Role of cleavage and shedding in human thyrotropin receptor function and trafficking.

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.     

Indigenous Peoples’ Interest in Wildlife-Based Enterprises in the Northern Territory,Australia

Despite high levels of poverty in Indigenous Australian communities, workforce participation is low for the few jobs available. Our research showed that, in contrast, there was a high level of interest in involvement in wildlife-based industries. Young men were particularly interested in animal-based industries whereas involvement in plant-based industries was more likely among middle-aged people of both sexes. People who had employment as land and sea managers (‘rangers’) were more likely than others to express interest in enterprise involvement. Importantly the level and type of interest differed between communities, reflecting differences in history and culture. The results, which are the first documentation of a quantitative analysis of Australian Indigenous peoples’ interest in wildlife-based enterprises, suggest that this is considered far more desirable than involvement in other types of work that might be available. It also suggests that any programmes facilitating such enterprises need to be tailored to the community for which they are being designed.     

Magnetic resonance and confocal imaging of solute penetration into the lens reveals a zone of restricted extracellular space diffusion

It has been proposed that in the absence of blood supply, the ocular lens operates an internal microcirculation system that delivers nutrients to internalized fiber cells faster and more efficiently than would occur by passive diffusion alone. To visualize the extracellular space solute fluxes potentially generated by this system, bovine lenses were organ cultured in artificial aqueous humor (AAH) for 4 h in the presence or absence of two gadolinium-based contrast agents, ionic Gd(3+), or a chelated form of Gd(3+), Gd-diethylenetriamine penta-acetic acid (Gd-DTPA; mol mass = 590 Da). Contrast reagent penetration into the lens core was monitored in real time using inversion recovery-spin echo (IR-SE) magnetic resonance imaging (MRI), while steady-state accumulation of [Gd-DTPA](-2) was also determined by calculating T1 values. After incubation, lenses were fixed and cryosectioned, and sections were labeled with the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal microscopy using standard and reflectance imaging modalities to visualize the fluorescent WGA label and gadolinium reagents, respectively. Real-time IR-SE MRI showed rapid penetration of Gd(3+) into the outer cortex of the lens and a subsequent bloom of signal in the core. These two areas of signal were separated by an area in the inner cortex that limited entry of Gd(3+). Similar results were obtained for Gd-DTPA, but the penetration of the larger negatively charged molecule into the core could only be detected by calculating T1 values. The presence of Gd-DTPA in the extracellular space of the outer cortex and core, but its apparent absence from the inner cortex was confirmed using reflectance imaging of equatorial sections. In axial sections, Gd-DTPA was associated with the sutures, suggesting these structures provide a pathway from the surface, across the inner cortex barrier to the lens core. Our studies have revealed inner and outer boundaries of a zone within which a narrowing of the extracellular space restricts solute diffusion and acts to direct fluxes into the lens core via the sutures.     

Functional genomics: identifying drug targets for parasitic diseases

The genomic sequences of parasitic diseases are rapidly becoming available and, recently, the full sequence of Plasmodium falciparum has been published. Much has been promised from this genomic revolution including the identification of new drug targets and novel chemotherapeutic treatments for the control of parasitic diseases. The challenge to use this information efficiently will require functional genomics tools such as bioinformatics, microarrays, proteomics and chemical genomics to identify potential drug targets, and to allow the development of optimized lead compounds. The information generated from these tools will provide a crucial link from genomic analysis to drug discovery.     

Human Somatostatin Receptor Genes: Localization of SSTR5 to Human Chromosome 20p11.2

The gene encoding the somatostatin receptor subtype designated as SSTR5 was mapped to human chromosome 20p11.2 by using fluorescence in situ hybridization to metaphase chromosomes. Fluorescence in situ hybridization using a probe for SSTR5 in combination with probes for neuroendocrine convertase-2 (NEC2), thrombomodulin (THBD), and brain glycogen phosphorylase (PYGB) established a physical order for these loci of 20pter-NEC2-SSTR5-THBD-PYGB-cen.     

A Community-Based Study of Factors Associated with Continuing Transmission of Lymphatic Filariasis in Leogane,Haiti

Seven rounds of mass drug administration (MDA) have been administered in Leogane, Haiti, an area hyperendemic for lymphatic filariasis (LF). Sentinel site surveys showed that the prevalence of microfilaremia was reduced to <1% from levels as high as 15.5%, suggesting that transmission had been reduced. A separate 30-cluster survey of 2- to 4-year-old children was conducted to determine if MDA interrupted transmission. Antigen and antifilarial antibody prevalence were 14.3% and 19.7%, respectively. Follow-up surveys were done in 6 villages, including those selected for the cluster survey, to assess risk factors related to continued LF transmission and to pinpoint hotspots of transmission. One hundred houses were mapped in each village using GPS-enabled PDAs, and then 30 houses and 10 alternates were chosen for testing. All individuals in selected houses were asked to participate in a short survey about participation in MDA, history of residence in Leogane and general knowledge of LF. Survey teams returned to the houses at night to collect blood for antigen testing, microfilaremia and Bm14 antibody testing and collected mosquitoes from these communities in parallel. Antigen prevalence was highly variable among the 6 villages, with the highest being 38.2% (Dampus) and the lowest being 2.9% (Corail Lemaire); overall antigen prevalence was 18.5%. Initial cluster surveys of 2- to 4-year-old children were not related to community antigen prevalence. Nearest neighbor analysis found evidence of clustering of infection suggesting that LF infection was focal in distribution. Antigen prevalence among individuals who were systematically noncompliant with the MDAs, i.e. they had never participated, was significantly higher than among compliant individuals (p<0.05). A logistic regression model found that of the factors examined for association with infection, only noncompliance was significantly associated with infection. Thus, continuing transmission of LF seems to be linked to rates of systematic noncompliance.     

Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA.

We determined previously that the selectable marker pSV2neo is preferentially inserted into chromosomal fragile sites in human x hamster hybrid cells in the presence of an agent (aphidicolin) that induces fragile-site expression. In contrast, cells transfected without fragile-site induction showed an essentially random integration pattern. To determine whether the integration of marker DNA at fragile sites affects the frequency of fragile-site expression, the parental hybrid and three transfectants (two with pSV2neo integrated at the fragile site at 3p14.2 [FRA3B] and specific hamster fragile sites [chromosome 1, bands q26-31, or mar2, bands q11-13] and one with pSV2neo integrated at sites that are not fragile sites) were treated with aphidicolin. After 24 h the two cell lines with plasmid integration at FRA3B showed structural rearrangements at that site; these rearrangements accounted for 43%-67% of the total deletions and translocations observed. Structural rearrangements were not observed in the parental cell line. After 5 d aphidicolin treatment, the observed excess in frequency of structural rearrangements at FRA3B in the cell lines with pSV2neo integration at 3p14 over that in the two lines without FRA3B integration was less dramatic, but nonetheless significant. Fluorescent in situ hybridization (FISH) analysis of these cells, using a biotin-labeled pSV2neo probe, showed results consistent with the gross rearrangements detected cytogenetically in the lines with FRA3B integration; however, the pSV2neo sequences were frequently deleted concomitantly with translocations.(ABSTRACT TRUNCATED AT 250 WORDS)