Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Vertebrate and nematode genes coding for yolk proteins are derived from a common ancestor.

One of the most obvious characteristics of the egg cells of oviparous animals is their large size resulting to a major extent from the deposition of nutritional reserves, mainly constituted of yolk proteins. In general, these are derived from a precursor called vitellogenin, which undergoes posttranslational modifications during secretion and during transport into and storage within the oocytes. Comparative analysis of the structural organization of the vitellogenin gene and of its product in different species shows that the vitellogenin gene is very ancient and that in vertebrates the gene may have more resemblance to the earliest gene than in invertebrates.

Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

Etude des gènes codant les protéines de gouttelettes lipidiques chez les patients avec la stéatose hépatique[Study of genes encoding proteins of lipid droplets in patients with fatty liver]

Introduction: La prévalence de la «non-alcoholic fatty liver disease (NAFLD)» dans les pays industrialisés augment de manière exponentielle. La NAFLD se développe d'une simple stéatose hépatique jusqu'à l'hépatite, puis à la cirrhose. De plus, la stéatose hépatique est fréquemment accompagnée par une résistance à l'insuline, une des causes principales du diabète. Les lipides intermédiaires, tels que céramides et diacylglycérols, ont été décrits comme induisant la résistance à l'insuline. Cependant, nous avons démontré dans notre modèle de stéatose hépatique, que les souris présentant une invalidation de la protéine «microsomal triglyceride transfer protein» (Mtpp) au niveau hépatique, ne développent pas de résistance à l'insuline. Ceci suggère fortement l'existence d'autres mécanismes susceptibles d'induire la résistance à l'insuline. Résultats: Grâce à une analyse de Microarray, nous avons observé une augmentation de l'expression des gènes «cell-death inducing DFFA-like effector c (CIDEC)», «lipid storage droplet protein 5 (LSDP5)» et «Bernardinelli-Seip congenital lipodystrophy 2 homolog (Seipin)» dans le foie des souris Mttp. Ces gènes ont récemment été identifiés comme des protéines localisées autour des gouttelettes lipidiques. Nous avons également constaté que la souris Mttp développe plutôt une microstéatose (petites gouttelettes lipidiques) qu'une macrostéatose qui est normalement observée chez les patients avec NAFLD. Nous avons étudié l'expression des gènes associés aux gouttelettes lipidiques chez les patients obèses avec stéatose hépatique, avec ou sans résistance à l'insuline. Comparés aux sujets sains sans stéatose hépatique, les patients avec la stéatose ont une expression significativement plus élevée. De manière intéressante, les patients avec résistance à l'insuline ont une diminution de ces expressions. Conclusion : Ces données suggèrent que les gènes des gouttelettes lipidiques sont impliqués dans le développement de la stéatose hépatique chez l'homme et peut-être contribue à la mise en place de la résistance à l'insuline.

Reserve substances and storage of Cyathea delgadii Sternb. spores

The spores used were collected from a population of trees of Cyathea delgadii Sternb. growing in a gallery forest at the Reserva Biológica e Estação Experimental de Moji Guaçu, São Paulo state, Brazil (22°18’S and 47°11’W). The germination of spores of Cyathea delgadii decreases with time when kept in closed bottles under storage at 4°C in darkness. Germination is still very high after storage for one year. Spores stored for three years do not germinate. The results also show a decrease in soluble proteins and an increase in starch after several months storage.

Storage substances in the androgametogenesis and mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae)

Storage substances such as starch grains, proteins and lipids were studied during the male gametogenesis and in the mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae). There are two cycles of amylogenesis and amylolyse. The first cycle lasts until the vacuolated stage when the starch is hydrolyzed and amorphous proteins are stored inside the single vacuole. The next cycle begins after mitosis with the formation of the vegetative and generative cells. At this point, the young vegetative cell stores many starch grains that are bigger than in the first cycle. During the maturation of the male gametophyte, the starch is hydrolyzed and it is absent in the mature pollen grain. Small lipid droplets surround the young generative cell after the mitosis of the androspore and are dispersed in the vegetative cytoplasm during its maturity. The relationship between the pollen storage substances and the ontogeny of the layers in the sporoderm, formation of the generative cell, and the male germ unit were discussed.

Honey proteins and their interaction with polyphenols

This project aimed to determine the protein prof i les and concent rat ion in honeys, ef fect of storage condi t ions on the protein content and the interact ion between proteins and polyphenols. Thi r teen honeys f rom di f ferent botanical or igins were analyzed for thei r protein prof i les using SDS-PAGE, protein concent rat ion and phenol ic content , using the Pierce Protein Assay and Fol in-Ciocal teau methods, respectively. Protein-polyphenol interact ions were analyzed by a combinat ion of the ext ract ion of honeys wi th solvents of di f ferent polar i t ies fol lowed by LCjMS analysis of the obtained f ract ions. Results demonst rated a di f ferent protein content in the tested honeys, wi th buckwheat honey possessing the highest protein concent rat ion. We have shown that the reduct ion of proteins dur ing honey storage was caused, partially, by the protein complexat ion wi th phenolics. The LCjMS analysis of the peak elut ing at retent ion t ime of 10 to 14 min demonst rated that these phenolics included f lavonoids such as Pinobanksin, Pinobanksin acetate, Apigenin, Kaemferol and Myricetin and also cinnamic acid.

