Cholesterol and Fatty Acids Regulate Dynamic Caveolin Trafficking through the Golgi Complex and between the Cell Surface and Lipid Bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Phototropism: Translating light into directional growth.

Phototropism allows plants to align their photosynthetic tissues with incoming light. The direction of incident light is sensed by the phototropin family of blue light photoreceptors (phot1 and phot2 in Arabidopsis), which are light-activated protein kinases. The kinase activity of phototropins and phosphorylation of residues in the activation loop of their kinase domains are essential for the phototropic response. These initial steps trigger the formation of the auxin gradient across the hypocotyl that leads to asymmetric growth. The molecular events between photoreceptor activation and the growth response are only starting to be elucidated. In this review, we discuss the major steps leading from light perception to directional growth concentrating on Arabidopsis. In addition, we highlight links that connect these different steps enabling the phototropic response.

Platforms for high-throughput screening of Wnt/Frizzled antagonists.

Signaling cascades initiated by Wnt lipoglycoproteins and their receptors of the Frizzled family regulate many aspects of animal development and physiology. Improper activation of this signaling promotes carcinogenic transformation and metastasis. Development of agents blocking the Wnt-Frizzled signaling is of prime interest for drug discovery. Despite certain progress no such agents are as yet brought to the market or even to clinical trials. One reason for these delays might be the use of suboptimal readout assays. In this article we overview existing and developing assay platforms to screen for Wnt-Frizzled antagonists. Among those, G protein-activating assays built on the emerging GPCR properties of Frizzleds are highlighted.

Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.

Special commentary : a call for intensive metabolic support.

PURPOSE OF REVIEW: This special commentary addresses recent clinical reviews regarding appropriate nutrition and metabolic support in the critical care setting. RECENT FINDINGS: There are divergent approaches between North America and Europe for the use of early nutrition support and combined enteral nutrition and parenteral nutrition support possibly due to the commercial availability of specific parenteral nutrients. The advent of intensive insulin therapy has changed the landscape of metabolic support in the intensive care unit, and previous notions about infective risk of parenteral nutrition will need to be re-addressed. Patients with brain failure may benefit from an intensive insulin therapy with a blood glucose target that is higher than that used in patients without brain failure. Patients with heart failure may benefit from the addition of nutritional pharmacology that targets proximate oxidative pathophysiological pathways. Intradialytic parenteral nutrition may be viewed as another form of supplemental parenteral nutrition when enteral nutrition is insufficient in patients on hemodialysis in the intensive care unit. SUMMARY: It is proposed that intensive metabolic support be routinely implemented in the intensive care unit based on the following steps: intensive insulin therapy with an appropriate blood glucose target, nutrition risk assessment, early and if needed combined enteral nutrition and parenteral nutrition to target 20-25 kcal/kg/day and 1.2-1.5 g protein/kg/day, and nutritional and metabolic monitoring.

Translation of polarity cues into asymmetric spindle positioning in "Caenorhabditis elegans"

