Characterization of pip5k3 fleck corneal dystrophy-linked gene in zebrafish.

PIKfyve is a kinase encoded by pip5k3 involved in phosphatidylinositols (PdtIns) pathways. These lipids building cell membranes have structural functions and are involved in complex intracellular regulations. Mutations in human PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy [Li, S., Tiab, L., Jiao, X., Munier, F.L., Zografos, L., Frueh, B.E., Sergeev, Y., Smith, J., Rubin, B., Meallet, M.A., Forster, R.K., Hejtmancik, J.F., Schorderet, D.F., 2005. Mutations in PIP5K3 are associated with François-Neetens mouchetee fleck corneal dystrophy. Am. J. Hum. Genet. 77, 54-63]. We cloned the zebrafish pip5k3 and report its molecular characterization and expression pattern in adult fish as well as during development. The zebrafish PIKfyve was 70% similar to the human homologue. The gene encompassed 42 exons and presented four alternatively spliced variants. It had a widespread expression in the adult organs and was localized in specific cell types in the eye as the cornea, lens, ganglion cell layer, inner nuclear layer and outer limiting membrane. Pip5k3 transcripts were detected in early cleavage stage embryos. Then it was uniformly expressed at 10 somites, 18 somites and 24 hpf. Its expression was then restricted to the head region at 48 hpf, 72 hpf and 5 dpf and partial expression was found in somites at 72 hpf and 5 dpf. In situ on eye sections at 3 dpf showed a staining mainly in lens, outer limiting membrane, inner nuclear layer and ganglion cell layer. A similar expression pattern was found in the eye at 5 dpf. A temporal regulation of the spliced variants was observed at 1, 3 and 5 dpf and they were also found in the adult eye.

Keeping the leaves green above us.

The plant immune system relies to a great extent on the highly regulated expression of hundreds of defense genes encoding antimicrobial proteins, such as defensins, and antiherbivore proteins, such as lectins. The expression of many of these genes is controlled by a family of mediators known as jasmonates; these cyclic oxygenated fatty acid derivatives are reminiscent of prostaglandins. The roles of jasmonates also extend to the control of reproductive development. How are these complex events regulated? Nearly 20 members of the jasmonate family have been characterized. Some, like jasmonic acid, exist in unmodified forms, whereas others are conjugated to other lipids or to hydrophobic amino acids. Why do so many chemically different forms of these mediators exist, and do individual jasmonates have unique signaling properties or are they made to facilitate transport within and between cells? Key features of the jasmonate signal pathway have been identified and include the specific activation of E3-type ubiquitin ligases thought to target as-yet-undescribed transcriptional repressors for modification or destruction. Several classes of transcription factor are known to function in the jasmonate pathway, and, in some cases, these proteins provide nodes that integrate this network with other important defensive and developmental pathways. Progress in jasmonate research is now rapid, but large gaps in our knowledge exist. Aimed to keep pace with progress, the ensemble of jasmonate Connections Maps at the Signal Transduction Knowledge Environment describe (i) the canonical signaling pathway, (ii) the Arabidopsis signaling pathway, and (iii) the biogenesis and structures of the jasmonates themselves.

Non-immunologic methods of diagnosis of babesiosis

The diagnosis of tick-borne diseases such as babesiosis still depends on observing the parasite in the infected erythrocyte. Microscopic observation is tedious and often problematic in both early and carrier infections. Better diagnostic methods are needed to prevent clinical disease, especially when susceptible cattle are being moved into disease enzootic areas. This study evaluates two techniques for early diagnosis of Babesia bovis infections in cattle, DNA probes specific for the organism and fluorescent probes specific nucleic acid. The radioisotopically labeled DNA probes are used in slot blot hybridizations whith lysed blood samples, not purified DNA. Thusfar, the probe is specific for B. bovis and can detect as few as 1000 B. bovis parasites in 10µl of blood. The specificity of the fluorescent probe depends on the characteristic morphology of the babesia in whole blood samples, as determined microscopically. The fluorescent probe detects as afew as 10,000 B. bovis parasites in 10*l as blood. The application of each method for alboratory and field use is discussed.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Basic biochemical investigations as rationale for the design of original antimalarial drugs. An example of phospholipid metabolism

The future of antimalarial chemotherapy is particulary alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaving resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate to phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity thereshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphblastoid cell line, demonstrating a total abscence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20-to 100-fold lower than their in acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.

Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement

Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE) whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF) , a 70 kDa glycoprotein attached to the membrane of NHue by a GPI anchor. IgG2a mAb anti-human DAF (IA10) immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vtro. Incubationof schistosomula with erytrocytes from patients with paroxsimal nocturnal hemoglobinuria (PNHE) or SRBC, wich are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polipeptide(s) present on the surface of the worm.

Histopathological alterations induced by non-viable cells and biochemical fractions from Paracoccidioides brasiliensis in mice

Non-viable cells and biochemical fractions from Paracoccidioides brasiliens were obtained for experimental inoculation in mice and posterior histopatological analysis. Dead total fungus, total fungus disrupted by sonorous waves, lipids of the fungus, supernatant of the lipid purification, integral and disrupted fungus free of lipids were obtained. The six preparations arised from masses of lyophilized yeasts of a recent isolate of P. brasiliensis (strain JT-1) and from a "Pool" equitably constituted by four strains maintained in laboratory for a long time (SN, 2, 18 and 192). Different doses of the 12 preparations were intraperitonially inoculated and histopathological analysis were done 30 days later. This analysis showed that all the inoculated preparations gave origin to inflamatory foci, except the one designated "supernatant of lipid purification". The alterations were detected exclusively in the liver of the animals and occurred from the smallest dose tested (1 mg), with exception of the lipids of the fungus, where the foci appeared only from a 3 mg dose onwards. No difference in the capacity of inducing histopathological alterations was found between the preparations obtained from the recent isolate (JT-1) and from the older ones ("Pool"). On the other hand, an increase of the number of inflammatory foci in function of the inoculated dose was observed.

Morphologic and morphometric aspects of oenocytes of Apis mellifera queens and workers in different phases of life

Oenocytes of adult workers and queens of Apis mellifera L. were studied in different ages or life stages, by means of morphometric and histologic techniques. In workers, the oenocytes were found in the head, near the mandibles and in the abdomen, immersed in the parietal fat body mainly below the sterna, close to the wax gland. In queens, two populations of oenocytes different in size and localization were found within the parietal and visceral fat body, respectively. The oenocytes of workers and queens show the presence of acid lipids and acid phosphatase. The role of these cells in the castes differences is discussed.

Moderate amounts of fructose consumption impair insulin sensitivity in healthy young men: a randomized controlled trial.

OBJECTIVE: Adverse effects of hypercaloric, high-fructose diets on insulin sensitivity and lipids in human subjects have been shown repeatedly. The implications of fructose in amounts close to usual daily consumption, however, have not been well studied. This study assessed the effect of moderate amounts of fructose and sucrose compared with glucose on glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: Nine healthy, normal-weight male volunteers (aged 21-25 years) were studied in this double-blind, randomized, cross-over trial. All subjects consumed four different sweetened beverages (600 mL/day) for 3 weeks each: medium fructose (MF) at 40 g/day, and high fructose (HF), high glucose (HG), and high sucrose (HS) each at 80 g/day. Euglycemic-hyperinsulinemic clamps with [6,6]-(2)H(2) glucose labeling were used to measure endogenous glucose production. Lipid profile, glucose, and insulin were measured in fasting samples. RESULTS: Hepatic suppression of glucose production during the clamp was significantly lower after HF (59.4 ± 11.0%) than HG (70.3 ± 10.5%, P < 0.05), whereas fasting glucose, insulin, and C-peptide did not differ between the interventions. Compared with HG, LDL cholesterol and total cholesterol were significantly higher after MF, HF, and HS, and free fatty acids were significantly increased after MF, but not after the two other interventions (P < 0.05). Subjects' energy intake during the interventions did not differ significantly from baseline intake. CONCLUSIONS: This study clearly shows that moderate amounts of fructose and sucrose significantly alter hepatic insulin sensitivity and lipid metabolism compared with similar amounts of glucose.

Energy expenditure by doubly labeled water: validation in humans and proposed calculation.

