Analysis of the active center of branching enzyme II from maize endosperm

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2–4) of the four highly conserved regions (regions 1–4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.     

Depression of carbon flow to the glycogen pool induced by nitrogen assimilation in intact cells of Anacystis nidulans

The influence of nitrate and ammonium assimilation on glycogen metabolism has been determined in intact Anacystis nidulans cell actively fixing CO₂. Assimilation of nitrate or ammonium resulted in significant decreases in both the incorporation into glycogen of newly fixed carbon and the accumulation of glycogen by the cells, the magnitude of these effects depending on the light intensity. The depression in glycogen synthesis induced by nitrogen assimilation was more marked at low light intensity, and especially when ammonium was the nitrogen source. Under these conditions, specific radioactivity of the glycogen pool was particularly high, indicating enhanced turnover of glycogen. Thus, in addition to a more general depressing effect of nitrogen assimilation on the carbon flow to glycogen, degradation of glycogen appears to be stimulated by ammonium assimilation at low (but not at high) light intensity.     

Diurnal Variations in [125I]Melatonin Binding by Rat Thymus Membranes: Effects of Continuous Light Exposure and Pinealectomy

Binding of melatonin by rat thymus membranes exhibited diurnal changes. Binding increased during the daytime and reached maximal values before entering the dark period. Then, binding decreased rapidly during the dark phase. In rats kept in light at night, binding of [¹²⁵I]melatonin by membranes was significantly higher than in animals that entered the normal dark period. Neonatal pinealectomy, which suppresses the circadian rhythm of melatonin, led to an increase in melatonin binding of 106%. Moreover, in animals maintained under continuous light exposure, which corresponds to functional pinealectomy, binding of melatonin by thymus membranes also increased in a time-dependent manner. The results support the hypothesis of a regulatory role of melatonin in the thymus in which melatonin downregulates its own binding sites.     

Efficiency of the photosynthetic apparatus in developing needles of Norway spruce (Picea abies L. Karst.)

The photosynthetic performance of developing spruce (Picea abies L. Karst.) needles was investigated. As revealed by previous reports, the biosynthesis of chlorophylls and carotenoids was not following the characteristic chloroplast ultrastructure building up during needle elongation process. The aim of our study was to investigate photosynthetic capability (evaluated by oxygen evolution and chlorophyll a fluorescence kinetics measurements), the dynamics of chloroplast pigments biosynthesis and the expression of major photosynthetic proteins as well as to find out possible correlation between components of issue. Low amounts of chlorophylls and carotenoids, LHC II and Rubisco LSU were detected in the embryonic shoot of vegetative buds. Although PS II was functional, oxygen production was not sufficient to compensate for respiration in the same developmental stage. The light compensation point of respiration was successively lowered during the needle elongation. Nevertheless the significant increase in photosynthetic pigments as well as the high level of expression of LHC II and Rubisco LSU proteins was observed in the later stages of needle development. Our results suggest that, besides light, some other environmental factors could be critical for producing fully functional chloroplasts in rapidly growing young needles.     

Chronic lymphocytic leukemia cells bind and present the erythrocyte protein band 3: possible role as initiators of autoimmune hemolytic anemia

The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4(+) T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.     

The neglected tropical diseases of Latin America and the Caribbean: a review of disease burden and distribution and a roadmap for control and elimination

The neglected tropical diseases (NTDs) represent some of the most common infections of the poorest people living in the Latin American and Caribbean region (LAC). Because they primarily afflict the disenfranchised poor as well as selected indigenous populations and people of African descent, the NTDs in LAC are largely forgotten diseases even though their collective disease burden may exceed better known conditions such as of HIV/AIDS, tuberculosis, or malaria. Based on their prevalence and healthy life years lost from disability, hookworm infection, other soil-transmitted helminth infections, and Chagas disease are the most important NTDs in LAC, followed by dengue, schistosomiasis, leishmaniasis, trachoma, leprosy, and lymphatic filariasis. On the other hand, for some important NTDs, such as leptospirosis and cysticercosis, complete disease burden estimates are not available. The NTDs in LAC geographically concentrate in 11 different sub-regions, each with a distinctive human and environmental ecology. In the coming years, schistosomiasis could be eliminated in the Caribbean and transmission of lymphatic filariasis and onchocerciasis could be eliminated in Latin America. However, the highest disease burden NTDs, such as Chagas disease, soil-transmitted helminth infections, and hookworm and schistosomiasis co-infections, may first require scale-up of existing resources or the development of new control tools in order to achieve control or elimination. Ultimately, the roadmap for the control and elimination of the more widespread NTDs will require an inter-sectoral approach that bridges public health, social services, and environmental interventions.     

