Variabilin, a chemotaxonomic marker for the family Irciniidae

The furanosesterterpene variabilin was isolated from the sponge Sarcotragus. From a chemical point of view, the family Irciniidae has been the source of furanosesterterpenes, and especially variabilin is an important chemotaxonomic marker for the family Irciniidae.     

Sharks checking in to the sponge hotel: First internal use of sponges of the genus Agelas and family Irciniidae by banded sand catsharks Atelomycterus fasciatus

Trawl surveys within and surrounding two northwestern Australian marine parks revealed banded sand catsharks Atelomycterus fasciatus (family Atelomycteridae) taking refuge within large sponges of the family Irciniidae (Demospongiae: Dictyoceratida) and the genus Agelas (Demospongiae: Agelasida: Agelasidae). Five sponges contained a total of 57 A. fasciatus, comprising both sexes and both immature and mature individuals ranging from 102 to 390 mm total length (TL). In the same surveys, only five A. fasciatus were captured unassociated with sponges, suggesting that sponges are an important microhabitat for A. fasciatus and may provide a daytime refuge from predators. A southerly range extension is also reported for this species.     

Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers

The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.     

Subunit-selective contribution to channel gating of the M4 domain of the nicotinic receptor

The muscle nicotinic receptor (AChR) is a pentamer of four different subunits, each of which contains four transmembrane domains (M1-M4). We recently showed that channel opening and closing rates of the AChR depend on a hydrogen bond involving a threonine at position 14' of the M4 domain in the alpha-subunit. To determine whether residues in equivalent positions in non-alpha-subunits contribute to channel gating, we mutated deltaT14', betaT14', and epsilonS14' and evaluated changes in the kinetics of acetylcholine-activated currents. The mutation epsilonS14'A profoundly slows the rate of channel closing, an effect opposite to that produced by mutation of alphaT14'. Unlike mutations of alphaT14', epsilonS14'A does not affect the rate of channel opening. Mutations in deltaT14' and betaT14' do not affect channel opening or closing kinetics, showing that conserved residues are not functionally equivalent in all subunits. Whereas alphaT14'A and epsilonS14'A subunits contribute additively to the closing rate, they contribute nonadditively to the opening rate. Substitution of residues preserving the hydrogen bonding ability at position 14' produce nearly normal gating kinetics. Thus, we identify subunit-specific contributions to channel gating of equivalent residues in M4 and elucidate the underlying mechanistic and structural bases.     

Allosteric modulators: the new generation of receptor antagonist

Allosteric antagonists modulate the affinity and/or efficacy of agonists for receptors. Although the manner in which this modulation can occur can mimic that of simple competitive antagonists, allosteric antagonists possess unique properties that can present seemingly capricious profiles of antagonism. These unique properties also offer potentially useful patterns for therapeutic utility. This review summarizes methods to detect allosteric antagonism and some special properties of these receptor modulators.     

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.     

Novel progesterone receptor modulators: 4-Aryl-phenylsulfonamides

We have developed a new series of progesterone receptor modulators based upon the 4-aryl-phenylsulfonamide. Initial work in the series afforded potent compounds with good properties, however an advanced intermediate proved to be genotoxic in a non-GLP Ames assay following metabolic activation. We subsequently solved this problem and identified advanced leads which demonstrated oral efficacy in rhesus monkey pharmacodynamic and kinetics models.     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Functional reconstitution of the glycine receptor

The functional reconstitution of the chloride channel coupled glycine receptor is described. Glycine receptors were purified from the cholate extract of rat spinal cord membranes by affinity chromatography and incorporated into phospholipid vesicles by the addition of phosphatidylcholine and removal of detergent by gel filtration. The reconstituted vesicles showed the same polypeptide composition as the purified receptor (proteins of Mr 48,000 and 58,000). The pharmacological characteristics of the glycine receptor were also preserved in the proteoliposomes, as demonstrated by the displacement of [3H]strychnine binding by several glycinergic ligands and by photoaffinity labeling experiments. In order to observe functional responses (i.e., specific agonist-induced anion translocation), we have developed an assay based on the fluorescence quenching of an anion-sensitive entrapped probe, SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium]. Reconstituted vesicles were loaded with the fluorescent probe during a freeze-thaw-sonication cycle in the presence of added liposomes containing cholesterol. In such a reconstituted system, glycine receptor agonists are able to increase the rate of anion influx into the vesicles. The action of agonists is blocked by the simultaneous presence of strychnine or other glycine antagonists. Our results show that the purified 48,000- and 58,000-dalton polypeptides reconstituted into phospholipid vesicles can bind ligands and promote specific ion translocation in a way similar to the glycine receptor in its native environment.     

Disorders of the inhibitory glycine receptor: the spastic mouse

The mutant mouse spastic suffers from a motor disorder of autosomal recessive inheritance which is characterized by tremor, myoclonic episodes, and a disturbed righting response. The most prominent alteration in the mutant is a substantial deficit of postsynaptic glycine receptor channels resulting in a dramatic reduction of glycinergic synaptic inhibition. Function and structure of the glycine receptor protein appear unaffected, which argues for a regulatory rather than a structural effect of the spastic mutation. It appears that other alterations in the spastic mouse are secondary to this fundamental disturbance in the balance of excitatory and inhibitory impulses. In particular, a significant increase in GABAA receptors of the lower parts of the CNS may serve a compensatory function, counteracting in part losses of glycinergic inhibition. Pharmacological experiments indeed show that facilitation of GABAA receptor-mediated inhibition alleviates symptoms of the spastic motor disorder. The recent cDNA cloning of glycine receptor subunits should help define the molecular mechanism by which the spastic gene causes the glycine receptor deficit.     

