Targeting and translocation of endothelial nitric oxide synthase

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca2+/calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co- and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca2+. These rapid receptor-mediated effects are seen not only for "classic" eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Feasibility of the use of recycled nutrients in fast growing pulpwood production

The application of pulp and paper mill (PPM) sludge in agriculture and forestry has been acknowledged as soil amendments and a plant nutrient source. The main objectives of this study were to evaluate the total cost of the use of recycled nutrients from PPM sludge in fast growing pulpwood production, and the financial profitability of fast growing pulpwood production with the use of these recycled nutrients. The investment and production costs of fast growing pulpwood plantation were directly acquired from a previous research, while the other data was compiled through different studies. The total cost of the use of PPM sludge was evaluated based on assumed factors. Discounted cash flow method was used to evaluate the financial profitability, using NPV and IRR as indicators. The results of estimated sludge nutrient contents were 16.2 g N, 2.9 g P, and 2.4 g K kg-1 of dry sludge. The sludge application rate was estimated at 1.36 Mg/ha in the first year. The total cost of the use of PPM sludge involved transport and spreading cost of US$49.15/dry ton. The fertilization cost applied in the financial model was designed in 3 different options and their results were as follows: option (1) was taken directly from the reference research (US$97/ha); option (2) was the use of sludge alone (US$66.75/ha); and option (3) was the use of sludge and TSP fertilizer (US$83.80/ha). The average NPV without discounting was US$248,180 while the IRRs ranged between approximately 3-4% with an average of 3.63%. Although option (2) and (3) contributed to higher IRRs compared to option (1), this increase was still not significant as the IRR was not sensitive to the total fertilization cost. The advantages are that this practice can be performed at a lower cost and the application rate can be still increased if necessary. It is better for forest plantations compared to agriculture and consequently supports reforestation program. In addition, it can be similarly applied in wood biomass production. A disadvantage is that the IRRs were not very favorable compared to the criterion of 11%. The sludge high in C:N ratio can cause nitrogen immobilization, and regulatory concerns may restrict and complicate the use of sludge landspreading and contribute to additional costs and processes.

Typical and atypical enteropathogenic Escherichia coli bacterial translocation associated with tissue hypoperfusion in rats

Although enteropathogenic Escherichia coli (EPEC) are well-recognized diarrheal agents, their ability to translocate and cause extraintestinal alterations is not known. We investigated whether a typical EPEC (tEPEC) and an atypical EPEC (aEPEC) strain translocate and cause microcirculation injury under conditions of intestinal bacterial overgrowth. Bacterial translocation (BT) was induced in female Wistar-EPM rats (200-250 g) by oroduodenal catheterization and inoculation of 10 mL 10(10) colony forming unit (CFU)/mL, with the bacteria being confined between the duodenum and ileum with ligatures. After 2 h, mesenteric lymph nodes (MLN), liver and spleen were cultured for translocated bacteria and BT-related microcirculation changes were monitored in mesenteric and abdominal organs by intravital microscopy and laser Doppler flow, respectively. tEPEC (N = 11) and aEPEC (N = 11) were recovered from MLN (100%), spleen (36.4 and 45.5%), and liver (45.5 and 72.7%) of the animals, respectively. Recovery of the positive control E. coli R-6 (N = 6) was 100% for all compartments. Bacteria were not recovered from extraintestinal sites of controls inoculated with non-pathogenic E. coli strains HB101 (N = 6) and HS (N = 10), or saline. Mesenteric microcirculation injuries were detected with both EPEC strains, but only aEPEC was similar to E. coli R-6 with regard to systemic tissue hypoperfusion. In conclusion, overgrowth of certain aEPEC strains may lead to BT and impairment of the microcirculation in systemic organs.

Nutrients in the cassava (Manihot esculenta Crantz) leaf meal at three ages of the plant

The high number of cassava cultivars adapted to many different regions provides a wide variation in the chemical composition of cassava leaves meal (CLM). Therefore, the contents of some nutrients in CLM from five cultivars at three ages of the plant were investigated in order to select the cultivars and ages with superior levels of these nutrients. When the plants were 12 months old, the highest levels of crude protein (CP), beta-carotene, iron, magnesium, phosphorus and sulfur were observed. The IAC 289-70 cv. showed the highest levels of magnesium, as well as considerable contents of CP, beta-carotene, iron, zinc and sulfur, which did not differ statistically from the cultivars showing the highest levels of these nutrients.