A new species of Brevilegnia (Saprolegniales, Straminipila) from Buenos Aires Province, Argentina

Brevilegnia ensenadensis sp. nov. is described from litter (floating twigs, leaves, and roots) in a man-made, polluted channel near a petroleum refinery, in Buenos Aires Province, Argentina. The species is illustrated and compared with other species of the genus; it can be identified by the preponderance of smooth or papillate oogonia, with a distinctive irregular inner wall, borne in bent or coiled oogonial stalks, which are terminal, lateral or of the intercalary position. The oogonia often are irregular in shape, and the oospheres mostly are immature. The single oospore is eccentric inside the great oogonia, and the species develops mainly diclinous antheridial branches and has fewer monoclinous and androgynous ones.     

Extracellular acidosis triggers the maturation of human dendritic cells and the production of IL-12

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.     

High-fat/high-cholesterol diet promotes a S1P receptor-mediated antiapoptotic activity for VLDL

Withdrawing growth factors or serum from endothelial cells leads to the activation of effector caspases 3 and 7, resulting in apoptotic cell death. HDL protects against caspase induction through sphingosine-1-phosphate (S1P) receptors. This anti-caspase activity of HDL is antagonized by VLDL from apolipoprotein E4 (apoE4) (genotype, APOE4/4; apolipoprotein, apoE) targeted replacement (TR) mice, but not by VLDL from TR APOE3/3 mice, and requires the binding of apoE4-VLDL to an LDL receptor family member. In the absence of HDL, apoE4-VLDL and apoE3-VLDL from TR mice have limited antiapoptotic activity. In contrast, we show here that a high-fat/high-cholesterol/cholate diet (HFD) radically alters this biological activity of VLDL. On HFD, both apoE3-VLDL and apoE4-VLDL (HFD VLDL) inhibit caspase 3/7 activation initiated by serum withdrawal. This activity of HFD VLDL is independent of an LDL receptor family member but requires the activation of S1P(3) receptors, as shown by the ability of pharmacological block of S1P receptors by VPC 23019 and by small interfering RNA-mediated downregulation of S1P(3) receptors to inhibit HFD VLDL anticaspase activity.     

Antimitotic activity of EA21b mammary-carcinoma extract

Many tumors produce factors that affect cell-cycle and cell proliferation. In the present study we have analyzed the effect of a mammary-tumor extract injection on the mitotic activity of several organs in young male C3H/S mice previously standardized for circadian periodicity. One-half of the animals received an intraperitoneal EA21b tumor extract dose at 16:00 h, while the other half received saline. Animals were sacrificed on the following day at 08:00, 12:00 or 16:00 h. 4 h after receiving an injection of colchicine by the same route. Samples of duodenum, kidney, liver, and submaxillary gland were excised and processed for hematoxylin-eosin staining. Mitotic indices, expressed as the number of colchicine-arrested metaphases per 1,000 nuclei, were assessed in convoluted tubule epithelium, duodenal crypt enterocytes, hepatocytes and submaxillary gland ductal and acinar sialocytes. All values were expressed as mean ± SEM. Statistical analyses were performed by ANOVA, Bonferroni and Student’s t-tests. In contrast to the mitotic indices reductions observed in renal convoluted tubules cells and duodenal crypt enterocytes, neither the submaxillary gland nor the liver were found to contain cell types whose mitotic activity was affected by the tumor extract. We conclude that EA21b mammary carcinoma contains one or more factors that inhibit the proliferation of selected populations of normal cells.