To further validate the doubly labeled water method for measurement of CO2 production and energy expenditure in humans, we compared it with near-continuous respiratory gas exchange in nine healthy young adult males. Subjects were housed in a respiratory chamber for 4 days. Each received 2H2(18)O at either a low (n = 6) or a moderate (n = 3) isotope dose. Low and moderate doses produced initial 2H enrichments of 5 and 10 X 10(-3) atom percent excess, respectively, and initial 18O enrichments of 2 and 2.5 X 10(-2) atom percent excess, respectively. Total body water was calculated from isotope dilution in saliva collected at 4 and 5 h after the dose. CO2 production was calculated by the two-point method using the isotopic enrichments of urines collected just before each subject entered and left the chamber. Isotope enrichments relative to predose samples were measured by isotope ratio mass spectrometry. At low isotope dose, doubly labeled water overestimated average daily energy expenditure by 8 +/- 9% (SD) (range -7 to 22%). At moderate dose the difference was reduced to +4 +/- 5% (range 0-9%). The isotope elimination curves for 2H and 18O from serial urines collected from one of the subjects showed expected diurnal variations but were otherwise quite smooth. The overestimate may be due to approximations in the corrections for isotope fractionation and isotope dilution. An alternative approach to the corrections is presented that reduces the overestimate to 1%.

Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition.

Pyochelin (Pch) and enantiopyochelin (EPch) are enantiomeric siderophores, with three chiral centers, produced under iron limitation conditions by Pseudomonas aeruginosa and Pseudomonas fluorescens , respectively. After iron chelation in the extracellular medium, Pch-Fe and EPch-Fe are recognized and transported by their specific outer-membrane transporters: FptA in P. aeruginosa and FetA in P. fluorescens . Structural analysis of FetA-EPch-Fe and FptA-Pch-Fe, combined with mutagenesis and docking studies revealed the structural basis of the stereospecific recognition of these enantiomers by their respective transporters. Whereas FetA and FptA have a low sequence identity but high structural homology, the Pch and EPch binding pockets do not share any structural homology, but display similar physicochemical properties. The stereospecific recognition of both enantiomers by their corresponding transporters is imposed by the configuration of the siderophore's C4'' and C2'' chiral centers. This recognition involves specific hydrogen bonds between the Arg91 guanidinium group and EPch-Fe for FetA and between the Leu117-Leu116 main chain and Pch-Fe for FptA. FetA and FptA are the first membrane receptors to be structurally described with opposite binding enantioselectivities for their ligands, giving insights into the structural basis of their enantiospecificity.

Behavior of endogenous tumor-associated macrophages assessed in vivo using a functionalized nanoparticle.

Tumor-associated macrophages (TAMs) invade the tumor stroma in many cancers, yet their role is incompletely understood. To visualize and better understand these critical cells in tumor progression, we screened a portfolio of rationally selected, injectable agents to image endogenous TAMs ubiquitously in three different cancer models (colon carcinoma, lung adenocarcinoma, and soft tissue sarcoma). AMTA680, a functionally derivatized magneto-fluorescent nanoparticle, labeled a subset of myeloid cells with an "M2" macrophage phenotype, whereas other neighboring cells, including tumor cells and a variety of other leukocytes, remained unlabeled. We further show that AMTA680-labeled endogenous TAMs are not altered and can be tracked noninvasively at different resolutions and using various imaging modalities, e.g., fluorescence molecular tomography, magnetic resonance imaging, and multiphoton and confocal intravital microscopy. Quantitative assessment of TAM distribution and activity in vivo identified that these cells cluster in delimited foci within tumors, show relatively low motility, and extend cytoplasmic protrusions for prolonged physical interactions with neighboring tumor cells. Noninvasive imaging can also be used to monitor TAM-depleting regimen quantitatively. Thus, AMTA680 or related cell-targeting agents represent appropriate injectable vehicles for in vivo analysis of the tumor microenvironment.

Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of Venezuela

Fluorescent (acridine orange) microscopical examination of capillary centrifuged blood (quantitative buffy coat [QBC®] analysis) and Giemsa stained thick blood smears (GTS) were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the States of Bolivar and Amazonas in southeastern and south Venezuela, respectively. Of a total of 198 GTS examined, 95 subjects (48%) showed parasitaemia. Among the 95 blood films with a positive GTS, 94 were judged positive by the QBC. However, positive QBC tubes were found in 29 out of 103 blood specimens with a negative GTS. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC-test was 99.2% and 72%, respectively. Young trophozoites of Plasmodium vivax and P. falciparum could not be distinguished with certainty. It is confirmed that the QBC offers many advantages compared with the standard diagnosis of malaria parasites, specifically in the speed of staining and ease of interpretation. However, in places where P. falciparum and P. vivax occur, species and stage differentiation should be confirmed with the GTS.

