Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.     

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.     

Antitumor mechanisms of oligodeoxynucleotides with CpG and polyG motifs in murine prostate cancer cells: decrease of NF-kappaB and AP-1 binding activities and induction of apoptosis

Previous studies have shown that CpG oligodeoxynucleotides (ODNs) have substantial immunostimulatory effects with anticancer applications. The antitumor applications that have been described previously are mediated through the CpG-induced activation of the host immune system, not through direct antitumor effects. Using cytostasis and cell proliferation assays, we demonstrated that specific ODNs inhibit the proliferation of RM-1 cells, a murine prostate cancer cell line. Flow cytometry analysis using propidium iodide (PI) nuclear staining confirmed the direct proapoptotic effect of ODNs on prostate cancer cells. This effect was dose dependent. Further studies using Western blot analysis and electrophoresis mobility shift assay (EMSA) revealed that the treatment of prostate cancer cells with specific ODNs activated the caspase pathway(s) and decreased the binding activities of AP-1 and NF-kappaB in a time-dependent manner. Evaluation of a panel of ODNs containing different DNA motifs demonstrated that the optimal proapoptotic sequences required polyG sequences but that CpG motifs were not essential. Finally, in vivo antitumor studies showed that the proapoptotic polyG motifs significantly inhibited prostate tumor growth. PolyG motifs inhibited tumor growth, and the effects were enhanced by CpG immune activating sequences. ODN containing both polyG and CpG motifs may have enhanced efficacy in tumor therapy through multiple mechanisms of action, including direct antitumor activities and immune activation.     

Familial mediterranean fever (FMF) in Moroccan Jews: Demonstration of a founder effect by extended haplotype analysis

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated “MEF”) is on 16p, with the gene order 16cen–D16S80–MEF–D16S94–D16S283–D16S291–16pter. Here we report the association of FMF susceptibility with alleles at D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94–D16S283–D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.     

Involvement of polyamines in the chilling tolerance of cucumber cultivars

The possible involvement of polyamines (PAs) in the chilling tolerance of cucumber (Cucumis sativus L. cv Jinchun No. 3 and cv Suyo) was investigated. Plants with the first expanded leaves were exposed to 3 degrees C or 15 degrees C in the dark for 24 h (chilling), and then transferred to 28 degrees C/22 degrees C under a 12-h photoperiod for another 24 h (rewarming). Chilling-tolerant cv Jinchun No. 3 showed a marked increase of free spermidine (Spd) in leaves, once during chilling and again during rewarming. Putrescine increased significantly during rewarming, but the increase of spermine was slight. Any of these PAs did not increase in chilling-sensitive cv Suyo during either period. PA-biosynthetic enzyme activities appear to mediate these differences between cultivars. Pretreatment of Spd to cv Suyo prevented chill-induced increases in the contents of hydrogen peroxide in leaves and activities of NADPH oxidases and NADPH-dependent superoxide generation in microsomes and alleviated chilling injury. Pretreatment of methylglyoxal-bis-(guanylhydrazone), a PA biosynthesis inhibitor, to chilled cv Jinchun No. 3 prevented Spd increase and enhanced microsomal NADPH oxidase activity and chilling injury. The results suggest that Spd plays important roles in chilling tolerance of cucumber, probably through prevention of chill-induced activation of NADPH oxidases in microsomes.     

Mutating away from your enemies: The evolution of mutation rate in a host–parasite system

The rate at which mutations occur in nature is itself under natural selection. While a general reduction of mutation rates is advantageous for species inhabiting constant environments, higher mutation rates can be advantageous for those inhabiting fluctuating environments that impose on-going directional selection. Analogously, species involved in antagonistic co-evolutionary arms races, such as hosts and parasites, can also benefit from higher mutation rates. We use modifier theory, combined with simulations, to investigate the evolution of mutation rate in such a host–parasite system. We derive an expression for the evolutionary stable mutation rate between two alleles, each of whose fitness depends on the current genetic composition of the other species. Recombination has been shown to weaken the strength of selection acting on mutation modifiers, and accordingly, we find that the evolutionarily attracting mutation rate is lower when recombination between the selected and the modifier locus is high. Cyclical dynamics are potentially commonplace for loci governing antagonistic species interactions. We characterize the parameter space where such cyclical dynamics occur and show that the evolution of large mutation rates tends to inhibit cycling and thus eliminates further selection on modifiers of the mutation rate. We then find using computer simulations that stochastic fluctuations in finite populations can increase the size of the region where cycles occur, creating selection for higher mutation rates. We finally use simulations to investigate the model behaviour when there are more than two alleles, finding that the region where cycling occurs becomes smaller and the evolutionarily attracting mutation rate lower when there are more alleles.     

Screening and characterization of yeasts for xylitol production

AIMS: To discover novel naturally occurring xylitol producing yeast species with potential for industrial applications. METHODS AND RESULTS: Exactly 274 strains were cultivated on both solid and liquid screening medium with xylose as the sole carbon resource. Five strains were selected on the basis of significant growth and high degree of xylose assimilation. Their phylogenetic position was confirmed by the PCR-RFLP and sequence analysis of the D1/D2 domain of the 5' end of the large subunit rDNA gene (5'-LSU rDNA). Enzymatic analysis was conducted to compare xylose metabolism in each strain. Candida guilliermondii Xu280 and Candida maltosa Xu316 were found to have high xylose consumption rates and xylitol yields in the batch fermentation under micro-aerobic condition. The effect of the different media with high initial xylose concentration on biosynthesis of xylitol by both strains was investigated. CONCLUSIONS: We have identified Candida spp. strains, which exhibit high levels of xylitol production from xylose suggesting that these may have potential for industrial applications. SIGNIFICANCE AND IMPACTS OF THE STUDY: Microbial species are of importance for xylitol production. Xylitol production involves complicated metabolic regulation including xylose transport, production of key enzymes and cofactor regeneration. Thus, screening of naturally occurring xylose-utilizing micro-organisms is a viable and effective mean to obtain xylitol producing organisms with industrial application. Moreover, the research on selected strains will contribute to a better understanding of regulatory properties of xylose metabolism in different yeasts.