Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G_q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.     

The FHIT gene product: tumor suppressor and genome “caretaker”

The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit-negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial–mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome “caretaker.” Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability.     

Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.     

PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans

Ichthyoses comprise a heterogeneous group of genodermatoses characterized by abnormal desquamation over the whole body, for which the genetic causes of several human forms remain unknown. We used a spontaneous dog model in the golden retriever breed, which is affected by a lamellar ichthyosis resembling human autosomal recessive congenital ichthyoses (ARCI), to carry out a genome-wide association study. We identified a homozygous insertion-deletion (indel) mutation in PNPLA1 that leads to a premature stop codon in all affected golden retriever dogs. We subsequently found one missense and one nonsense mutation in the catalytic domain of human PNPLA1 in six individuals with ARCI from two families. Further experiments highlighted the importance of PNPLA1 in the formation of the epidermal lipid barrier. This study identifies a new gene involved in human ichthyoses and provides insights into the localization and function of this yet uncharacterized member of the PNPLA protein family.     

Razorback sucker movements and habitat use in the San Juan River inflow, Lake Powell, Utah, 1995-1997

Seventeen subadult, hatchery-reared razorback suckers ( Xyrauchen texanus ; ( ￣x = 456 mm total length) were implanted with sonic transmitters and tracked for 23 months in the lower 89.6 km of the San Juan River (San Juan arm of Lake Powell, Utah). Fish were released at 2 sites, and 9 made extensive up- and downstream movements ( ￣x = 47.8 km; contact was lost with 4, and 4 others presumably died or lost their transmitters). The San Juan arm is primarily inundated canyon; however, most fish contacts occurred in shallow coves and shoreline with thick stands of flooded salt cedar in the upper inflow area. Eight fish frequented the Piute Farms river/lake mixing zone, and at least 4 moved upstream into the San Juan River. Seven fish were found in 2 aggregations in spring (3 fish in Neskahi Bay in 1996 and 4 fish just downstream of Piute Farms in 1997), and these may have been associated with spawning activity. Continued presence of razorback suckers in the Piute Farms area and lower San Juan River suggests the San Juan inflow to Lake Powell could be used as an alternate stocking site for reintroduction efforts.     

Ecological opportunity and sexual selection together predict adaptive radiation

A fundamental challenge to our understanding of biodiversity is to explain why some groups of species undergo adaptive radiations, diversifying extensively into many and varied species, whereas others do not. Both extrinsic environmental factors (for example, resource availability, climate) and intrinsic lineage-specific traits (for example, behavioural or morphological traits, genetic architecture) influence diversification, but few studies have addressed how such factors interact. Radiations of cichlid fishes in the African Great Lakes provide some of the most dramatic cases of species diversification. However, most cichlid lineages in African lakes have not undergone adaptive radiations. Here we compile data on cichlid colonization and diversification in 46 African lakes, along with lake environmental features and information about the traits of colonizing cichlid lineages, to investigate why adaptive radiation does and does not occur. We find that extrinsic environmental factors related to ecological opportunity and intrinsic lineage-specific traits related to sexual selection both strongly influence whether cichlids radiate. Cichlids are more likely to radiate in deep lakes, in regions with more incident solar radiation and in lakes where there has been more time for diversification. Weak or negative associations between diversification and lake surface area indicate that cichlid speciation is not constrained by area, in contrast to diversification in many terrestrial taxa. Among the suite of intrinsic traits that we investigate, sexual dichromatism, a surrogate for the intensity of sexual selection, is consistently positively associated with diversification. Thus, for cichlids, it is the coincidence between ecological opportunity and sexual selection that best predicts whether adaptive radiation will occur. These findings suggest that adaptive radiation is predictable, but only when species traits and environmental factors are jointly considered.     

A method for distinguishing Tetrodotoxin from Saxitoxin,by comparing their relative stabilities when heated in acid solution

Resumen La tetrodotoxina pierde toxicidad rapidamente cuando se calienta en solución acida por debajo de pH 2, mientras que la saxitoxina es estable. Se puede distinguir facilmente entre estos venenos calentándolos a 100°C en solution pH 1.0 durante 20–30 min.

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This work was carried out whileC. J. Waterfield was on industrial training leave from Bath University of Technology. We wish to thank MissJ. Tulloch for her assistance.     

Modifications of female sex chromatin (Barr body) in rat neurons after reserpine administration

Summary 3 groups of rats were sacrificed 30 min, 4 h and 24 h after reserpine (10 mg/kg, i. p.) injection. Toluidine blue stained sections showed that in the motor neurons of the spinal cord the drug, at 4 h, had induced a migration of the Barr body from the nucleolus to the nuclear membrane and an increase in its size and RNA concentration. From our findings we suggest that reserpine may have an activating role on X-linked genes.Supported by grant 0230-135 of the National Research Foundation of Greece.     

Variation in FTO contributes to childhood obesity and severe adult obesity

We identified a set of SNPs in the first intron of the FTO (fat mass and obesity associated) gene on chromosome 16q12.2 that is consistently strongly associated with early-onset and severe obesity in both adults and children of European ancestry with an experiment-wise P value of 1.67 x 10(-26) in 2,900 affected individuals and 5,100 controls. The at-risk haplotype yields a proportion of attributable risk of 22% for common obesity. We conclude that FTO contributes to human obesity and hence may be a target for subsequent functional analyses.