Abstract: Asymmetric cell division is important to generate tissue diversity. The Caenorhabditis elegans embryo is well suited to study the mechanisms of asymmetric cell division. In wild type one-cell stage embryos, the spindle sets up along the anterior-posterior axis (AP). During anaphase, the spindle elongates. While the anterior spindle pole is relatively immobile, the posterior spindle pole moves towards the posterior cortex during anaphase leading to an asymmetric spindle position. As a result, the first cleavage gives rise to a large anterior blastomere and a smaller posterior one, which differs also in cell fate determinants. This posterior spindle displacement occurs in response to polarity cues set up along the AP axis by the PAR proteins and is due to imbalanced pulling forces acting on the two spindle poles, with net forces acting on the posterior spindle pole being more extensive than those at the anterior one. The project of my thesis was to characterize the involvement of two new components, gpr-1 and gpr-2, in spindle positioning. These genes encode essentially identical proteins containing a GoLoco motif characteristic of proteins interacting with α subunits of heterotrimeric G protein (Gα). In gpr-1/2(RNAi) embryos and in embryos lacking simultaneously two α subunits, goa-1 and gpa-16, (Ga(RNAi) embryos), there is a minimal posterior displacement of the spindle during anaphase, and the first division is equal. I found that the pulling forces acting on the two spindle poles is weak and equal in gpr-1/2(RNAi) and Gα (RNAi) embryos. I found that GPR-1/2 acts downstream of polarity cues for generation of pulling forces. Furthermore, I showed that GPR-1/2 distribution was enriched at the posterior cortex during metaphase whereas GOA-1 and GPA-16 were uniformly distributed at the cell cortex throughout the cell cycle. Gα subunits oscillate between GDP- and GTP-bound forms. Gα signaling is turned on by GDP/GTP exchange catalyzed by guanine nucleotide exchange factors (GEFs) and turned off by hydrolysis of GTP catalyzed by GTPase activating proteins (GAPs). A third class of proteins, the guanine dissociation inhibitors (GDIs), binds the GDP-bound form of Gα subunits and inhibits nucleotide exchange. I found that GPR-1/2 acts as a GDI for GOA-1. Taken together, my findings suggest a model in which differential activation of Gα subunits along the AP axis may translate into generation of differential pulling forces on the anterior and posterior spindle poles, and, thus, asymmetric cell division. Résumé L'embryon du nématode Caenorhabditis elegans est un modèle approprié pour étudier les mécanismes de la division asymétrique. Chez l'embryon précoce, le fuseau mitotique se forme le long de l'axe antéro-postérieur (A/P) et au centre de l'embryon, le pôle antérieur restant relativement immobile alors que le pôle postérieur du fuseau se déplace vers le cortex postérieur au cours de l'anaphase conduisant à une position excentrée du fuseau. 11 en résulte une première division qui génère un blastomère antérieur et postérieur de grande et petite taille respectivement et qui diffèrent en facteurs développementaux. Ce déplacement postérieur se produit en réponse de la polarité établie par la distribution polarisée des protéines PAR et est le résultat de la génération de forces inégales tirant sur les deux pôles du fuseau, les forces agissant sur le pôle postérieur du fuseau étant plus grandes. Le projet de ma thèse était d'identifier la fonction de deux nouveaux constituants, gpr-1 et gpr-2 dans le positionnement asymétrique du fuseau. Ces gènes codent essentiellement pour la même protéine qui contient un motif GoLoco, caractéristique des protéines interagissant avec la sous-unité alpha des protéines G hétérotrimériques. Chez l'embryon gpr-1/2(RNAi) et chez les embryons dépourvus d'activité de deux sous-unités alpha, goa-1 et gpa-16, (Gα(RNAi)), j'ai montré qu'il y avait un déplacement minimal du fuseau vers le pôle postérieur au cours de l'anaphase et la première division est symétrique en raison de forces faibles et égales agissant sur les deux pôles du fuseau. J'ai également montré que gpr-1/2 était requis en aval des signaux établissant la polarité pour générer les forces responsables du positionnement asymétrique du fuseau. De plus, j'ai montré que GPR-1/2 était enrichi au pôle postérieur lors de la métaphase alors que GOA-1 et GPA-16 étaient localisés de façon uniforme au cortex de l'embryon précoce. Gas oscillent entre une forme liée au GDP et une forme liée au GTP. La signalisation des Gas est activée par l'échange GDP/GTP qui est catalysé par des protéines GEFs. La signalisation des Gas est désactivée par l'hydrolyse du GTP qui est catalysée par des protéines GAPs. Une troisième classe de protéines, GDIs lie la forme GDP et inhibe l'échange de nucléotides. J'ai montré que GPR-1/2 agissait comme un GDI pour GOA-1. Mes résultats suggèrent un modèle dans lequel une activation différentielle des Gα le long de l'axe A/P pourrait générer des forces différentielles sur le pôle antérieur et postérieur du fuseau.

Distribution and risk factors for Plasmodium and helminth co-infections: a cross-sectional survey among children in Bagamoyo district, coastal region of Tanzania.

