Marine sponges as microbial fermenters

The discovery of phylogenetically complex, yet highly sponge-specific microbial communities in marine sponges, including novel lineages and even candidate phyla, came as a surprise. At the same time, unique research opportunities opened up, because the microorganisms of sponges are in many ways more accessible than those of seawater. Accordingly, we consider sponges as microbial fermenters that provide exciting new avenues in marine microbiology and biotechnology. This review covers recent findings regarding diversity, biogeography and population dynamics of sponge-associated microbiota, and the data are discussed within the larger context of the microbiology of the ocean.     

Nuclear and cytoplasmic binding components for vitamin D metabolites.

Specific binding of 1α,25-dihydroxyvitamin D₃ to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D₃ complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D₃-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed.Macromolecular binding components with a high affinity for 25-hydroxyvitamin D₃ (K_d = 4.5 × 10⁻⁹ M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D₃ receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D₃ at higher concentrations than 25-hydroxyvitamin D₃, they are not observed to transfer either 25-hydroxyvitamin D₃ or 1α,25-dihydroxyvitamin D₃ to the nucleus, $\text{in vitro}$.     

Retrocyclin-2: structural analysis of a potent anti-HIV theta-defensin

Retrocyclins are circular mini-defensins with significant potential as agents against human immunodeficiency virus, influenza A, and herpes simplex virus. Retrocyclins bind carbohydrate-containing surface molecules such as gp120 and CD4 with high affinity (Kd, 10-100 nM), promoting their localization on cell membranes. The structural features important for activity have yet to be fully elucidated, but here, we have determined the first three-dimensional structure of a retrocyclin, namely, one of the most potent forms, retrocyclin-2. In the presence of SDS micelles, a well-defined beta-hairpin braced by three disulfide bonds that defines the cystine ladder motif is present. By contrast, a well-defined structure could not be determined in aqueous solution, suggesting that the presence of SDS micelles stabilizes the extended conformation of retrocyclin-2. Translational diffusion measurements indicate that retrocyclin-2 interacts with the SDS micelles, and such a membrane-like interaction may be an important feature in the mechanism of action of these antimicrobial peptides. Analytical ultracentrifugation and the NMR data indicated that retrocyclin-2 self-associates to form a trimer in a concentration-dependent manner. The ability to self-associate may contribute to the high-affinity binding of retrocyclins for glycoproteins by increasing the valency and enhancing the ability of retrocyclins to cross-link cell surface glycoproteins.     

Cooperative transition in the conformation of 24-mer tarantula hemocyanin upon oxygen binding

Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula hemocyanin is approximately 2, matching the number of allosteric units/hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.