Fatty acids and nutrients in the flour made from tilapia (Oreochromis niloticus) heads

The objective of this work was to analyze the fatty acid composition and nutrient potential of flour made from tilapia heads, which are normally discarded during the filleting operation. Significant differences were found between the proximate composition (moisture, ash, protein and total lipids) of the in natura tilapia and the flour, due to the drying process. The predominant fatty acids in the heads (in natura and in the flour) were palmitic acid (1,999 mg.100 g-1 and 7,699 mg.100 g-1, respectively), oleic acid (3,128 mg.100 g-1 and 11,447 mg.100g-1, respectively), and linoleic acid (1,018 mg.100 g-1 and 3,784 mg.100 g-1, respectively). The results lead us to conclude that tilapia head flour offers high levels of protein (38.41%), total lipids (35.46%), and ash (minerals) (19.38%). The content of omega-3 (731 mg.100 g-1) were proved to be satisfactory. Also, n-6/n-3 ratio was 6.15 and PUFA/SFA ratio was 0.47, which are in agreement with the recommended levels. Thus, tilapia heads can be used as a low-cost raw material for food fit for human consumption.

Influence of the use of rice bran extract as a source of nutrients on xylitol production

Xylose-to-xylitol bioconversion using 2.5 or 10% (v/v) rice bran extract was performed to verify the influence of this source of nutrients on Candida guilliermondii metabolism. Semisynthetic medium (SM) and sugarcane bagasse hemicellulosic hydrolysate detoxified with ion-exchange resins (HIE) or with alteration in pH combined with adsorption onto activated charcoal (HAC) were fermented in 125 mL Erlenmeyer flasks at 30 ºC and 200 rpm for 72 hours. Activated charcoal supplemented with 2.5% (v/v) rice bran extract was fermented by C. guilliermondii in a MULTIGEN stirred tank reactor using pH 5.0 and 22.9/hour oxygen transfer volumetric coefficient. Higher values of xylitol productivity (0.70, 0.71, and 0.62 g.Lh-1) and xylose-to-xylitol conversion yield (0.71, 0.69, and 0.63 g.g-1) were obtained with 2.5% (v/v) rice bran in semisynthetic medium, ion-exchange resins, and activated charcoal, respectively. Moreover, during batch fermentation, the xylitol volumetric productivity and fermentation efficiency values obtained were 0.53 g.Lh-1 and 61.1%, respectively.

Changes in the magnitude and pattern of translocation of photoassimilated ¹CO in soybean plants following an acute exposure to gamma radiation /

Young soybean plants (Glycine ~. L. cultivar Harosoy '63), grown under controlled conditions, were exposed to gamma radiation on a single occasion. One hour following exposure to 3,750 rads, the mature trifoliate leaf of the soybean plant was isolated in a closed system and permitted to photoassimilate approximately 1-5 pCi of 14C02 for 15 minutes. After an additional 45 minute-period, the plant was sacrificed and the magnitude of translocation and distribution pattern of 14C determined. In the non-irradiated plants 18~ of the total 14C recovered was outside the fed leaf blades and of this translocated 14c, 28~ was above the node of the fed leaf, 38~ in the stem below the node, 28~ in the roots and 7~ in the petiole. As well, in the irradiated plants, a smaller per cent (6~) of the total 14 C recovered was exported out of the source leaf blades. Of this translocated 14c , a smaller per cent (20~) was found in the apical region above the node of the source leaf and a higher per cent (45~) was recovered from the stem below the node and in the petiole (11~). The per cent of exported 14 C recovered from the root was unaffected by the radiation. Replacement of the shoot apex with 20 ppm IAA immediately following irradiation, only J partially increased the magnitude of translocation but did completely restore the pattern of distribution to that observed in the non-irradiated plants. From supplementary studies showing a radiationinduced reduction of photosynthetic rates in the source leaf and a reduction of the cumulative stem and leaf lengths in the apical sink region, the observed effects of radiation on the translocation process have been correlated to damage incurred by the source and sink regions. These data suggest that the reduction in the magnitude of translocation is the result of damage to both the source and sink regions rather than the phloem conducting tissue itself, whereas the change in the pattern of translocation is probably the result of a reduced rate of 14C-assimilate movement caused by a radiation-induced decrease of sink metabolism, especially the decrease in the metabolism of the apical sink.

