Solution NMR of supramolecular complexes: providing new insights into function

Solution NMR spectroscopy is an extremely powerful technology for the study of biomolecular dynamics and site-specific molecular interactions. An important limitation in the past has been molecule size, with molecular weights of targets seldom exceeding 50 kDa. New labeling technology and NMR experiments are changing this paradigm so that applications for investigating supramolecular complexes are starting to become feasible. Here we describe a strategy developed in our laboratory that involves the use of labeled methyl groups of isoleucine, leucine and valine residues in proteins as probes, along with experiments that significantly enhance the lifetimes of the resulting signals. We describe the application of these methods to a number of systems with molecular weights in the hundreds of kilodaltons.     

The lens: a classical model of embryonic induction providing new insights into cell determination in early development

The lens was the first tissue in which the concept of embryonic induction was demonstrated. For many years lens induction was thought to occur at the time the optic vesicle and lens placode came in contact. Since then, studies have revealed that lens placodal progenitor cells are specified already at gastrula stages, much earlier than previously believed, and independent of optic vesicle interactions. In this review, I will focus on how individual signalling molecules, in particular BMP, FGF, Wnt and Shh, regulate the initial specification of lens placodal cells and the progressive development of lens cells. I will discuss recent work that has shed light on the combination of signalling molecules and the molecular interactions that affect lens specification and proper lens formation. I will also discuss proposed tissue interactions important for lens development. A greater knowledge of the molecular interactions during lens induction is likely to have practical benefits in understanding the causes and consequences of lens diseases. Moreover, knowledge regarding lens induction is providing fundamental important insights into inductive processes in development in general.     

Alpha-synuclein interacts with Glucocerebrosidase providing a molecular link between Parkinson and Gaucher diseases

The presynaptic protein α-synuclein (α-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between α-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that α-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the α-syn C-terminal residues, 118-137. This α-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 μm in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for α-syn, as does the inhibitor conduritol-β-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the α-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.     

N-acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.     

Evidence supporting a model of voltage-dependent uptake of auxin into Cucurbita vesicles

The accumulation of [¹⁴C]indole-3-acetic acid (IAA), of [³H]tetra-phenyl phosphonium ion as a membrane potential probe, and of [¹⁴C]butyric acid as probe for pH gradients was studied with membrane vesicles from etiolated hypocotyls of Cucurbita pepo. Ion gradients (K⁺, H⁺) were applied in the presence and absence of specific ionophores e.g. valinomycin or carbonylcyanide m-chlorophenylhydrazone. In all cases tested, the accumulation of [¹⁴C]IAA equals neither potential probe nor pH-probe accumulation, but represents. an intermediate between the two. Auxin molecules seem to be taken up as positively charged ions and a pH gradient is required for accumulation. The uptake mechanism thus appears to be a specific, carrier-mediated cotransport of the anion of IAA and no less than two protons. The initial rates of auxin uptake by the saturable influx carrier, of permeation through the membrane, and of efflux by the phytotropin-affected efflux carrier were analysed.Abbreviations BA butyric acid - CCCP carbonylcyanid-3-chlorophenylhydrazone - CPD 2-carboxylphenyl-3-phenylpropan-1,3-dion - IAA indole-3-acetic acid - IAA anion of IAA - IAAH undissociated form of IAA - 2-NAA 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - TPP⁺ tetra-phenyl phosphonium ion     

An insight into synthetic Schiff bases revealing antiproliferative activities in vitro

Schiff bases or azomethines are among the most important groups of biomolecules. These compounds have been found to reveal both remarkable biological activities and a variety of valuable practical applications. An interest in the exploration of novel series of synthetic Schiff bases has undoubtedly been growing due to their proven utility as attractive lead structures for the design of novel cytotoxic and cytostatic agents with a mechanism of action that sometimes differs from that of clinically authorized anticancer agents. Therefore, in the present paper we have focussed our attention on the collected synthetic simple Schiff bases of aldimine- and ketimine-types revealing anticancer activities in vitro, that have been described in the scientific literature during the last decade, and on structural variations whose affect the antiproliferative activity in sets of the designed molecules.     

Structural insights into a new homodimeric self-activated GTPase family

The human XAB1/MBDin GTPase and its close homologues form one of the ten phylogenetically distinct families of the SIMIBI (after signal recognition particle, MinD and BioD) class of phosphate-binding loop NTPases. The genomic context and the partners identified for the archaeal and eukaryotic homologues indicate that they are involved in genome maintenance--DNA repair or replication. The crystal structure of PAB0955 from Pyrococcus abyssi shows that, unlike other SIMIBI class G proteins, these highly conserved GTPases are homodimeric, regardless of the presence of nucleotides. The nucleotide-binding site of PAB0955 is rather rigid and its conformation is closest to that of the activated SRP G domain. One insertion to the G domain bears a strictly conserved GPN motif, which is part of the catalytic site of the other monomer and stabilizes the phosphate ion formed. Owing to this unique functional feature, we propose to call this family as GPN-loop GTPase.     

Synergism between the antiproliferative activities of arabinosyladenine and N6-benzyladenosine

N6-Benzyladenosine is a competitive inhibitor of adenosine deaminase from L-1210 cells in axenic culture as well as a potent antiproliferative agent in vitro and in vivo. Potentiation of the growth inhibitory activity of 9-beta-D-arabinosyladenine (ara-A) was observed in the L-1210 system with maximum synergism with a mixture of 16 micron ara-A and 10 micron benzyladenosine. Kinetic studies with L-1210 cell lysates showed values for Km of 0.25 mM ara-A and Ki of 0.23 mM benzyladenosine. It is suggested that ara-A and benzyladenosine in suitable combination may be expected to demonstrate enhanced clinical chemotherapeutic effectiveness.     

