L-Carnitine induces recovery of liver lipid metabolism in cancer cachexia

Cancer cachexia causes metabolic alterations with a marked effect on hepatic lipid metabolism. l-Carnitine modulates lipid metabolism and its supplementation has been proposed as a therapeutic strategy in many diseases. In the present study, the effects of l-carnitine supplementation on gene expression and on liver lipid metabolism-related proteins was investigated in cachectic tumour-bearing rats. Wistar rats were assigned to receive 1 g/kg of l-carnitine or saline. After 14 days, supplemented and control animals were assigned to a control (N), control supplemented with l-carnitine (CN), tumour-bearing Walker 256 carcinosarcoma (TB) and tumour-bearing supplemented with l-carnitine (CTB) group. The mRNA expression of carnitine palmitoyltransferase I and II (CPT I and II), microsomal triglyceride transfer protein (MTP), liver fatty acid-binding protein (L-FABP), fatty acid translocase (FAT/CD36), peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and organic cation transporter 2 (OCTN2) was assessed, and the maximal activity of CPT I and II in the liver measured, along with plasma and liver triacylglycerol content. The gene expression of MTP, and CPT I catalytic activity were reduced in TB, who also showed increased liver (150%) and plasma (3.3-fold) triacylglycerol content. l-Carnitine supplementation was able to restore these parameters back to control values (p < 0.05). These data show that l-carnitine preserves hepatic lipid metabolism in tumour-bearing animals, suggesting its supplementation to be of potential interest in cachexia.

Interaction of miltefosine with intercellular membranes of stratum corneum and biomimetic lipid vesicles

Miltefosine (MT) is an alkylphospholipid approved for breast cancer metastasis and visceral leishmaniasis treatments, although the respective action mechanisms at the molecular level remain poorly understood. In this work, the interaction of miltefosine with the lipid component of stratum corneum (SC), the uppermost skin layer, was studied by electron paramagnetic resonance (EPR) spectroscopy of several fatty acid spin-labels. In addition, the effect of miltefosine on (i) spherical lipid vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and (ii) lipids extracted from SC was also investigated, by EPR and time-resolved polarized fluorescence methods. In SC of neonatal Wistar rats, 4% (w/w) miltefosine give rise to a large increase of the fluidity of the intercellular membranes, in the temperature range from 6 to about 50 degrees C. This effect becomes negligible at temperatures higher that ca. 60 degrees C. In large unilamelar vesicles of DPPC no significant changes could be observed with a miltefosine concentration 25% molar, in close analogy with the behavior of biomimetic vesicles prepared with bovine brain ceramide, behenic acid and cholesterol. In these last samples, a 25 mol% molar concentration of miltefosine produced only a modest decrease in the bilayer fluidity. Although miltefosine is not a feasible skin permeation enhancer due to its toxicity, the information provided in this work could be of utility in the development of a MT topical treatment of cutaneous leishmaniasis. Published by Elsevier B.V.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

Association of postalimentary lipemia with atherosclerotic manifestations

We identified different lipemic and metabolic responses after the ingestion of a standardized meal by healthy adults and related them to atherosclerotic markers. Samples from 60 normolipidemic adults were collected before and after a liquid meal (40 g fat/m² body surface) at 0, 2, 4, 6, and 8 h for measurements of lipids, free fatty acids (FFA), insulin, cholesteryl ester transfer protein (CETP), autoantibodies to epitopes of oxidized LDL (oxLDL Ab), lipolytic activities, and apolipoprotein E polymorphism. Mean carotid intima-media thickness (cIMT) was determined by Doppler ultrasound. The volunteers were classified into early (N = 39) and late (N = 31) triacylglycerol (TAG) responders to the test meal. Late responders showed lower HDL cholesterol concentration at fasting and in the TAG peak, lower insulin and higher FFA concentrations compared to early responders. Multivariate regression analyses showed that mean cIMT was associated with gender (male) and age in early responders and by cholesterol levels at the 6th hour in late responders. oxLDL Ab were explained by lipoprotein lipase and negatively by hepatic lipase and oxLDL Ab (fasting period) by CETP (negative) and FFA (positive). This study is the first to identify a postalimentary insulin resistance state, combined with a reduced CETP response exclusively among late responders, and the identification of the regulators of postalimentary atherogenicity. Further research is required to determine the metabolic mechanisms described in the different postalimentary phenotypes observed in this study, as well as in different pathological states, as currently investigated in our laboratory.