Characterization of Lipid Droplets and Functional Analysis of Lipid Droplet-Associated Proteins in Dictyostelium discoideum

Lipid droplets (LDs) are the universal storage form of fat as a reservoir of metabolic energy in animals, plants, bacteria and single celled eukaryotes. Dictyostelium LD formation was investigated in response to the addition of different nutrients to the growth medium. LDs were induced by adding exogenous cholesterol, palmitic acid (PA) as well as growth in bacterial suspension, while glucose addition fails to form LDs. Among these nutrients, PA addition is most effective to stimulate LD formation, and depletion of PA from the medium caused LD degradation. The neutral lipids incorporated into the LD-core are composed of triacylglycerol (TAG), steryl esters, and an unknown neutral lipid (UKL) species when the cells were loaded simultaneously with cholesterol and PA. In order to avoid the contamination with other cellular organelles, the LD-purification method was modified. The isolated LD fraction was analysed by mass spectrometry and 100 proteins were identified. Nineteen of these appear to be directly involved in lipid metabolism or function in regulating LD morphology. Together with a previous study, a total of 13 proteins from the LD-proteome were confirmed to localize to LDs after the induction with PA. Among the identified LD-proteins, the localization of Ldp (lipid droplet membrane protein), GPAT3 (glycerol-3-phosphate acyltransferase 3) and AGPAT3 (1-acylglycerol-3-phosphate-acyltransferase 3) were further verified by GFP-tagging at the N-termini or C-termini of the respective proteins. Fluorescence microscopy demonstrated that PA-treatment stimulated the translocation of the three proteins from the ER to LDs. In order to clarify DGAT (diacylglycerol acyltransferase) function in Dictyostelium, the localization of DGAT1, that is not present in LD-proteome, was also investigated. GFP-tagged DGAT1 localized to the ER both, in the presence and absence of PA, which is different from the previously observed localization of GFP-tagged DGAT2, which almost exclusively binds to LDs. The investigation of the cellular neutral lipid level helps to elucidate the mechanism responsible for LD-formation in Dictyostelium cells. Ldp and two short-chain dehydrogenases, ADH (alcohol dehydrogenase) and Ali (ADH-like protein), are not involved in neutral lipid biosynthesis. GPAT, AGPAT and DGAT are three transferases responsible for the three acylation steps of de novo TAG synthesis. Knock-out (KO) of AGPAT3 and DGAT2 did not affect storage-fat formation significantly, whereas cells lacking GPAT3 or DGAT1 decreased TAG and LD accumulation dramatically. Furthermore, DGAT1 is responsible for the accumulation of the unknown lipid UKL. Overexpression of DGAT2 can rescue the reduced TAG content of the DGAT1-KO mutant, but fails to restore UKL content in these cells, indicating that of DGAT1 and DGAT2 have overlapping functions in TAG synthesis, but the role in UKL formation is unique to DGAT1. Both GPAT3 and DGAT1 affect phagocytic activity. Mutation of GPAT3 increases it but a DGAT1-KO decreases phagocytosis. The double knockout of DGAT1 and 2 also impairs the ability to grow on a bacterial lawn, which again can be rescued by overexpression of DGAT2. These and other results are incorporated into a new model, which proposes that up-regulation of phagocytosis serves to replenish precursor molecules of membrane lipid synthesis, whereas phagocytosis is down-regulated when excess fatty acids are used for storage-fat formation.  

Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins

This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP) whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling It Included seven batches/trials three were supplemented with each type of the dairy protein used three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g(-1) of final product A control experiment was prepared without supplementing the milk base Processed milk bases were fermented at 42 C until pH 4 5 by using a starter culture blend that consisted of Streptococcus thermophilus Lactobacillus delbrueckii subsp bulgaricus and Bifidobacterium (Humans subsp lactis The kinetics of acidification was followed during the fermentation period as well the physico-chemical analyses enumeration of viable bacteria and theological characteristics of the yoghurts Models were adjusted to the results (kinetic responses counts of viable bacteria and theological parameters) through three regression models (linear quadratic and cubic special) applied to mixtures The results showed that the addition of milk proteins affected slightly acidification profile and counts of S thermophilus and B animal`s subsp lactis but it was significant for L delbrueckii subsp bulgaricus Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the theological properties of probiotic yoghurts taking into account the kinetics of acidification and enumeration of viable bacteria (C) 2010 Elsevier Ltd All rights reserved