A relationship between photosynthesis and translocation in plants stressed by ionizing radiation

Since previous investigations have shown that low levels of ionizing radiation can induce a reduction in the rates of apparent photosynthesis and in the magnitude of photoassimilated l4C exported out of a leaf, the present studies were designed and conducted to determine the relationship, if any, between the radiation effects on these two physiological processes. The experiments were particularly designed to determine if the radiation-induced reduction in export is the result of the reduction in photosynthesis and hence availability of materials for translocation or the result of a reduction in the amount of energy available for the vein loading process. This study has shown that the radiation-induced reduction in l4C export out of a leaf is likely related to a loss of energy available for the vein loading process rather than a reduction in the supply of materials available for export due to reduced C02 uptake. The process of photophosphorylation was shown to be reduced by exposure to radiation to an extent similar to the reduction in the export of l4C which was also observed. Both of these processes returned to their pre-irradiation rates 120 minutes following radiatruon exposure. The rate of photosynthetic C02 uptake was also reduced by radiation exposur~ howeve~ this process did not return to the control level nor was the extent of reduction as large as observed for photophosphorylation and photoassimilate export. The observed relationship between the reductions of export and photoph~sphorylation pointed to the utilization of photosynthetically produced ATP in the vein loading process. The radiation-induced reduction in the export of l4C was observed at the highest light intensity used in this study which would also imply the involvement of the photophosphorylation process as an energy seurce for vein loading. The lack of radiation-induced reduction in export at low light intensities was interpreted as being due to the utilization of respiratory derived ATP, a process known to be insensitive to radiation at the levels used in this study, as the energy source for the vein loading process. Studies using plants not stressed by radiation showed that there was an increase in export of 14C with higher light intensities. In summary, the data has been interpreted as showing that at high light intensities the ATP, produced by photophosphorylation, is available for use in the vein loading process. The site of ATP utilization could not be determined from the data obtained in this study but possible sites have been indicated from the work done by other physiologists and are discussed in the thesis.

Proton translocation by cytochrome c oxidase reconstituted into proteoliposomes

Cytochrome c oxidase .inserted into proteoliposomes translocates protons with a stoichiometry of approx-, imately 0.4-0.6 H+/e- in the presence of valinomycin plus pottasium. The existance .ofsuchproton translocation is .supportedby experiments with lauryl maltoside which abolished the pulses but~~d not inhibit cyt. c binding .or oxidase turnover. Pulses with K3FeCN6 did not induce acidification further supporting vectorial proton transport by cyt ..aa3 . Upon lowering the ionic strength and pulsing with ferrocytochrome c, H+/eratios increased. This increase is attributed to scaler proton release consequent upon cyt.c-phospholipid binding. Oxygen pulses at low ionic strength however did not exhibit this large scaler increase in H+/e- ratios.A-small increase was observed upon .02 pul'sing at·low ionic strengt.h. This increase was KeN and, ,pcep sensitive and thus possibly due to a redox linked scaler deprotonation. Increases in the H+/e- ratio also occurred ifp~lses ,were performed in the presence of nonactin rather.than valinomycin. The fluorescent pH indicator pyranine was internally trapped inaa3 conta~ning "proteoliposomes. Internal alkalinization, as mon,itored by pyranine fluorescence leads to a of approx.imately 0.35 units, which is proportional to electron flux. This internal alkalinization was also DCCD sensitive, being inhibited by approximately 50%. This 50% inhibition of internal alkalinization supports the existance of vectorial proton transport.

