The release of Hsp70 from A431 carcinoma cells is mediated by secretory-like granules

In our earlier work we have demonstrated that the treatment of squamous carcinoma cell line A431 with a pharmacological inhibitor of phospholipase C activity, U73122, resulted in fast release of stress-inducible heat shock protein 70 (Hsp70) into the extracellular medium (Evdonin et al., Cancer Cell Int., 4, 2, 2004). The purpose of the present study was to identify cellular organelles involved in the release of Hsp70 from A431 cells. We determined that Hsp70 is present in granules located at the periphery of cells, which had been treated with U73122 or subjected to heat shock. An inhibitor of the classical protein export pathway, brefeldin A was found to prevent the U73122-induced appearance of Hsp70 in the extracellular medium and in the peripheral granules. These findings suggest that vesicular transport is involved in Hsp70 release. The Hsp70-containing granules did not carry markers specific for lipid bodies, endosomes, or lysosomes. However, they were positive for a marker of secretory granules, i.e. chromogranin A. The levels of extracellular Hsp70 and chromogranin A were found to increase simultaneously. The secretory-like granule-dependent transport of Hsp70 was also studied in minimally transformed human HaCaT keratinocytes. We found that after U73122 and heat stress treatment, HaCaT cells secreted Hsp70 in a manner similar to A431 cells. Collectively our results suggest that human keratinocyte-derived cells release Hsp70 in the extracellular medium through a pathway involving secretory-like granules.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.     

Mast Cell Proteoglycans

Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease. A hallmark morphological feature of mast cells is their large content of cytoplasmic secretory granules, filled with numerous secretory compounds, including highly negatively charged heparin or chondroitin sulfate proteoglycans of serglycin type. These anionic proteoglycans provide the basis for the strong metachromatic staining properties of mast cells seen when applying various cationic dyes. Functionally, the mast cell proteoglycans have been shown to have an essential role in promoting the storage of other granule-contained compounds, including bioactive monoamines and different mast cell-specific proteases. Moreover, granule proteoglycans have been shown to regulate the enzymatic activities of mast cell proteases and to promote apoptosis. Here, the current knowledge of mast cell proteoglycans is reviewed.     

P-bodies and mitochondria: Which place in RNA interference?

Micro-RNAs (miRNAs) are major actors of RNA interference (RNAi), a regulation pathway which leads to translational repression and/or degradation of specific mRNAs. They provide target specificity by incorporating into the RISC complex and guiding its binding to mRNA. Since the discovery of RNAi, many progresses have been made on the mechanism of action of the RISC complex and on the identification of target mRNAs. However, the regulation of RNAi has been poorly investigated so far. Recently, various studies have revealed physical and functional relationships between RNAi, P-bodies and mitochondria. This review intends to recapitulate these data and discuss their potential importance in cell metabolism.     

Biodegradation of dimethyl phthalate by Sphingomonas sp. isolated from phthalic-acid-degrading aerobic granules

This study isolated nine strains of aerobic phenol-degrading granules. These isolates (I1–I9) were characterized using 16S rRNA gene sequencing, with γ-Proteobacteria as the dominant strains in the aerobic granules. While most strains demonstrated either high phenol-degrading capabilities or auto-aggregation capabilities, three isolates, I2, I6, and I8 showed both features. These findings contradict the previous view that auto-aggregation and phenol degradation are mutually exclusive in aerobic granules. Strains I2 and I8 independently formed single-culture aerobic granules except for I3. Anti-microbial activity test results indicated that strains I2 and I8 inhibited growth of strain I3. However, co-culturing I3 with I2 or I8 helped to form granules.     

An enzyme family reunion — similarities,differences and eccentricities in actions on <Emphasis Type="Italic">α</Emphasis>-glucans

α-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the “test of time” blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY_3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the “robotics mechanism” of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new “eccentric” functions, are there new families out there, and why are crystal structures of “simple” enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications.     

Plant stress granules and mRNA processing bodies are distinct from heat stress granules

Similar to the situation in mammalian cells and yeast, messenger ribonucleo protein (mRNP) homeostasis in plant cells depends on rapid transitions between three functional states, i.e. translated mRNPs in polysomes, stored mRNPs and mRNPs under degradation. Studies in mammalian cells showed that whenever the dynamic exchange of the components between these states is disrupted, stalled mRNPs accumulate in cytoplasmic aggregates, such as stress granules (SGs) or processing bodies (PBs). We identified PBs and SGs in plant cells by detection of DCP1, DCP2 and XRN4, as marker proteins for the 5'-->3' mRNA degradation pathway, and eIF4E, as well as the RNA binding proteins RBP47 and UBP1, as marker proteins for stored mRNPs in SGs. Cycloheximide-inhibited translation, stress treatments and mutants defective in mRNP homeostasis were used to study the dynamic transitions of mRNPs between SGs and PBs. SGs and PBs can be clearly discriminated from the previously described heat stress granules (HSGs), which evidently do not contain mRNPs. Thus, the role of HSGs as putative mRNP storage sites must be revised.