Endogenous thyrotropin-releasing hormone in the anterior pituitary: sites of activity as identified by immunocytochemical staining.

Thyrotropin-releasing hormone (TRH) immunoreactivity was localized in the rat anterior pituitary with rabbit anti-TRH sera and the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. Stain was present in secretory granules of cells possessing morphological characteristics of thyrotropes, gonadotropes and lactotropes. Antibody absorption studies with anti-TRH sera absorbed with TRH, 3 diastereoisomeric analogues of TRH, gonadotropin-releasing hormone (GnRH), bovine serum albumin, thyrotropin, prolactin, adrenocorticotropin, luteinizing hormone, follicle stimulating hormone were performed to determine the specificity of the staining reaction. Only absorption with TRH resulted in a significant reduction in staining intensity. In vitro experiments were then begun with hemipituitaries to ascertain if intrapituitary TRH might originate by sequestration of exogenous, plasma membrane bound TRH or by de novo synthesis. The results suggest that anterior pituitary TRH is of endogenous origin.     

Population trends of large non-migratory wild herbivores and livestock in the Masai Mara ecosystem, Kenya, between 1977 and 1997

The total of all non-migratory wildlife species in the Masai Mara ecosystem has declined by 58% in the last 20 years. This decline ranges from 49% in small brown antelopes to 72% in medium brown antelopes. In individual wildlife species, the decline ranges from 52% in Grant's gazelle to 88% in the warthog. Declines of over 70% have been recorded in buffalo, giraffe, eland and waterbuck. Only elephant, impala and ostrich have not shown any significant decline or increase. Overall, there has not been any significant difference in decline of all wildlife population sizes inside and outside the reserve, except for Thomson's gazelle and warthog. Livestock have not significantly declined over the entire analysis period. However, livestock and cattle populations significantly declined during the 1983–88 period. Donkey declined by 67%, while shoats (goats and sheep) remained stable. In the case of wildlife, land use and vegetation changes, drought effects and poaching are considered to be among the potential factors that may have been responsible for the decline; the decline in livestock during the 1983–84 period was probably due to drought effects.     

The interplay of supercoiling and thymine dimers in DNA

Thymine dimers are a major mutagenic photoproduct induced by UV radiation. While they have been the subject of extensive theoretical and experimental investigations, questions of how DNA supercoiling affects local defect properties, or, conversely, how the presence of such defects changes global supercoiled structure, are largely unexplored. Here, we introduce a model of thymine dimers in the oxDNA forcefield, parametrized by comparison to melting experiments and structural measurements of the thymine dimer induced bend angle. We performed extensive molecular dynamics simulations of double-stranded DNA as a function of external twist and force. Compared to undamaged DNA, the presence of a thymine dimer lowers the supercoiling densities at which plectonemes and bubbles occur. For biologically relevant supercoiling densities and forces, thymine dimers can preferentially segregate to the tips of the plectonemes, where they enhance the probability of a localized tip-bubble. This mechanism increases the probability of highly bent and denatured states at the thymine dimer site, which may facilitate repair enzyme binding. Thymine dimer-induced tip-bubbles also pin plectonemes, which may help repair enzymes to locate damage. We hypothesize that the interplay of supercoiling and local defects plays an important role for a wider set of DNA damage repair systems.     

Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices

A single serine point mutation (S374A) in the adenosine A_2A receptor (A_2AR) C-terminal tail reduces A_2AR-D₂R heteromerization and prevents its allosteric modulation of the dopamine D₂ receptor (D₂R). By means of site directed mutagenesis of the A_2AR and synthetic transmembrane (TM) α-helix peptides of the D₂R we further explored the role of electrostatic interactions and TM helix interactions of the A_2AR-D₂R heteromer interface. We found evidence that the TM domains IV and V of the D₂R play a major role in the A_2AR-D₂R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A_2AR and D₂R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A_2AR-D₂R heteromers. The mutation of two negatively charged aspartates in the A_2AR C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A_2AR-D₂R interaction and lost the ability of antagonistic allosteric modulation over the A_2AR-D₂R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A_2AR and the intracellular loop 3 (IL3) of D₂R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A_2AR-D_2LR heteromer interface together with D_2LR TM segments IV/V interacting with A_2AR TM-IV/V or TM-I/VII.     

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET² signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser–Ser–Ser) and YGS (Tyr–Gly–Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.     

Dopamine D2 and D4 receptor heteromerization and its allosteric receptor-receptor interactions

Dopamine D₂ and D₄ receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D₂R–D_4.xR (D_4.2R, D_4.4R or D_4.7R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET¹ techniques with the D₂R and D_4.7R receptors being the least effective in forming heteromers. Allosteric receptor–receptor interactions in D₂R–D_4.2R and D₂R–D_4.4 R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor–receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor–receptor interaction interfaces.     

Dopamine D4 receptor oligomerization--contribution to receptor biogenesis

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.