Regulación de la movilidad celular por Profilina 1

Mi proyecto de tesis se basaba en el estudio del papel de profilina 1 en la formación de lamelas, para ello generamos una proteína recombinante y transducible, con el objetivo de poder modificar los niveles endógenos de profilina. Objetivos: i-caracterización bioquímica los tres sitios de union conocidos de la proteína de transducción, el sitio de unión a fosfo-inocitoles (PIP), el de unión a actina (Ac) y el de unión a poli-prolinas (PLP). ii-estudio de la polimerización in-vitro de actina - PTD4-Profilina1 iii-estudio de las proteínas componentes de lamelas inducidas por PTD4-Profilina1. Plan de trabajo: i-Para comprobar la funcionalidad los 3 sitios de unión fueron necesarias las primeras 6 semanas, ya que en primer lugar había que expresar y purificar el peptido Srv2, necesario para el ensayo de PLP. En segundo lugar, se obtuvieron los datos de las concentraciones adecuadas de lípidos para el ensayo de fosfo-inocitoles y por ultimo, se purifico la actina necesaria para el ensayo de unión a actina. Una vez establecida la funcionalidad de la proteína, se procedió a: ii-el estudio de polimerización in-vitro, que llevo 2 semanas. Demostrando que in-vitro era capaz de inhibir la polimerización de una manera similar a la endógena. Una vez terminados estos ensayos, se procedio a: iii-la caracterización inmunohistoquímica de las proteínas componentes de la lamela que fue llevado a cabo en 4 semanas. Para ello se usaron anticuerpos contra: alfa-actinina, talina, vinculina, ENA/Vasp y paxillina. Conclusiones: i-las propiedades bioquímicas de la PTD4-Profilina1 son similares a las de la profilina endógena. ii-los estudios de polimerización indican que la polimerización se produce de manera similar a la endogena. iii-los ensayos de inmunohistoquímica sugieren que, talina esta ausente y que las demás están presentes aunque en menor concentración y con otra distribución comparadas con los controles.

The effect of ethanol on fat storage in healthy subjects.

BACKGROUND: Ethanol can account for up to 10 percent of the energy intake of persons who consume moderate amounts of ethanol. Its effect on energy metabolism, however, is not known. METHODS: We studied the effect of ethanol on 24-hour substrate-oxidation rates in eight normal men during two 48-hour sessions in an indirect-calorimetry chamber. In each session, the first 24 hours served as the control period. On the second day of one session, an additional 25 percent of the total energy requirement was added as ethanol (mean [+/- SD], 96 +/- 4 g per day); during the other session, 25 percent of the total energy requirement was replaced by ethanol, which was isocalorically substituted for lipids and carbohydrates. RESULTS: Both the addition of ethanol and the isocaloric substitution of ethanol for other foods reduced 24-hour lipid oxidation. The respective mean (+/- SE) decreases were 49.4 +/- 6.7 and 44.1 +/- 9.3 g per day (i.e., reductions of 36 +/- 3 percent and 31 +/- 7 percent from the oxidation rate during the control day; P less than 0.001 and P less than 0.0025). This effect occurred only during the daytime period (8:30 a.m. to 11:30 p.m.), when ethanol was consumed and metabolized. Neither the addition of ethanol to the diet nor the isocaloric substitution of ethanol for other foods significantly altered the oxidation of carbohydrate or protein. Both regimens including ethanol produced an increase in 24-hour energy expenditure (7 +/- 1 percent with the addition of ethanol, P less than 0.001; 4 +/- 1 percent with the substitution of ethanol for other energy sources, P less than 0.025). CONCLUSIONS: Ethanol, either added to the diet or substituted for other foods, increases 24-hour energy expenditure and decreases lipid oxidation. Habitual consumption of ethanol in excess of energy needs probably favors lipid storage and weight gain.