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Proteine im Wein

Untersucht wird die Proteinzusammensetzung verschiedener Weinsorten der Anbaugebiete Rheinhessen, Rheingau und Pfalz. Es erfolgt erstmalig die Identifizierung der Proteine eines Rotweins (Portugieser 2005 aus der Pfalz). Hierzu werden die Proteine mittels Dialyse und Gefriertrocknung konzentriert, auf einer SDS-PAGE aufgetrennt und mittels ESI-Q-TOF-Massenspektrometrie identifiziert. Von den identifizierten Proteinen des Rotweins stammen zwölf aus der Weinbeere und sechs werden im Laufe der Weinbereitung durch die Hefe ein-gebracht. Der Großteil der über die Weinbeere in den Wein gelangten Proteine ist der Gruppe der Pathogenese bezogenen Proteine zuzuordnen. Ein Vergleich der Proteinzusammensetzung verschiedener Rotweine, Weißweine und Rosé-weine zeigt, dass ein gemeinsames Proteinspektrum in allen Weinsorten enthalten ist, es je-doch auch Unterschiede hinsichtlich der Proteinzusammensetzung und Konzentration der ein-zelnen Proteine zwischen den Rebsorten, insbesondere roten und weißen, gibt. In Portugieser Rotwein des Jahrgangs 2005 aus der Pfalz sowie in Dornfelder Rotwein der Jahrgänge 2002, 2003, 2004 und 2005 kann das als Allergen beschriebene Lipid Transfer Pro-tein (Isoform 4) nachgewiesen werden. In den untersuchten Weißweinsorten Riesling, Sau-vignon Blanc, Morio Muskat sowie Gewürztraminer ist dieses nicht bzw. nur in sehr geringen Mengen enthalten. Ebenfalls nur in Rotwein wird eine Klasse IV Endochitinase identifiziert, die als Allergen in jungem Rotwein bereits beschrieben wurde. Außerdem bedingen Chitin-asen zusammen mit den Thaumatin-ähnlichen Proteinen die Proteininstabilität. Thaumatin-ähnliche Proteine stellen in allen Weinsorten den größten Anteil der Proteine dar. Viele der Weinproteine liegen in glykosylierter Form vor, was durch die Perjodsäurefärbung und den Lektinblot gezeigt werden kann. Mit der Detektion von Thaumatin-ähnlichen Proteinen, Lipid Transfer Proteinen sowie einer Endochitinase werden potentielle Allergene im Wein nachgewiesen sowie strukturell mit be-kannten Allergenen verglichen. Die Kenntnis der Proteinzusammensetzung im Wein bildet die Grundlage für weiterführende Untersuchungen zur Weinstabilität und zur Allergenität von Weinproteinen.

Entwicklung und Optimierung eines Mikroarray zur Erfassung der genetischen Prädisposition von Atherosklerose

