Methylotrophic methanogens were readily enriched with monomethylamine (MMA) in water samples obtained from throughout the stratified but oxygenated water column of Chesapeake Bay, USA. Of the 3 different populations of methylotrophic methanogens enriched, 02-and H2S-tolerant methanogens enriched in bacterially reduced seawater in glass were twice as prevalent as those enrichments either reduced chemically in glass or reduced bacterially in semipermeable polycarbonate flasks. Thirty-three obligately anaerobic, but 02and H2S-tolerant, methanogenic cultures from the dominant group were fingerprinted immunologically using antibody probes. The antigenic fingerprints divided these cultures into 4 clusters. One cluster was related to Methanosarcina barkeri RlM3, a second was related to M. mazei S6, and a third was related to other species of Methanosarcina. None of these were identical to known species and can be considered new immunotypes. These new immunotypes, related to described species, were found throughout the water column. A fourth cluster was unrelated to any extant genus of methanogens, was absent from surface waters, and was restricted to the pycnocline and deeper waters. This group of new methanogens constituted 24 % of the pycnocline cultures and 39 % of those from bottom waters. Stratification, which is a prerequisite for the development or concentration of methane-cycle bacteria in the pycnocline. apparently allows unique water-column methanogens to selectively enrich in particulates in the pycnocline that can increase in bottom waters before sedimentation. The observed distribution of known and new taxa of methanogens in the 3 water layers of Chesapeake Bay is illustrated by a model.

Hsp60 can be released by tumor cells to promote growth. Hsp60 exits mitochondria, reaches the blood and distant targets by mechanisms not yet understood. We determined Hsp60 levels and secretion in normal and tumor cell lines in cell lysates, medium, exosomes, membranes, lipid rafts, and other materials, by ultracentrifugation, electrophoresis, Western blotting, ELISA, EM, and enzymatic tests. Exosomes were purified and characterized by EM and by presence of ALIX, Hsp90, Hsp70, ATPase, and AChE. The tumor, not the normal cells secreted Hsp60, in the absence of cell death, involving lipid rafts‐exosome pathways; Hsp60 associated with lipid‐rafts and cell and exosomal membranes. A working model predicts: Hsp60 gets in the cytosol from mitochondria or from synthesis in the cytosol without entering the organelle, arrives at the cell membrane and binds lipid rafts and, through lipid‐raft mediated endocytosis, is included in early endosomes and then in multivesicular bodies and, lastly, in exosomes for secretion. Hsp60 in exosomes may then reach components of the innate immune system and trigger inflammation; endothelial cells to elicit neoangiogenesis; and other tumor cells to block apoptosis and promote cancer survival.

Hsp60 is a Group I chaperonin highly conserved during evolution that in normal human cells is localized mainly in the mitochondrial matrix but stress causes changes in its levels and localization. We studied with a battery of techniques human mucosa from: a) normal colon (NM); b) hyperplastic polyps (HP); c) tubular adenomas with dysplasia (TAD); d) Crohn's disease (CD); e) ulcerative colitis (UC); and f) adenocarcinomas (AC). We observed patterns of Hsp60 levels and subcellular localization which depended on the pathology. Hsp60 levels in TAD, CD and UC (all pre‐tumoral conditions) were higher than in NM and HP but lower than in AC that showed the highest levels. Hsp60 was localized mainly in epithelial cells and, sparsely also in lamina propria. In epithelial cells of all the conditions examined (except NM and HP) Hsp60 was abundantly present not only in mitochondria but also in the cytosol, the latter being a pre‐requisite for its secretion. In conclusion, Hsp60 levels and localization patterns distinctively reflect the health status of these tissues and help to distinguish pathologic conditions.Grant Funding Source: University of Palermo

Hsp60 and Hsp10 are considered typical mitochondrial chaperonins, residing inside the organelle, but works in the last years showed they can also be outside mitochondria and cells. The exact extramitochondrial locations of the chaperonins in the various normal cells and tissues under diverse conditions remain to be ascertained. Consequently, we undertook to examine the subcellular distribution of Hsp60 and Hsp10 in lung cells in order to provide a baseline for comparison between normal and pathological tissues. We used a battery of methods (light and electron microscopies, immunocytochemistry, subcellular fractionation‐purification plus electrophoresis, Western blotting) to study two cell types, lung fibroblasts and epithelial cells, unstressed and exposed to the stressor cigarette smoke extract, which is pertinent to lung pathology. Here we focus on Hsp10, which was found in the mitochondria, cytosol, and nucleus, with their levels changing after stress. Nuclear Hsp10 has not been reported before and was demonstrated in various ways, including immunoelectromicroscopy with specific antibodies.Grant Funding Source: University of Palermo