Hemojuvelin: a new link between obesity and iron homeostasis

The adipose tissue may play an active role in systemic iron regulation and this role may be determinant in obese patients. Indeed, we reported previously that hepcidin, the iron-regulatory hormone, is expressed in adipose tissue and its messenger RNA (mRNA) expression is increased in adipose tissue of morbidly obese patients. The objectives of this study were to characterize the status of hemojuvelin (HJV), another iron-regulatory protein, within the adipose tissue of morbidly obese patients. Since cell-associated HJV acts as a coreceptor of bone morphogenetic protein (BMP) to enhance hepcidin expression in liver cells, we investigated the possible involvement of this pathway in adipose tissue in regulating hepcidin expression. HJV expression was studied in adipose tissue of morbidly obese patients. Soluble HJV blood concentrations were assessed. Hepcidin regulation through BMP pathway was investigated in cultured adipocytes. HJV was expressed both at mRNA and protein levels in adipose tissue. Moreover, its mRNA expression was highly increased in adipose tissue of obese patients and correlated with mRNA hepcidin expression levels. Interestingly, HJV expressed by adipose tissue may be effective since cultured adipocytes increased their hepcidin expression when challenged with BMP2 through Smad effectors. In addition, blood concentrations of soluble HJV were significantly increased. In conclusion, adipose tissue may influence iron homeostasis in obese patients by expressing major iron-regulatory proteins and the BMP signaling pathway could be involved in regulating hepcidin expression in this tissue.     

Omega-pyridiniumalkylethers of steroidal phenols: new compounds with potent antibacterial and antiproliferative activities

Novel omega-pyridiniumalkylethers of two steroidal phenols were synthesized as compounds with potential antimicrobial activity. 3-Hydroxy-estra-1,3,5(10)-triene-17-one and 1-hydroxy-4-methyl-estra-1,3,5(10)-triene-17-one were reacted with omega,omega'-dibromoalkanes to omega-bromoalkoxy-estra-1,3,5(10)-trienes followed by reaction with pyridine to obtain the desired steroidal omega-pyridiniumalkoxy compounds as bromides. Their antimicrobial activity against strains of multiresistant Staphylococcus aureus (MRSA), a vancomycin resistant Enterococcus faecalis and fast growing mycobacteria depends clearly on the length of the alkyl chain. A strong broadband activity has been found for the compounds with eight or 10 C-atoms; in some cases better than ciprofloxacin or cetylpyridinium salts. In addition, the antiproliferative and cytotoxic activity depends on the chain length, too. The differentiation between antibacterial and cytotoxic activity is better for the steroid hybrid molecules than the cetylpyridinium salts. These new compounds can serve as lead compounds for further optimization.     

Evidence of a link between survival and pair fidelity across multiple tit populations

Although they have the potential to strongly influence individual fitness and the dynamics and productivity of populations, the survival consequences of pairing outcomes and the influence of current pairing outcomes on those in the future have rarely been addressed. Previously, we have shown that pair fidelity increases both survival and future pair fidelity in a population of great tits Parus major. The aim of this study was to explore the generality of our previous findings by evaluating the influence of current paring outcomes on survival and on future pairing outcomes in two different species and in different populations. We addressed our aims within a multievent capture–mark–recapture (MECMR) statistical framework, which accounts for differences in recapture rates and uncertainty in the assignment of pair status (i.e. whether an individual is breeding with the same partner or not). We applied the framework to breeding records of two great tit populations and one blue tit Cyanistes caeruleus population. We detected survival benefits (i.e. increased survival) of pair fidelity in all three populations. These were similar in both great tit populations, but higher for male great tits than for male blue tits. We found that age‐dependence in the rate of pair fidelity was shared between different populations and species, but did not detect any influence of current pair status on future pair status. Our study highlights the importance of considering survival when studying the fitness benefits of pair fidelity. Some of the differences in pair fidelity rates and survival benefits of pair fidelity are likely the result of long‐term and short‐term demographic and environmental factors in the population. We advocate the use of the MECMR framework used here for further exploration of these differences.     

Phosphoproteomics: new insights into cellular signaling

Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far.     

Evidence of a link between taboos and sacrifices and resource scarcity of ritual plants

Background

One of the main obstacles for the mainstreaming of religious traditions as tools for the conservation of nature is the limited applicability of research results in this field. We documented two different restrictions implemented by local people (taboos and sacrifices) related to the use of ritual plants in Benin (West Africa) and Gabon (Central Africa).

Methods

To see whether these restrictions reflected plant scarcity from an etic perspective (official threat status) and an emic viewpoint (perceived scarcity by local people), we conducted 102 interviews with traditional healers and adepts of traditional faiths.

Results

We documented a total of 618 ritual plants, from which 52 species were used in both countries. In Benin, the use of 63 of the 414 ritual plant species was restricted; while in Gabon 23 of the 256 ritual plants were associated with taboos and sacrifices. In Benin, restricted plants were significantly more often officially threatened, perceived as scarce, and actively protected than non-restricted plants. In the more forested and less densely populated Gabon, plants that were perceived as scarce were more often associated to local restrictions than officially threatened species.

Conclusions

These results prove the presence of a form of adaptive management where restrictions are related to resource scarcity and protection of ritual plant species. By providing baseline data on possibly endangered species, we demonstrate how plant use in the context of religious traditions can yield important information for conservation planning.

Electronic supplementary material

The online version of this article (doi:10.1186/1746-4269-11-5) contains supplementary material, which is available to authorized users.     

Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes

Chromosoma - The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful...