hfAIM: A reliable bioinformatics approach for in silico genome-wide identification of autophagy-associated Atg8-interacting motifs in various organisms

Most of the proteins that are specifically turned over by selective autophagy are recognized by the presence of short Atg8 interacting motifs (AIMs) that facilitate their association with the autophagy apparatus. Such AIMs can be identified by bioinformatics methods based on their defined degenerate consensus F/W/Y-X-X-L/I/V sequences in which X represents any amino acid. Achieving reliability and/or fidelity of the prediction of such AIMs on a genome-wide scale represents a major challenge. Here, we present a bioinformatics approach, high fidelity AIM (hfAIM), which uses additional sequence requirements—the presence of acidic amino acids and the absence of positively charged amino acids in certain positions—to reliably identify AIMs in proteins. We demonstrate that the use of the hfAIM method allows for in silico high fidelity prediction of AIMs in AIM-containing proteins (ACPs) on a genome-wide scale in various organisms. Furthermore, by using hfAIM to identify putative AIMs in the Arabidopsis proteome, we illustrate a potential contribution of selective autophagy to various biological processes. More specifically, we identified 9 peroxisomal PEX proteins that contain hfAIM motifs, among which AtPEX1, AtPEX6 and AtPEX10 possess evolutionary-conserved AIMs. Bimolecular fluorescence complementation (BiFC) results verified that AtPEX6 and AtPEX10 indeed interact with Atg8 in planta. In addition, we show that mutations occurring within or nearby hfAIMs in PEX1, PEX6 and PEX10 caused defects in the growth and development of various organisms. Taken together, the above results suggest that the hfAIM tool can be used to effectively perform genome-wide in silico screens of proteins that are potentially regulated by selective autophagy. The hfAIM system is a web tool that can be accessed at link: http://bioinformatics.psb.ugent.be/hfAIM/.     

Relationships between foliage and conducting xylem in Picea abies (L.) Karst.

Summary The relationship of leaf biomass and leaf area to the conductive area of stems and branches was investigated in Picea abies. A total of 30 trees were harvested to determine if these relationships were different in different crown zones and in trees growing with and without competition for light. Two methods were compared. In the first, data were accumulated from crown zones situated at the top of trees to the bottom; in the second, data were used from individual crown zones. The results indicated that the latter method is much more sensitive in detecting differences in the relationship of leaf biomass or leaf area to conductive area. The analysis also indicated that ratios such as leaf area/sapwood area are frequently size-dependent. This size-dependency can in some cases result in the differences being abscured, but more often leads to the false impression that the relationship between the variables changes. The relationship between leaf biomass and leaf area and conductive area of stems or branches was different in different crown zones and under different growth conditions. The slopes of these regressions appear to increase with decreasing transpirational demand and decrease with increasing hydraulic conductivity. The intercepts are probably related to the amount of identified sapwood actually involved in water conductance.     

Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.     

Tolerance of extremely halophilic archaebacteria towards bromide

The tolerance of halophilic archaebacteria towards bromide was tested in view of the fact that bromide occurs in natural brines in concentrations of up to 66 mM. It was found that, while concentrations of up to 0.8–1M are tolerated well by all halobacterial types examined, great differences exist between species with respect to bromide tolerance. WhileHalobacterium (H. salinarium, H. halobium, andH. sodomense) andNatronobacterium species are only moderately tolerant,Haloarcula (H. vallismortis, H. marismortui), andHaloferax species (H. mediterranei, H. gibbonsii) tolerate higher concentrations.Haloferax volcanii proved extremely tolerant and showed growth in bromide media at very low chloride concentrations (below 50 mM). No correlation was found between bromide tolerance and the bromide concentration in the habitat from which the strains were isolated. Iodide proved much more toxic than bromide. Bromide-tolerant strains also proved relatively resistant to growth inhibition by iodide.     

Co-operation between the p53 protein tumor antigen and platelet-poor plasma in the induction of cellular DNA synthesis

Plasmids containing DNA sequences coding for p53 were microinjected into quiescent Swiss 3T3 cells. Three constructs were used, carrying either the whole gene sequence, a full-length cDNA, or a hybrid between the gene and the cDNA. All of them stimulated DNA synthesis when cells were incubated with platelet-poor plasma (PPP) following injection. The p53 gene stimulated DNA synthesis to a lesser extent, also in the absence of PPP. Several negative results were obtained with different plasmids, including deletion mutants in the p53 coding region. However, a deletion mutant in which the p53 reading frame ended in the middle of the coding part of the p53 gene still stimulated DNA synthesis in co-operation with PPP. The stimulation of DNA synthesis induced by p53 cDNA was more synchronous and more limited than that induced by serum. The present data suggest that p53 may act as a competence factor in cell cycle progression.     

Reclassification of <Emphasis Type="Italic">Halomonas caseinilytica</Emphasis> Wu et al. 2008 as a later synonym of <Emphasis Type="Italic">Halomonas sinaiensis</Emphasis>—Comments on the proposal by Hwang et al., Antonie van Leeuwenhoek 109:1345–1352, 2016

Hwang et al. (Antonie van Leeuwenhoek 109:1345–1352, 2016) proposed the reclassification of Halomonas caseinilytica (Wu et al. 2008) as a later synonym of Halomonas sinaiensis, based on the publication of the latter name in 2007 by Romano et al. However, the name H. sinaiensis was validly published only in 2011. Therefore the proposal by Hwang et al. is not appropriate; instead, the name H. sinaiensis can be proposed as a later synonym of H. casinilytica.     

Human and porcine early kidney precursors as a new source for transplantation

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.     

Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.