. A high proportion of the glutamate decarboxylase activity was present in the P₂ fractions of the substantia nigra and caudate nucleus and, although the synaptosomal (P₂B) fraction contained the enzyme, significant amounts were found in the mitochondrial (P₂C) fraction. This may have been due to some contamination of the mitochondria with small synaptosomes. Succinate dehydrogenase showed a conventional bimodal distribution between synaptosomes and mitochondria with a concentration in the latter.     

Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

A key event in signal transduction in many eukaryotes is activation of polyphosphoinositide-specific phospholipase C (PIC). This enzyme hydrolyses the plasma membrane-associated lipid, phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P₂) which leads to the production of the two second messenger molecules: inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃) and 1,2-diacylglycerol (DG). In plants, an enzyme which functionally resembles mammalian PIC is known to exist in the plasma membrane, but little is understood about how its activity is regulated. The recent discovery of several plant proteins with 30–40% homology to the mammalian actin- and phosphoinositide-binding protein, profilin, has prompted an investigation as to whether these proteins (plant profilins) are able to interact with polyphosphoinositides and, if so, whether such interactions have physiological relevance for signal transduction via the plant phosphoinositide system.
In this study it is demonstrated that a direct and highly specific interaction does exist between plant profilin and polyphosphoinositides and that these interactions dramatically affect the ability of plant plasma membrane phosphoinositide phospholipase C to utilize phosphoinositides for second messenger production. These data are the first to demonstrate a functional role of plant profilin in controlling polyphosphoinositide turnover and also provide the first evidence for a direct effect of an actin-binding protein on a membrane-associated signalling enzyme. These findings indicate a novel mechanism for control of plant phosphoinositide turnover, and suggest a possible link between plant cell activation, second messenger production and modulation of cytoskeletal dynamics.     

Enhanced potassium uptake and phosphatidylinositol -phosphate turnover by hypertonic mannitol shock

A hypertonic mannitol shock enhanced K⁺ uptake by Beta vulgaris L. (cv. early flat Egyptian) storage tissue slices and also increased the inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃) content of the slices as well as of Sorghum bicolor L. (cv. Hazera) and Vigna radiata L. (cv. unknown) roots. K⁺ uptake by B. vulgaris slices could be enhanced, in the absence of mannitol, by application of effectors that mimic products of the phosphatidylinositol 4,5-bisphosphate (PIP₂) turnover cycle. Maximal Ins (1,4,5)P₃ content was found 10 min after hypertonic induction and maximal K⁺ uptake was obtained 10 min later. The hypertonic mannitol shock, administered to intact B. vulgaris slices, also enhanced the phosphorylation of a 39 kDa protein in the plasmalemma.     

Functional Modulation of P2×2 Receptors by Cyclic AMP-Dependent Protein Kinase

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P_2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P_2×2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P_2×2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P_2×2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser⁴³¹) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P_2×2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P_2×2 receptor and ATP-mediated physiological effects in the nervous system.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.     

Depletion of Vipp1 in Synechocystis sp. PCC 6803 affects photosynthetic activity before the loss of thylakoid membranes

A vipp1 mutant of Synechocystis sp. PCC 6803 could not be completely segregated under either mixotrophic or heterotrophic conditions. A vipp1 gene with a copper-regulated promoter (P _petE -vipp1 ) was integrated into a neutral platform in the genome of the merodiploid mutant. The copper-induced expression of P _petE -vipp1 allowed a complete segregation of the vipp1 mutant and observation of the phenotype of Synechocystis 6803 with different levels of vesicle-inducing protein in plastids 1 (Vipp1). When P _petE -vipp1 was turned off by copper deprivation, Synechocystis lost Vipp1 and photosynthetic activity almost simultaneously, and at a later stage, thylakoid membranes and cell viability. The photosystem II (PSII)-mediated electron transfer was much more rapidly reduced than the PSI-mediated electron transfer. By testing a series of concentrations, we found that P _petE -vipp1 cells grown in medium with 0.025 μM Cu²⁺ showed no reduction of thylakoid membranes, but greatly reduced photosynthetic activity and viability. These results suggested that in contrast to a previous report, the loss of photosynthetic activity may not have been due to the loss of thylakoid membranes, but may have been caused more directly by the loss of Vipp1 in Synechocystis 6803.     

Evidence for an association of prion protein and sphingolipid-mediated signaling

The physiological function of the cellular prion protein (PrP^c) is unclear. PrP^c associates with lipid rafts, highly glycolipid-rich membrane domains containing a large variety of signaling molecules, e.g., sphingolipids (SL). In this study, we investigated possible connections between PrP^c and sphingolipid-associated signaling pathways. Using PrP^c-wt and PrP^c-k.o. hippocampal cell lines and mouse brains we showed higher activity of neutral and acid sphingomyelinase (SMase) in PrP^c-k.o.-groups, while ceramide and sphingomyelin-levels were unchanged. Furthermore, despite lower basal expression levels of sphingosine kinase (SphK) in PrP^c-k.o.-groups, the levels of its metabolite sphingosine-1-phosphate were increased, whereas S1P₃-receptor expression was higher in PrP^c-wt-groups again. In addition, we detected enhanced activity of phospholipase D1, an enzyme that seems to be suitable to act as a connector between the S1P₃ receptor and continuative signaling. Finally, evidence for an impact on downstream signaling cascades, especially activation of the PI3K/Akt pathway, was found. In summary, our data suggest that PrP^c is involved in sphingolipid-associated signaling, modulating pathways that exert anti-apoptotic functions, hence indicating that PrP^c plays a role in neuroprotection.     

The effect of variation of thermal processing on the microbial spoilage of chub-packed luncheon meat

Process pasteurization values for reference temperature 70°C (P₇₀) were calculated from the temperature profiles of 250 g luncheon meat chubs cooked under experimental conditions. A simple equation relating Process P₇₀-value and the time and temperature of cooking was derived. With minimal cooking (P₇₀= 40) the surviving microflora (10³/g) was dominated by species of Lactobacillus, Brochothrix and Micrococcus. These organisms were destroyed by more intensive cooking (P₇₀= 105), leaving a flora (10²/g) composed of Bacillus and Micrococcus species. The spoilage that developed after 14 d storage at 25°C reflected the severity of the heat treatment received by each chub: with P₇₀ between 40 and 90, a Streptococcus spoilage sequence occurred; with P₇₀ between 105 and 120, a Bacillus/Streptococcus spoilage sequence occurred; with P₇₀ of 135 and above, a Bacillus spoilage sequence occurred. Cooking to a P₇₀= 75 was adequate to reduce the surviving microflora to the 10²/g level associated with current good manufacturing practice.