Physical-chemical analysis of the cerebrospinal fluid of healthy dogs submitted to different storage periods and temperatures

Disfunções envolvendo o sistema nervoso são de grande importância na Medicina Veterinária, pois tratam-se de enfermidades de elevada incidência e com poucos subsídios auxiliares no seu diagnóstico, prognóstico e na avaliação de terapias empregadas. Ainda hoje, o diagnóstico baseia-se, em grande parte, no histórico e no exame clínico neurológico. Dessa forma, a análise dos constituintes do fluido cefalorraquidiano torna-se uma das poucas alternativas de acesso clínico ao sistema nervoso central (SNC). Mesmo com a grande utilidade do exame físico-químico e citoscópico do liquor na neurologia veterinária, poucos são os estudos sobre a estabilidade dos seus constituintes sob estocagem. Dessa forma, o presente trabalho teve como finalidade verificar a influência da temperatura e do tempo de conservação nas características físico-químicas do liquor de cães hígidos. Para tanto, foram coletadas amostras de LCR, através da punção da cisterna cerebelo-medular de cães clinicamente sadios, as quais foram submetidas à análise da densidade específica, do pH, da glicorraquia, das proteínas totais e das atividades das enzimas creatina quinase (CK) e aspartato aminotransferase (AST), após conservá-las em diferentes temperaturas (25°C, 4°C e -4°C) e por diferentes períodos de tempo (logo após a colheita, 24 horas, 48 horas, uma semana e um mês). Dentre os resultados obtidos, foi possível verificar, principalmente, que houve estabilidade dos parâmetros estudados por até um mês de estocagem nas amostras mantidas sob a temperaturas de congelamento de -4°C.

Storage sites in seeds of Caesalpinia echinata and C. ferrea (Leguminosae) with considerations on nutrients flow

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fat body in some genera of leaf-cutting ants (Hymenoptera : Formicidae). Proteins, lipids and polysaccharides detection

The comparative histochemical analysis of the fat body of workers belonging to the basal species Cyphomyrmex rimosus and Mycetarotes parallelus and to derived species Acromyrmex disciger and Atta laevigata revealed that this tissue is constituted mainly by cells denominated trophocytes and oenocytes. The trophocytes of all species studied here were characterized mainly by the proteins and lipids synthesis and storage, being the derived species the ones who have presented higher quantity of lipids in the trophocytes when compared to the trophocytes of basal species. In workers M. parallelus and A. laevigata, besides proteins and lipids, there has being observed the presence of polysaccharides, however, in C. rimosus and A. disciger these elements were detected in lower quantities. The histochemical studies of the oenocytes of basal and derived species revealed significant presence of proteins as well as lipids in these cells. In the oenocytes of derived species A. disciger and A. laevigata a higher quantity of lipidic inclusions has being observed, when compared to the basal species. (c) 2005 Elsevier Ltd. All rights reserved.

Changes during chilled storage of whole tilapia and short-term frozen storage of tilapia fillets

This study evaluated the physicochemical changes in Nile tilapia (n = 82, 373.71 ± 61.91 g) refrigerated for up to 92 h and in the frozen fillets. The tilapias were captured with nets, slaughtered by ice and water shock (1:1) in a temperature of approximately 2°C for 30 min, and stored refrigerated at 4°C in polystyrene boxes containing ice. The fish were filleted, and filets were weighed and frozen. The drip loss and protein were determined after 23 days of frozen storage. After 4 h of storage, all fish were in full rigor mortis. The pH of the muscles decreased for up to 45 h of the storage period. The fillets obtained from tilapia stored for more than 72 h lost more weight and protein. Thus, the filleting or processing of tilapia should be done before 72 h of cold storage, since deterioration of the fish starts to occur after this period. Copyright © Taylor & Francis Group, LLC.

Studies toward the identification of transcription factors in cassava storage root

Fatores de transcrição desempenham importantes funções em vários processos fisiológicos. Nos últimos anos, muitos fatores de transcrição têm sido isolados de plantas, emergindo como poderosas ferramentas na manipulação de características agronômicas. No presente trabalho, iniciamos estudos para isolar fatores de transcrição de mandioca (Manihot esculenta Crantz), importante cultura tropical e subtropical. Nossos resultados revelaram três tipos de proteínas diferencialmente expressas na raiz de reserva de mandioca (Manihot esculenta Crantz):e imunologicamente relacionadas com o fator de transcrição opaco-2 de milho. Experimentos de Southwestern mostraram duas proteínas capazes de interagir in vitro com uma seqüência de DNA do gene be2S1 de castanha-do-brasil.

Disappearance of fat body proteins during soldier differentiation in the neotropical termite Heterotermes tenuis (Isoptera: Rhinotermitidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)