Die Entstehung der Atherosklerose ist ein komplexer Vorgang, der sich durch Ablagerung von Lipiden an der Gefäßwand sowie durch immunologische und inflammatorische Prozesse auszeichnet. Neben konventionellen Risikofaktoren wie Alter, Geschlecht, Rauchen, HDL-Cholesterin, Diabetes mellitus und einer positiven Familienanamnese werden zur Bestimmung des atherosklerotischen Risikos neue Biomarker der inflammatorischen Reaktion untersucht. Ziel dieser Arbeit war die Entwicklung einer Methode zur Diagnostik des Atheroskleroserisikos. Es wurde eine neuartige Chip-Technologie eingesetzt, um das Risiko für eine potentiell drohende atherosklerotische Erkrankung abzuschätzen. Dabei wurde ausgenutzt, dass molekulare Veränderungen in Genen bestimmte Krankheitsbilder auslösen können. rnEs wurde ein molekularbiologischer Test entwickelt, welcher die Untersuchung von genetischen Variationen aus genomischer DNA ermöglicht. Dafür fand die Entwicklung einer Multiplex-PCR statt, deren Produkt mit der Chip-Technologie untersucht werden kann. Dazu wurden auf einem Mikroarray Sonden immobilisiert, mit deren Hilfe genspezifische Mutationen nachgewiesen werden können. So wurden mehrere Gene mit einem geringen Aufwand gleichzeitig getestet. rnDie Auswahl der entsprechenden Marker erfolgte anhand einer Literaturrecherche von randomisierten und kontrollierten klinischen Studien. Der Mikroarray konnte für zwölf Variationen in den acht Genen Prostaglandinsynthase-1 (PTGS1), Endotheliale NO-Synthase (eNOS), Faktor V (F5), 5,10-Methylentetrahydrofolsäure-Reduktase (MTHFR), Cholesterinester-Transferprotein (CETP), Apolipoprotein E (ApoE), Prothrombin (F2) und Lipoproteinlipase (LPL) erfolgreich etabliert werden. Die Präzision des Biochips wurde anhand der Echtzeit-PCR und der Sequenzierung nachgewiesen. rnDer innovative Mikroarray ermöglicht eine einfache, schnelle und kosteneffektive Genotypisierung von wichtigen Allelen. Viele klinisch relevante Variationen für Atherosklerose können nun in nur einem Test überprüft werden. Zukünftige Studien müssen zeigen, ob die Methode eine Vorhersage über den Ausbruch der Erkrankung und eine gezielte Therapie ermöglicht. Dies wäre ein erster Schritt in Richtung präventive und personalisierter Medizin für Atherosklerose.rn

Weinproteine - Allergenität und Trubbildung

Wein ist eine komplexe Lösung bestehend aus verschiedensten Komponenten wie Alkohol, Polyphenolen, Polysacchariden, Sulfiten und auch Proteinen. Auch wenn Proteine nur in geringen Mengen im Wein enthalten sind, beeinflussen sie die Qualität maßgeblich. Hier ist zum einen deren potentielle Unverträglichkeit bis hin zur Allergie zu nennen, und zum anderen der Einfluss der Weinproteine auf die Trübung. Im Rahmen einer epidemiologischen Studie der Arbeitsgruppe Fronk/Decker wurde festgestellt, dass es in der Weinregion Mainz ein starkes Interesse gibt die Ursache einer Weinunverträglichkeit zu untersuchen. Für weiterführende Untersuchungen wurde im Rahmen meiner Arbeit das Lipid Transfer Protein (LTP), welches als einziges Allergen der Traube bekannt ist, aus Trauben und Wein in hohem Reinheitsgrad isoliert. Es konnte gezeigt werden, dass dessen Struktur bei der Weinherstellung nicht maßgeblich verändert wurde. In einer klinischen Studie mit 29 Probanden wurde die potentielle Allergenität von Weinproteinen, im Besonderen des LTPs untersucht. Allerdings konnte bei den untersuchten Probanden keine echte IgE-Antikörper-vermittelte Allergie auf das LTP nachgewiesen werden. Daher wird die Ursache der beschriebenen Unverträglichkeiten bei anderen Weininhaltsstoffen oder auch auf pollenassoziierten Kreuzreaktionen vermutet. Bei der Entstehung einer Weintrübung sind zahlreiche Inhaltstoffe beteiligt. Die Rolle der Proteine ist in diesem Zusammenhang noch nicht abschließend geklärt. In dieser Arbeit wurde die Komplexität der Proteinzusammensetzung in Abhängigkeit von Lage, Jahrgang, Rebsorte sowie Behandlungsmaßnahmen gezeigt. Hinsichtlich der Stabilisierung und Trübungsrelevanz der Weinproteine konnte mittels biochemischer, bioinformatischer und biophysikalischer Methoden gezeigt werden, dass nur ein Teil der im Wein enthaltenen Thaumatin-ähnlichen Proteine und Chitinasen an der Trubbildung beteiligt sind. Die Invertase hingegen denaturiert erst ab einer Temperatur von ca. 83 °C und aggregiert in der Trübung. Somit führt dieses Protein bei Wärmetests zu Bentonitbedarfsermittlung in diesem Temperaturbereich zu einer Überschätzung. Die Versuche zur temperaturabhängigen Aggregation von Proteinen zeigen, wie wichtig die Berücksichtigung der Umgebungsfaktoren bei der Trubbildung ist. So konnten unterschiedliche Wechselwirkungen im Puffer- und realen Weinsystem von potentiell trübungsstabilisierenden Polysacchariden mit den Weinproteinen detektiert werden. Für das Arabinogalactan beispielsweise wurde in den Versuchen im Weinsystem eine destabilisierende Wirkung gefunden, während es bei den Versuchen im Puffersystem eine positive Wirkung auf die Stabilisierung der Probe zeigte. Es zeigte sich, dass die verschiedenen Weininhaltsstoffe in einer komplexen Wechselwirkung zueinander stehen und somit eine molekulare Interpretation erschweren.

Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

NADPH P450 oxidoreductase: structure, function, and pathology of diseases

Cytochrome P450 oxidoreductase (POR) is an enzyme that is essential for multiple metabolic processes, chiefly among them are reactions catalyzed by cytochrome P450 proteins for metabolism of steroid hormones, drugs and xenobiotics. Mutations in POR cause a complex set of disorders that often resemble defects in steroid metabolizing enzymes 17α-hydroxylase, 21-hydroxylase and aromatase. Since our initial reports of POR mutations in 2004, more than 200 different mutations and polymorphisms in POR gene have been identified. Several missense variations in POR have been tested for their effect on activities of multiple steroid and drug metabolizing P450 proteins. Mutations in POR may have variable effects on different P450 partner proteins depending on the location of the mutation. The POR mutations that disrupt the binding of co-factors have negative impact on all partner proteins, while mutations causing subtle structural changes may lead to altered interaction with specific partner proteins and the overall effect may be different for each partner. This review summarizes the recent discoveries related to mutations and polymorphisms in POR and discusses these mutations in the context of historical developments in the discovery and characterization of POR as an electron transfer protein. The review is focused on the structural, enzymatic and clinical implications of the mutations linked to newly identified disorders in humans, now categorized as POR deficiency.

Characterization of three human sec14p-like proteins: alpha-tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Dusty punch cards and an eternal enigma: high-density lipoproteins and atherosclerosis

Epidemiological, clinical, and experimental evidence has accumulated during the last decades suggesting that high-density lipoproteins (HDLs) may protect from atherosclerosis and its clinical consequences. However, more than 55 years after the first description of the link between HDL and heart attacks, many facets of the biochemistry, function, and clinical significance of HDL remain enigmatic. This applies particularly to the completely unexpected results that became available from some recent clinical trials of nicotinic acid and of inhibitors of cholesteryl ester transfer protein (CETP). The concept that raising HDL cholesterol by pharmacological means would decrease the risk of vascular disease has therefore been challenged.

Mechanisms of Ligand–Protein Interaction in Sec-14-like Transporters Investigated by Computer Simulations

We review our recent work on protein-ligand interactions in vitamin transporters of the Sec-14-like protein. Our studies focused on the cellular-retinaldehyde binding protein (CRALBP) and the alpha-tocopherol transfer protein (alpha-TTP). CRALBP is responsible for mobilisation and photo-protection of short-chain cis-retinoids in the dim-light visual cycle or rod photoreceptors. alpha-TTP is a key protein responsible for selection and retention of RRR-alpha-tocopherol, the most active isoform of vitamin E in superior animals. Our simulation studies evidence how subtle chemical variations in the substrate can lead to significant distortion in the structure of the complex, and how these changes can either lead to new protein function, or be used to model engineered protein variants with tailored properties. Finally, we show how integration of computational and experimental results can contribute in synergy to the understanding of fundamental processes at the biomolecular scale.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.