L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Lateral gene transfer in Salmonella

Comparative genomics and microarrays reveal that the genomes of different Salmonella enterica serovars are distinguished from each other by the presence or absence of hundreds of genes. The distribution of these variable genome regions is often not clonal. Therefore, lateral gene transfer (LGT) plays an important role in diversity among Salmonella. Overall, almost one quarter of the entire S. enterica sv Typhimurium genome may have been introduced by LGT.     

Feeding territoriality and the reproductive consequences in brown skuas<Emphasis Type="Italic"> Catharacta antarctica lonnbergi</Emphasis>

In the maritime Antarctic, brown skuas (Catharacta antarctica lonnbergi) show two foraging strategies: some pairs occupy feeding territories in penguin colonies, while others can only feed in unoccupied areas of a penguin colony without defending a feeding territory. One-third of the studied breeding skua population in the South Shetlands occupied territories of varying size (48 to >3,000 penguin nests) and monopolised 93% of all penguin nests in sub-colonies. Skuas without feeding territories foraged in only 7% of penguin sub-colonies and in part of the main colony. Females owning feeding territories were larger in body size than females without feeding territories; no differences in size were found in males. Territory holders permanently controlled their resources but defence power diminished towards the end of the reproductive season. Territory ownership guaranteed sufficient food supply and led to a 5.5 days earlier egg-laying and chick-hatching. Short distances between nest and foraging site allowed territorial pairs a higher nest-attendance rate such that their chicks survived better (71%) than chicks from skua pairs without feeding territories (45%). Due to lower hatching success in territorial pairs, no difference in breeding success of pairs with and without feeding territories was found in 3 years. We conclude that skuas owning feeding territories in penguin colonies benefit from the predictable and stable food resource by an earlier termination of the annual breeding cycle and higher offspring survivorship.Research licence: Umweltbundesamt Bonn 13.4-94003-1/5-7.     

Two approaches for metabolic pathway analysis?

Metabolic pathway analysis is becoming increasingly important for assessing inherent network properties in (reconstructed) biochemical reaction networks. Of the two most promising concepts for pathway analysis, one relies on elementary flux modes and the other on extreme pathways. These concepts are closely related because extreme pathways are a subset of elementary modes. Here, the common features, differences and applicability of these concepts are discussed. Assessing metabolic systems by the set of extreme pathways can, in general, give misleading results owing to the exclusion of possibly important routes. However, in certain network topologies, the sets of elementary modes and extreme pathways coincide. This is quite often the case in realistic applications. In our opinion, the unification of both approaches into one common framework for metabolic pathway analysis is necessary and achievable.     

Ontogeny of the ventral nerve cord in malacostracan crustaceans: a common plan for neuronal development in Crustacea, Hexapoda and other Arthropoda?

This review sets out to summarize our current knowledge on the structural layout of the embryonic ventral nerve cord in decapod crustaceans and its development from stem cell to the mature structure. In Decapoda, neuronal stem cells, the neuroblasts, mostly originate from ectodermal stem cells, the ectoteloblast, via a defined lineage. The neuroblasts undergo repeated asymmetric division and generate ganglion mother cells. The ganglion mother cells later divide again to give birth to ganglion cells (neurons) and there is increasing evidence now that ganglion mother cells divide again not only once but repeatedly. Various other aspects of neuroblast proliferation such as their temporal patterns of mitotic activity and spatial arrangement as well as the relation of neurogenesis to the development of the segmental appendages and maturation of motor behaviors are described. The link between cell lineage and cell differentiation in Decapoda so far has only been established for the midline neuroblast. However, there are several other identified early differentiating neurons, the outgrowing neurites of which pioneer the axonal scaffold within the neuromeres of the ventral nerve cord. The maturation of identified neurons as examined by immunohistochemistry against their neurotransmitters or engrailed, is briefly described. These processes are compared to other Arthropoda (including Onychophora, Chelicerata, Diplopoda and Hexapoda) in order to shed light on variations and conserved motifs of the theme 'neurogenesis'. The question of a 'common plan for neuronal development' in the ventral nerve cords of Hexapoda and Crustacea is critically evaluated and the possibility of homologous neurons arising through divergent developmental pathways is discussed.     

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.     

OrCGDB: a database of genes involved in oral cancer

The Oral Cancer Gene Database (OrCGDB; http://www.tumor-gene. org/Oral/oral.html) was developed to provide the biomedical community with easy access to the latest information on the genes involved in oral cancer. The information is stored in a relational database and accessed through a WWW interface. The OrCGDB is organized by gene name, which is linked to information describing properties of the gene. This information is stored as a collection of findings ('facts') that are entered by the database curator in a semi-structured format from information in primary publications using a WWW interface. These facts include causes of oncogenic activation, chromosomal localization of the gene, mutations associated with the gene, the biochemical identity and activity of the gene product, synonyms for the gene name and a variety of clinical information. Each fact is associated with a MEDLINE citation. The user can search the OrCGDB by gene name or by entering a textword. The OrCGDB is part of a larger WWW-based tumor gene database and represents a new approach to catalog and display the research literature.     

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.