Sex chromosome translocation and speciation : a Drosophila genetic model

Although exceptions may be readily identified, two generalizations concerning genetic differences among species may be drawn from the available allozyme and chromosome data. First, structural gene differences among species vary widely. In many cases, species pairs do not differ more than intraspecific populations. This suggests that either very few or no gene substitutions are required to produce barriers to reproduction (Avise 1976). Second, chromosome form and/or number differs among even closely related species (White 1963; 1978; Fredga 1977; Wright 1970). Many of the observed chromosomal differences involve translocational rearrangements; these produce severe fitness depression in heterozygotes and were, thus, long considered unlikely candidates for the fixation required of genetic changes leading to speciation (Wright 1977). Nonetheless, the fact that species differences are frequently translocational argues convincingly for their fixation despite prejudices to the contrary. Haldane's rule states that in the F of interspecific crosses, the heterogametic sex is absent or sterile in the preponderance of cases (Haldane 1932). This rule definitely applies in the genus Dr°sophila (Ehrman 1962). Sex chromosome translocations do not impose a fitness depression as severe as that imposed by autosomal translocations, and X-Y translocations may account for Haldane's rule (Haldane 1932). Consequently a study of the fit ness parameters of an X·yL and a yS chromosome in Drosophila melanogaster populations was initiated by Tracey (1972). Preliminary results suggested that x.yL//YSmales enjoyed a mating advantage with X·yL//X·yL females, that this advantage was frequency dependent, that the translocation produced sexual isolation and that interactions between the yL, yS and a yellow marker contributed to the observed isolation (Tracey and Espinet 1976; Espinet and Tracey 1976). Encouraged by the results of these prelimimary studies, further experiments were performed to clarify the genetic nature of the observed sexual isolation, S the reality of the y frequency dependent fitness .and the behavioural changes, if any, produced by the translocation. The results of this work are reported herein. Although the marker genes used in earlier studies, sparkling poliert an d yellow have both been found to affect activity,but only yellow effects asymmetric sexual isolation. In addition yellow effects isolation through an interaction with the T(X-y) chromosomes, yS also effects isolation, and translocational strains are isolated from those of normal karyotype in the absence of marker gene differences. When yS chromosomes are in competition with y chromosomes on an X.yL background, yS males are at a distinct advantage only when their frequency is less than 97%. The sex chromosome translocation alters the normal courtship pattern by the incorporation of circling between vibration and licking in the male repertoire. Finally a model of speciation base on the fixation of this sex chromosome translocation in a geographically isolated gene pool is proposed.

Influence of dietary nutrients on life history-related traits of black flies and mosquitoes

The sugar-feeding ecology of dipteran vectors has recently been targeted because it presents opportunities to inoculate common food sources for these dipterans with entomopathogenic bacteria as a means of controlling the population of host-seeking adult dipteran vectors. Whereas this approach to vector control holds some promise, differences in the nutrient composition and concentration in sugary food sources can influence the food selection pattern of dipteran vectors and potentially confound the outcomes of field trials on the efficacy of entomopathogenic bacteria as vector control agents. Further, nutrient components of bacteria-inoculated artificial diets may present unintended effects of extending the survivorship or fecundity of the target population and potentially render the whole approach counterproductive. The present study investigated the diet-specific factors that influence the foraging decisions of female Simulium venustum/verecundum (Diptera: Simuliidae) and female Anopheles stephensi (Diptera: Culicidae) on artificial nectar and honeydew. Paired choice experiments showed that the black flies forage more frequently from high calorie diets, which contained melezitose, or those diets that contained amino acids, compared to low calorie melezitose-free diets or amino acid-free diets. The mosquitoes however displayed a more random diet selection pattern. The effects of sugary diets on certain life-history traits considered to be important to the ecological fitness of the black flies and mosquitoes were also investigated. Sugary diets had no significant effect on the survivorship and fecundity of the black flies, but they influenced the resistance of Leucocytozoon-infected flies to the parasite. Amino acid-containing diets appeared to extend the survival of mosquitoes, and also allowed them to take more vertebrate blood when they blood fed.

Caractérisation de la translocation (12;13)(p12;q12-14) et l’insertion (X;6)(p11.23;q21q23.3) dans des leucémies aiguës pédiatriques

Par une stratégie de dépistage combinant le caryotype et l’hybridation in situ en fluorescence (FISH), une insertion (X;6) présente chez des jumelles avec une leucémie myéloïde aiguë (LMA) et une translocation (12;13) dans deux cas de LMA et un cas de leucémie lymphoblastique aiguë (LLA) ont été mis en évidence. L’insertion (X;6) n’est pas rapportée et serait un variant de la translocation (X;6) rapportée dans 4 cas de LMA, dont un associe un gène de fusion MYB-GATA1. Nous avons mis en évidence la dérégulation de l’expression de ces gènes dans le cas d’insertion sans la présence de fusion MYB-GATA1. De plus, dans le premier cas de translocation (12;13) identifié, ETV6 serait fusionné à CDX2 ou FLT3. Le deuxième cas associe la délétion des gènes miR-15a et miR-16-1 à une fusion d’ETV6 et le troisième cas impliquerait une fusion ETV6- FOXO1.