BACKGROUND: Plasmodium and soil transmitted helminth infections (STH) are a major public health problem, particularly among children. There are conflicting findings on potential association between these two parasites. This study investigated the Plasmodium and helminth co-infections among children aged 2 months to 9 years living in Bagamoyo district, coastal region of Tanzania. METHODS: A community-based cross-sectional survey was conducted among 1033 children. Stool, urine and blood samples were examined using a broad set of quality controlled diagnostic methods for common STH (Ascaris lumbricoides, hookworm, Strongyloides stercoralis, Enterobius vermicularis, Trichuris trichura), schistosoma species and Wuchereria bancrofti. Blood slides and malaria rapid diagnostic tests (mRDTs) were utilized for Plasmodium diagnosis. RESULTS: Out of 992 children analyzed, the prevalence of Plasmodium infection was 13% (130/992), helminth 28.5% (283/992); 5% (50/992) had co-infection with Plasmodium and helminth. The prevalence rate of Plasmodium, specific STH and co-infections increased significantly with age (p < 0.001), with older children mostly affected except for S. stercoralis monoinfection and co-infections. Spatial variations of co-infection prevalence were observed between and within villages. There was a trend for STH infections to be associated with Plasmodium infection [OR adjusted for age group 1.4, 95% CI (1.0-2.1)], which was more marked for S. stercoralis (OR = 2.2, 95% CI (1.1-4.3). Age and not schooling were risk factors for Plasmodium and STH co-infection. CONCLUSION: The findings suggest that STH and Plasmodium infections tend to occur in the same children, with increasing prevalence of co-infection with age. This calls for an integrated approach such as using mass chemotherapy with dual effect (e.g., ivermectin) coupled with improved housing, sanitation and hygiene for the control of both parasitic infections.

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

A cascata dos fosfoinositídeos

Inositol is a polyalcohol required for the proper formation of cell membranes. In the body, its plays an important role in the transmission of nerve impulses, its also helps in the transporting of fats within the body. In mammals, inositol exists as phosphorylated derivatives, various phosphoinositides, and in its free form. Agonist stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is the first step in the transmembrane signalling mechanism when cells respond to external stimuli. Under control of activated phospholipase C (PLC) via G-protein, two second messengers D-myo-inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and diacylglycerol are released into the cell. From Ins(1,4,5)P3, enzymatic process under phosphatases or kinases control affords subsequent inositol phosphate metabolites. During the last decade the synthesis of modified inositol phosphate derivatives has been strongly investigated. This paper reviews principal aspects about synthesis and biological functions of these biomolecules.

Use of grass carp (Ctenopharyngodon idella) as a biological control agent for submerged aquatic macrophytes

This study aimed to evaluate feed preference and control efficacy of grass carp (Ctenopharyngodon idella) on the aquatic macrophytes Ceratophyllum demersum, Egeria densa and Egeria najas. An experiment was carried out at mesocosms conditions with 2,000 liters capacity and water residence time of 2.8 days. C. demersum, E. densa e E. najas biomasses were offered individually with sixty g and coupled in similar quantities of 30 g of each species, evaluated during 81 days, envolving 6 treatments. (1 - C. demersum, 2 - E. najas, 3 -E. densa, 4 - C. demersum + E. najas, 5 - C. demersum + E. densa and 6 - E. najas + E. densa). When offered individually, E. najas and C. demersum presented the same predation rate by grass carp, which was higher than E. densa predation rate. When plants were tested in pairs, the order of feed preference was C. demersum > E. najas > E. densa. E. najas and C. demersum percentage control ranged from 73 to 83%. No relation between biomass consumption and grass carp body weight gain was observed, probably due to differences in nutritional quality among macrophyte species according to fish necessities. Therefore, it is concluded that the use of grass carp is one excellent technique to control submersed macrophytes in Brazil.

Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin

On the basis of our report that a glycolipoprotein fraction (GLP) extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1) GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2) Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3) GLP inhibits Na,K-ATPase from intact cells, and 4) GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 µg protein GLP/ml) all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 µg protein GLP/ml for rat and rabbit, respectively), indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ± SEM; control: 23.8 ± 1.8; GLP, 88 µg protein/ml: 8.2 ± 0.9), demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 µg/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed µg protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2­14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD50 141.25 µg protein/ml) and to mice injected intraperitoneally (LD50 201.8 ± 8.6 mg protein/kg), in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s) of a protein nature which may be involved in the chemical defense mechanism of this sea hare.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.