Methylomonas albis sp. nov. and Methylomonas fluvii sp. nov.: Two cold-adapted methanotrophs from the river Elbe and emended description of the species Methylovulum psychrotolerans.

Three strains of methanotrophic bacteria (EbAT, EbBT and Eb1) were isolated from the River Elbe, Germany. These Gram-negative, rod-shaped or coccoid cells contain intracytoplasmic membranes perpendicular to the cell surface. Colonies and liquid cultures appeared bright-pink. The major cellular fatty acids were 12:0 and 14:0, in addition in Eb1 the FA 16:1ω5t was also dominant. Methane and methanol were utilized as sole carbon sources by EbBT and Eb1, while EbAT could not use methanol. All strains oxidize methane using the particulate methane monooxygenase. Both strains contain an additional soluble methane monooxygenase. The strains grew optimally at 15-25 °C and at pH 6 and 8. Based on 16S rRNA gene analysis recovered from the full genome, the phylogenetic position of EbAT is robustly outside any species clade with its closest relatives being Methylomonas sp. MK1 (98.24%) and Methylomonas sp. 11b (98.11%). Its closest type strain is Methylomonas methanica NCIMB11130 (97.91%). The 16S rRNA genes of EbBT are highly similar to Methylomonas methanica strains with Methylomonas methanica R-45371 as the closest relative (99.87% sequence identity). However, average nucleotide identity (ANI) and digital DNA-DNA-hybridization (dDDH) values reveal it as distinct species. The DNA G + C contents were 51.07 mol% and 51.5 mol% for EbAT and EbBT, and 50.7 mol% for Eb1, respectively. Strains EbAT and EbBT are representing two novel species within the genus Methylomonas. For strain EbAT we propose the name Methylomonas albis sp. nov (LMG 29958, JCM 32282) and for EbBT, we propose the name Methylomonas fluvii sp. nov (LMG 29959, JCM 32283). Eco-physiological descriptions for both strains are provided. Strain Eb1 (LMG 30323, JCM 32281) is a member of the species Methylovulum psychrotolerans. This genus is so far only represented by two isolates but Eb1 is the first isolate from a temperate environment; so, an emended description of the species is given.

Siphonodictyal B1 from a marine sponge increases intracellular calcium levels comparable to the Ca2+-ATPase (SERCA) inhibitor thapsigargin.

Siphonodictyal B1 is a sesquiterpene-hydroquinone isolated from the Caribbean coral reef bioeroding sponge Siphonodictyon coralliphagum. Siphonodictyal B1 increased intracellular calcium levels in neuroendocrine cells (PC12) in the presence and absence of extracellular calcium using Fura-2 as a calcium-sensitive dye. The calcium rise was comparable in amplitude and timing to the application of the sarco-endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin from the terrestrial plant Thapsia garganica. The effects of thapsigargin and siphonodictyal B1 on intracellular calcium levels were not distinguishable in pharmacological experiments conducted with caffeine, ryanodine, muscarine, and thapsigargin in calcium-free and calcium-containing buffer, although thapsigargin was effective at lower concentrations. Thapsigargin is a sesquiterpene-lactone and has no structural similarities to siphonodictyal B1. We conclude that thapsigargin and siphonodictyal B1 share SERCAs as cellular targets. Siphonodictyal B1 may be involved in the process of bioeroding the calcium carbonate endoskeleton of the scleractinian corals attacked by S. coralliphagum.

Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye.

The alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.

Disturbance of voltage-induced cellular calcium entry by marine dimeric and tetrameric pyrrole--imidazole alkaloids.

Twelve brominated pyrrole-imidazole alkaloids from the Caribbean sponges Stylissa caribica and Agelas wiedenmayeri were tested for interactions with cellular calcium homeostasis using PC12 cells. Massadine (half maximal concentration: 5.32 +/- 0.007microM), stylissadines A (4.48 +/- 1.1microM) and B (4.6 +/- 1.6microM) as well as tetrabromostyloguanidine (15.6 +/- 0.004microM) reduced voltage-dependent calcium entry in PC12 cells as measured with Fura II as calcium indicator. Dibromopalau'amine and mauritiamine reduced voltage-dependent calcium entry but no half maximal concentration can be calculated from our results. Monomeric brominated pyrrole alkaloids such as stevensine, cyclooroidin, oxocyclostylidol, 4-bromopyrrole-2-carboxy-N(epsilon)-lysine, and 4-bromopyrrole-2-carboxyarginine showed no or only minor effects. Ageladine A itself showed fluorescence in a similar range as Fura II and therefore no data are reported here. Based on the results a structure-activity relationship could be established. Absolutely necessary for an activity seem to be a lipophilic (brominated side chain) and a hydrophilic (amino-imidazole core) substructure. The combination of these substructures may be on one hand responsible for the membrane solubility (dibromopyrrole moieties) and on the other hand for the interaction with the hydrophilic area of the calcium channel (amino-imidazole moieties) to accomplish the alkaloids neurotoxic potential.