A glycoprotein hormone expressed in corticotrophs exhibits unique binding properties on thyroid-stimulating hormone receptor

Corticotroph-derived glycoprotein hormone (CGH), also referred to as thyrostimulin, is a noncovalent heterodimer of glycoprotein hormone alpha 2 (GPHA2) and glycoprotein hormone beta 5 (GPHB5). Here, we demonstrate that both subunits of CGH are expressed in the corticotroph cells of the human anterior pituitary, as well as in skin, retina, and testis. CGH activates the TSH receptor (TSHR); (125)I-CGH binding to cells expressing TSHR is saturable, specific, and of high affinity. In competition studies, unlabeled CGH is a potent competitor for (125)I-TSH binding, whereas unlabeled TSH does not compete for (125)I-CGH binding. Binding and competition analyses are consistent with the presence of two binding sites on the TSHR transfected baby hamster kidney cells, one that can interact with either TSH or CGH, and another that binds CGH alone. Transgenic overexpression of GPHB5 in mice produces elevations in serum T(4) levels, reductions in body weight, and proptosis. However, neither transgenic overexpression of GPHA2 nor deletion of GPHB5 produces an overt phenotype in mice. In vivo administration of CGH to mice produces a dose-dependent hyperthyroid phenotype including elevation of T(4) and hypertrophy of cells within the inner adrenal cortex. However, the distinctive expression patterns and binding characteristics of CGH suggest that it has endogenous biological roles that are discrete from those of TSH.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.     

The difference is in the start: impact of timing and start procedure on sprint running performance

The difference is in the start: impact of timing and start procedure on sprint running performance. The purpose of this study was to compare different sprint start positions and to generate correction factors between popular timing triggering methods on 40-m/40-yd sprint time. Fourteen female athletes (17 ± 1 years), personal best 100 m: 13.26 (±0.68) seconds and 11 male athletes (20 ± 5 years), personal best 100 m: 11.58 (±0.74) seconds participated. They performed 2 series of 3 40-m sprints in randomized order: (a) start from the block, measured by means of Brower audio sensor (BAS) and Dartfish video timing (DVT), (b) 3-point start, measured by using hand release pod (HR) and DVT, and (c) standing start, triggered by both photocell across starting line (SFC), and foot release (FR) plus DVT. Video analysis was performed by 2 independent observers and averaged. Simultaneous measurements at national athletics competitions demonstrated that DVT and BAS were equivalent to Omega Timing within the limits of precision of video timing (±0.01 seconds). Hand and floor timer triggering showed small but significant biases compared with movement captured from video (0.02-0.04 seconds), presumably because of sensitivity of pressure thresholds. Coefficient of variation for test-retest timing using different starting positions ranged from 0.7 to 1.0%. Compared with block starts reacting to gunfire, HR, SFC, and FR starts yielded 0.17 ± 0.09, 0.27 ± 0.12, and 0.69 ± 0.11 second faster times, respectively, over 40 m (all p < 0.001) because of inclusion or exclusion of reaction time, plus momentum, and body position differences at trigger moment. Correction factors for the conversion of 40 m/40 yd and 40 yd/40 m were 0.92 and 1.08, respectively. The correction factors obtained from this study may facilitate more meaningful comparisons of published sprint performances.     

The evolution of alternative developmental pathways: footprints of selection on life-history traits in a butterfly

Developmental pathways may evolve to optimize alternative phenotypes across environments. However, the maintenance of such adaptive plasticity under relaxed selection has received little study. We compare the expression of life-history traits across two developmental pathways in two populations of the butterfly Pararge aegeria where both populations express a diapause pathway but one never expresses direct development in nature. In the population with ongoing selection on both pathways, the difference between pathways in development time and growth rate was larger, whereas the difference in body size was smaller compared with the population experiencing relaxed selection on one pathway. This indicates that relaxed selection on the direct pathway has allowed life-history traits to drift towards values associated with lower fitness when following this pathway. Relaxed selection on direct development was also associated with a higher degree of genetic variation for protandry expressed as within-family sexual dimorphism in growth rate. Genetic correlations for larval growth rate across sexes and pathways were generally positive, with the notable exception of correlation estimates that involved directly developing males of the population that experienced relaxed selection on this pathway. We conclude that relaxed selection on one developmental pathway appears to have partly disrupted the developmental regulation of life-history trait expression. This in turn suggests that ongoing selection may be responsible for maintaining adaptive developmental regulation along alternative developmental pathways in these populations.     

Mutational Analysis of the Class IIa Bacteriocin Curvacin A and Its Orientation in Target Cell Membranes

To analyze the orientation in target cell membranes of the pediocin-like bacteriocin (antimicrobial peptide) curvacin A, 55 variants were generated by site-directed mutagenesis and their potencies against four different target cells determined. The result suggest that the somewhat hydrophilic short central helix (residues 19 to 24), along with the N-terminal β-sheet-like structure (residues 1 to 16), inserts in the interface region of the target cell membrane, with Ala22 close to the hydrophobic core of the membrane. The following hinge region, with Gly28 as an important residue, may then form a turn wherein Gly28 becomes positioned near the border between the interface and the hydrophobic regions, thus permitting the longer and more-hydrophobic C-terminal helix (residues 29 to 41) to insert into the hydrophobic core of the membrane. This helix contains three glycine residues (G33, G37, and G40) that form a putative helix-helix-interacting GxxxGxxG motif. The replacement of any of these glycines with a larger residue was very detrimental, suggesting their possible involvement in helix-helix interactions with a membrane-embedded receptor protein.     

Characterization of the self-splicing products of two complex Naegleria LSU rDNA group I introns containing homing endonuclease genes.

The two group I introns Nae.L1926 and Nmo.L2563, found at two different sites in nuclear LSU rRNA genes of Naegleria amoebo-flagellates, have been characterized in vitro. Their structural organization is related to that of the mobile Physarum intron Ppo.L1925 (PpLSU3) with ORFs extending the L1-loop of a typical group IC1 ribozyme. Nae.L1926, Nmo.L2563 and Ppo.L1925 RNAs all self-splice in vitro, generating ligated exons and full-length intron circles as well as internal processed excised intron RNAs. Formation of full-length intron circles is found to be a general feature in RNA processing of ORF-containing nuclear group I introns. Both Naegleria LSU rDNA introns contain a conserved polyadenylation signal at exactly the same position in the 3' end of the ORFs close to the internal processing sites, indicating an RNA polymerase II-like expression pathway of intron proteins in vivo. The intron proteins I-NaeI and I-NmoI encoded by Nae.L1926 and Nmo.L2563, respectively, correspond to His-Cys homing endonucleases of 148 and 175 amino acids. I-NaeI contains an additional sequence motif homologous to the unusual DNA binding motif of three antiparallel beta sheets found in the I-PpoI endonuclease, the product of the Ppo.L1925 intron ORF.