Protein-tyrosine phosphorylation in Bacillus subtilis

In recent years bacterial protein-tyrosine kinases have been found to phosphorylate a growing number of protein substrates, including RNA polymerase sigma factors, UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. The activity of these protein substrates was affected by tyrosine phosphorylation, indicating that this post-translational modification could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this field was done in Bacillus subtilis, and we here present the current state of knowledge on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specificity and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology.     

The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae)

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8× depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373 bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,657 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperzia chloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophyte chloroplast genome data also enable a better reconstruction of the basal tracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, inferred amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.     

Perinatal asphyxia pathophysiology in pig and human: a review

In utero fetuses are evidently exposed to several factors that cause an interruption of the oxygen flow through the umbilical cord causing asphyxia leading to hypoxia and metabolic acidosis. These conditions are important causes of intra-partum and neonatal mortality. The main objective of this review is to provide current information regarding the pathophysiology of asphyxia in piglets around parturition; the physiological mechanisms invoked by affected piglets to compensate perinatal hypoxemia are discussed. This review also addresses some similarities and differences of asphyxia between piglets and other mammals, including human neonates. Metabolic acidosis and hypoxia are sequela to asphyxia and can cause profound health effects in postnatal performance because of an abnormal suckling, a reduced absorption of colostrum and inadequate passive transfer of neonatal immunity. Acidosis also cause hypothermia, increased mortality and reduced survival in neonates. One of the first deleterious effects of intrauterine hypoxia is the expulsion of meconium into the amniotic sac leading to meconium staining of the skin, and in severe cases, meconium aspiration into the lungs. Even though there have been technological changes and improvements in husbandry, piglet mortality due to asphyxia remains a major problem. One potential alternative to reduce neonatal mortality in pigs is the monitoring of fetal stress during birth and the implemention of strategies such as the Apgar score, that is often used in human pediatrics. It is also important to consider the physiological, behavioral and biochemical changes that take place during parturition which subsequently impact the vitality, maturity and development of neonatal pigs. Understanding the pathophysiology of fetal hypoxia should help practitioners and farmers implement more effective delivery techniques aimed at reducing neonatal mortality and improving postnatal performance.     

Adaptations required for mitochondrial import following mitochondrial to nucleus gene transfer of ribosomal protein S10

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphipathic alpha-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal.     

The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease

Ho endonuclease initiates a mating type switch by making a double-strand break at the mating type locus, MAT. Ho is marked by phosphorylation for rapid destruction by functions of the DNA damage response, MEC1, RAD9, and CHK1. Phosphorylated Ho is recruited for ubiquitylation via the SCF ubiquitin ligase complex by the F-box protein, Ufo1. Here we identify a further DNA damage-inducible protein, the UbL-UbA protein Ddi1, specifically required for Ho degradation. Ho interacts only with Ddi1; it does not interact with the other UbL-UbA proteins, Rad23 or Dsk2. Ho must be ubiquitylated to interact with Ddi1, and there is no interaction when Ho is produced in mec1 or Deltaufo1 mutants that do not support its degradation. Ddi1 binds the proteasome via its N-terminal ubiquitin-like domain (UbL) and interacts with ubiquitylated Ho via its ubiquitin-associated domain (UbA); both domains of Ddi1 are required for association of ubiquitylated Ho with the proteasome. Despite being a nuclear protein, Ho is exported to the cytoplasm for degradation. In the absence of Ddi1, ubiquitylated Ho is stabilized and accumulates in the cytoplasm. These results establish a role for Ddi1 in the degradation of a natural ubiquitylated substrate. The specific interaction between Ho and Ddi1 identifies an additional function associated with DNA damage involved in its degradation.     

Branched aminoglycosides: biochemical studies and antibacterial activity of neomycin B derivatives

The C5'-OH group in neomycin B was glycosylated with a variety of mono- and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity and interaction with the aminoglycosides modifying enzyme APH(3')-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3')-IIIa.     

Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.     

Structure-based mutational analyses in FGF7 identify new residues involved in specific interaction with FGFR2IIIb

Receptor binding specificity is an essential element in regulating the diverse activities of fibroblast growth factors (FGFs). FGF7 is ideal to study how this specificity is conferred at the structural level, as it interacts exclusively with one isoform of the FGF-receptor (FGFR) family, known as FGFR2IIIb. Previous mutational analysis suggested the importance of the beta4/beta5 loop of FGF7 in specific receptor recognition. Here a theoretical model of FGFR2IIIb/FGF7 complex showed that this loop interacts with the FGFR2IIIb unique exon. In addition, the model revealed new residues that either directly interact with the FGFR2IIIb unique exon (Asp63, Leu142) or facilitate this interaction (Arg65). Mutations in these residues reduced both receptor binding affinity and biological activity of FGF7. Altogether, these results provide the basis for understanding how receptor-binding specificity of FGF7 is conferred at the structural level.