Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Evidence for the presence of lipopolysaccharide in Treponema phagedenis (biotype Reiterii) but not in Treponema pallidum (Nichols)

Abstract Immunoblotting profiles of whole or protease-K-digested organisms with homologous antisera demonstrated the presence of a characteristic ladder pattern of smooth LPS in Treponema phagedenis . Periodic acid silver staining of SDS-PAGE gels confirmed these findings. However, when heterologous or homologous serum was reacted with Treponema pallidum , no such pattern or cross-reactions were observed. The significance of apparent absence of LPS in T. pallidum is discussed.     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

The rise of coastal Middle Bronze Age Levant – A multidisciplinary approach for investigating in Sidon,Lebanon

Objectives

The Levantine Middle Bronze Age (MBA, circa 2000–1500 BCE) marks a period of increased trade and regional interaction, spurred on by technological developments. In light of previous research exhibiting limited mobility in Sidon, further investigation was conducted using biodistance analysis to understand local population history and site development.

Materials and Methods

Dental nonmetric traits, a proxy for genetic information, were explored using ASUDAS on a sub-sample of primary inhumations (n = 35). The biodistance matrix was generated using Gower distance measures, and further tested using PERMDISP, PERMANOVA, Mantel test and hierarchical cluster analysis. The data was also contrasted to ⁸⁷Sr/⁸⁶Sr and δ¹⁸O as well as δ¹³C and δ¹⁵N values.

Results

There were no significant diachronic differences in isotopes values, and there was biological continuity (n = 35, Mantel test r = 0.11, p = 0.02, comparing local phases and biodistance). The analysis also suggested of a sub-group of individuals with biological proximity shared a more limited range of mobility and dietary habits.

Conclusions

The isotopes (⁸⁷Sr/⁸⁶Sr, δ¹⁸O, δ¹³C, δ¹⁵N) and biodistance analysis conducted on the Sidon College site skeletal assemblage exhibits stability and continuity of the people, despite the site's increasing role in the maritime network. This continuity may have been a key factor in Sidon's success, allowing it to accumulate wealth and resources for centuries to come.

     

Body size increase in insular rodent populations: a role for predators?

Insular mammalian populations living in areas of small size are often characterized by a drastic change in body mass compared to related continental populations or species. Generally, small mammals (less than 100 g) evolve into giant forms while large mammals (up to 100 g) evolve into dwarf forms. These changes, coupled with changes in other life, behavioural, physiological or demographic traits are referred to generally as the insular syndrome. We tested in this study the relative contribution of three factors — area of island, numbers of competitor species and number of predator species — to changes in body size of the woodmouse (Apodemus sylvaticus) in the Western Mediterranean Sea. Our results, based on a comparative analysis using the phylogenetic independent contrasts method, indicate that the increase in body size is related both to the decrease of island size and to the lower number of predator species. A decrease of competitor species does not seem to have an important effect.     

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis

The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.     

Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis

Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis. Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined. The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis. An unexpectedly high cross-reactivity was revealed between the EF-Tu of B. subtilis and the antiserum against the EF-Tu of E. coli. A comparison of the predicted amino acid sequences from the tuf-genes of E. coli and B. subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate - MCF microcomplement fixation - T type strain     

Uplift of the roof of africa and its bearing on the evolution of mankind

Evidence concerning the geomorphological evolution of the Western Rift Valley, sedimentation within the valley and comparison of the fossil mammalian faunas of Western Uganda and East Africa indicate that the mountain ranges which now flank the Western Rift were uplifted in three or more stages beginning during the upper Miocene and that they reached climatically important altitudes during the upper Pliocene, at which time they began to modify regional climatic patterns in East Africa. Their main effect was the xerification of conditions over much of the region east of the mountains. The regional climatic effects due to the mountain ranges were themselves modified by global climatic changes related to the onset of the Glacial Period, the two phenomena combining to yield the Present day climatic regime of East Africa. As the climate changed, so did the flora and fauna. Faunal response was of three main kinds: a) dispersal into East Africa of pre-existing forms already adapted to more xeric conditions (many bovids, some cercopithecids), b) autochthonous evolution of forms adapted to mesic environments into forms adapted to more xeric conditions (suids, elephantids, some bovids, hominids), c) displacement of species ranges of those lineages unable to adapt to changing conditions (i.e. local extinctions) (Anancus, Brachypotherium). Autochthonous evolvers, including hominids, adopted two main strategies reflected in their hard anatomy: a) dietary shift (suids, proboscideans, bovids and later Pliocene hominids) and b) locomotor changes (early Pliocene hominids).