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.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Feeding preference by the male blowfly Phormia regina for some natural foods and its relation to carbohydrate, amino acid, and alcohol content

The male blowfly, Phormia regina Meigen shows preference among a variety of natural and artificial foods. In a laboratory study preference was observed among sweet and fermenting juices as well as between liver and fish. The preferences between fresh and fermenting juices were based not only on component sugars, but also on other components, namely, amino acids, alcohol salts and presumably other chemical compounds. The preference between decaying liver and fish was probably based partly on the composition and concentration of amino acids in these foods. A variety of amino acids were found in foods and some of these amino acids were shown to be excitatory, some inhibitory and others neutral. Salts were also observed to be present in the juices; and in a laboratory study NaCl at lower concentrations acted synergestically with 0.1 M sucrose and at higher concentrations repulsive. It is suggested that feeding preference is based on several chemicals which behave either in an excitatory, additive, neutral, inhibitory or synergistic manner. The ratio of excitants to inhibitants is important in deciding the direction of preference.

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Résumé P. regina Meigen mâle préfere certains aliments naturels ou artificiels. Au laboratoire ses préférences parmi les jus sucrés sont les suivantes: orange > miel > pomme > sucrose 0.1 M; et parmi les aliments en décomposition: sucrose 0.1 M > foie > poisson. Les jus frais sont préférés aux jus fermentés. Les préférences ne dépendent pas seulement de la nature des sucres, mais aussi des acides aminés, de l'alcool, des sels et probablement d'autres substances chimiques. Le foie contient à la fois le plus grand nombre et la concentration la plus élevée en acides aminés, ce qui explique partiellement le goût pour le foie. Expérimentalement on peut classer les acides aminés en stimulants, inhibiteurs et neutres. Des sels ont été notés dans les jus, mais aucune analyse qualitative et quantitative n'en a été faite. Mélangé à du sucrose à 0.1 M, NaCl agit en synergie aux faibles concentrations, devient neutre à 0.175 M et à des concentrations supérieures le sucrose seul est préféré au mélange avec NaCl. Cependant, un mélange de NaCl à 0.25 M (bien qu'une telle concentration soit peu probable dans des jus végétaux) avec du sucrose à 0.1 M était préféré à l'eau. Il en est déduit que les préférences alimentaires. dans la nature sont orientées par plusieurs substances chimiques qui se comportent d'une façon stimulante, additive, neutre, inhibitrice ou synergique, et que les préférences alimentaires sont relatives. Le rapport de stimulants sur inhibiteurs est important dans la détermination du sens de la préférence.

     

In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

Biogeography of Iberian freshwater fishes revisited: the roles of historical versus contemporary constraints

Aim The question of how much of the shared geographical distribution of biota is due to environmental vs. historical constraints remains unanswered. The aim of this paper is to disentangle the contribution of historical vs. contemporary factors to the distribution of freshwater fish species. In addition, it illustrates how quantifying the contribution of each type of factor improves the classification of biogeographical provinces. Location Iberian Peninsula, south‐western Europe (c. 581,000 km²). Methods We used the most comprehensive data on native fish distributions for the Iberian Peninsula, compiled from Portuguese and Spanish sources on a 20‐km grid‐cell resolution. Overall, 58 species were analysed after being categorized into three groups according to their ability to disperse through saltwater: (1) species strictly intolerant of saltwater (primary species); (2) species partially tolerant of saltwater, making limited incursions into saltwaters (secondary species); and (3) saltwater‐tolerant species that migrate back and forth from sea to freshwaters or have invaded freshwaters recently (peripheral species). Distance‐based multivariate analyses were used to test the role of historical (basin formation) vs. contemporary environmental (climate) conditions in explaining current patterns of native fish assemblage composition. Cluster analyses were performed to explore species co‐occurrence patterns and redefine biogeographical provinces based on the distributions of fishes. Results River basin boundaries were better at segregating species composition for all species groups than contemporary climate variables. This historical signal was especially evident for primary and secondary freshwater fishes. Eleven biogeographical provinces were delineated. Basins flowing to the Atlantic Ocean north of the Tagus Basin and those flowing to the Mediterranean Sea north of the Mijares Basin were the most dissimilar group. Primary and secondary freshwater species had higher province fidelity than peripheral species. Main conclusions The results support the hypothesis that historical factors exert greater constraints on native freshwater fish assemblages in the Iberian Peninsula than do current environmental factors. After examining patterns of assemblage variation across space, as evidenced by the biogeographical provinces, we discuss the likely dispersal and speciation events that underlie these patterns.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

Prophylactic and curative effects of Bacopa monniera in gastric ulcer models.

Bacopa monniera Wettst. (BM, syn. Herpestis monniera L; Scrophulariaceae), is an Ayurvedic drug used as a rasayana. Its fresh juice was earlier reported to have significant antiulcerogenic activity. In continuation, methanolic extract of BM (BME) standardized to bacoside-A content (percentage-38.0 ± 0.9), when given in the dose of 10–50 mg/kg, twice daily for 5 days, showed dose-dependent anti-ulcerogenic on various gastric ulcer models induced by ethanol, aspirin, 2 h cold restraint stress and 4 h pylorus ligation. BME in the dose of 20 mg/kg, given for 10 days, twice daily showed healing effects against 50% acetic acid-induced gastric ulcers. Further work was done to investigate the possible mechanisms of its action by studying its effect on various mucosal offensive acid-pepsin secretion and defensive factors like mucin secretion, mucosal cell shedding, cell proliferation and antioxidant activity in rats. BME 20 mg/kg showed no effect on acid-pepsin secretion, increased mucin secretion, while it decreased cell shedding with no effect on cell proliferation. BME showed significant antioxidant effect per se and in stressed animals. Thus, the gastric prophylactic and curative effects of BME may be due to its predominant effect on mucosal defensive factors.     

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996     

Gradients in maize roots: local elongation and pH

The elongation rate, the gradient of the local elongation rate and the surface pH of maize roots were measured over 12 h. A data bank was constituted by storing these values. By sorting these results on the basis of different elongation rates, different classes of root were obtained. Two classes were chosen: the low-growth roots and the high-growth roots. The mean growth of these two root classes was stable with time and differed significantly from one another. The surface pH of the elongation zone was the same for the roots of these two classes, but the roots selected for their higher growth rate had a larger acid efflux in this zone.