Lipid compounds of freshwater sponges: family Spongillidae,class Demospongiae

More than 100 novel, unusual and rare fatty acids, lipids and sterols have been isolated from freshwater sponges. The structures, biogenesis, synthesis and bioactivity of some lipid compounds of freshwater sponge species are reviewed.     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ? or α7 subunits. The replacement of M3 in α1 by ?M3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ? chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼ 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

CCN family of proteins: critical modulators of the tumor cell microenvironment

The CCN family of proteins consisting of CCN1 (Cyr61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3) are considered matricellular proteins operating essentially in the extracellular microenvironment between cells. Evidence has also been gradually building since their first discovery of additional intracellular roles although the major activity is triggered at the cell membrane. The proteins consist of 4 motifs, a signal peptide (for secretion} followed consecutively by the IGFBP, VWC, TSP1 and CT (C-terminal cysteine knot domain) motifs, which signify their potential binding partners and functional connections to a variety of key regulators of physiological processes. With respect to cancer it is now clear that, whereas certain members can facilitate tumor behavior and progression, others can competitively counter the process. It is therefore clear that the net outcome of biological interactions in the matrix and what gets signaled or inhibited can be a function of the interplay of these CCN 1–6 proteins. Because the CCN proteins further interact with other key proteins, like growth factors in the matrix, the balance is not only important but can vary dynamically with the physiological states of tumor cells and the surrounding normal cells. The tumor niche with its many cell players has surfaced as a critical determinant of tumor behavior, invasiveness, and metastasis. It is in this context that CCN proteins should be investigated with the potential of being recognized and validated for future therapeutic approaches.     

Age-related changes of glycine receptor at the rat hippocampus: from the embryo to the adult

Glycinergic inhibitory transmission has been described in spinal cord, but rather disregarded in the brain. The spatial-temporal characterization of glycine receptors (GlyR) in the hippocampus over development is herein reported. GlyR expression increases from late embryonic stage (E18) to 7 days postnatal (P7) and decreases from P7 on. Quantitative real-time PCR showed that GlyR subunit expression changes over neuronal maturation with a preponderance of α2 and α3, over α1 and β. In immature stages, GlyR delineate the cell body of neurons at the Dentate Gyrus and Cornus Ammonis 1 and 3 (CA1/CA3) and are composed of α2 and α3 subunits. At P7, synaptic GlyRα2β can already be observed in the dendritic areas of Dentate Gyrus and of CA1/CA3. In the mature hippocampus, synaptic GlyR decrease and, although a few synaptic GlyRα1β can still be detected in the dendritic layers, extrasynaptic α2/α3-containing GlyR and somatic localized GlyRα3 are the most abundant. Our results point towards an important function of a slow tonic activation of extrasynaptic GlyR, over a fast phasic activation of synaptic GlyRα1β. We clearly show that GlyR are widely expressed in hippocampus and that their subcellular localization and subunit composition change over development.     

What lies beneath: natural products from marine organisms as nuclear receptor modulators

The consequences for organism fitness of mutations in a given protein are often thought to be determined to a significant extent by epistasis, that is, by the fact that the effect of a mutation may be strongly dependent on the previous mutational background. Actually, a given mutation could be deleterious or beneficial depending on the background, a situation known as 'sign epistasis'. Under pervasive sign epistasis, many mutational trajectories towards a 'fitter protein' will show a 'dip' in fitness and, it has been previously suggested, only a few trajectories will be available to Darwinian selection. In this issue of the Biochemical Journal, Zhang et al. explore how this simple picture needs to be modified when two rather general and important features are taken into account, namely that many proteins are promiscuous and that living organisms must survive and thrive in environments that change continuously. The multidimensional nature of epistasis for a protein involved in several tasks, together with the fact that different tasks may become critical for organism survival as environmental conditions change, is shown by Zhang et al. to contribute to eliminating fitness dead-ends in protein sequence space. Consequently, many alternative mutational trajectories should allow protein optimization for enhanced organism fitness under changing environmental conditions.     

Sesquiterpenoids from Atractylodes macrocephala act as farnesoid X receptor and progesterone receptor modulators

Two sesquiterpenoids, atractylenolide II and III, were isolated and identified from Atractylodes macrocephala (Asteraceae) to be subsequently evaluated for their activity against farnesoid X receptor (FXR) and progesterone receptor (PR) by transient transfection reporter assays. These sesquiterpenoids did not exert significant agonistic effect but antagonized the activity of chenodeoxycholic acid (CDCA), an endogenous FXR agonist, for FXR driven SHP promoter transactivation. Additionally, they transactivated CYP7A1 gene promoter activity by antagonizing FXR. Apart from acting as a FXR antagonist, atractylenolide III also showed agonistic activity against PR. All these results demonstrated that atractylenolide II and III are the active components of Atractylodes macrocephala to exert specific pharmacologic effects.     

Genotoxicity of the some selective estrogen receptor modulators: a review

The objective of this article is to review genotoxicological profile of the major selective estrogen receptor modulators, including clomiphene, tamoxifen, toremifene, raloxifene. These drugs have been used for infertility treatment and breast cancer prevention in high risk-women. However, some studies reported that especially tamoxifen is a genotoxic agent and is related with endometrial cancer. Our review indicate that clomiphene and tamoxifen were found as genotoxic agent in majority of the tests. However published reports showed that toremifene is a weakly genotoxic agent. The genotoxic effects of raloxifene are still poorly known. Further genotoxicity studies should be conducted especially for raloxifene.