Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C-terminal NPY analogues

1 Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors.

The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica

WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaPCT domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be a-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.

The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

Molecular modeling of N-terminal domains of NMDA-receptor. Study of ligand binding with N-terminal domains

Using the molecular-graphic complex Sybyl6.7.2, computational construction of spatial models for N-terminal domains (of NR1- and NR2B-subunits) of NMDA-receptor was conducted. On the basis of the constructed models and also CoMFA method the conclusion is made about presence of the binding site for the compounds similar to iphenprodyl in two N-terminal domains of NR1- and NR2B-subunits. The obtained data can be used for constructing new ligands.

Refractory cachexia and truth-telling about terminal prognosis; A qualitative study

The purpose of this paper is to examine the consequences that medical practitioners’ decisions about whether or not to be candid about terminal prognosis have for those suffering from refractory cachexia and their families. It presents the findings of a qualitative study which used focus groups and semi-structured interviews of a volunteer sample of doctors, nurses and dieticians in a cancer centre of a large teaching hospital in Northern Ireland. Respondents reported that some physicians tended to avoid discussing terminal prognosis in a direct manner with their patients. Nurses and dieticians tended to be reluctant to engage in conversations about weight loss with patients with cachexia. One of the reasons they reported for their lack of acknowledgement of weight loss concerned the close association between refractory cachexia and terminal prognosis. Because they viewed the telling of bad news as an exclusive prerogative of medical practitioners, they did not feel in a position to discuss cachexia because they were concerned that this had the potential to raise end-of-life issues that lay outside the boundaries of their professional role. This meant patients and their families were provided with little information about how to cope with the distressing consequences of cachexia.

Development and application of a multiple linear regression model to consider the impact of weekly waste container capacity on the yield from kerbside recycling programmes

The paper describes the development and application of a multiple linear regression model to identify how the key elements of waste and recycling infrastructure, namely container capacity and frequency of collection affect the yield from municipal kerbside recycling programmes. The overall aim of the research was to gain an understanding of the factors affecting the yield from municipal kerbside recycling programmes in Scotland. The study isolates the principal kerbside collection service offered by 32 councils across Scotland, eliminating those recycling programmes associated with flatted properties or multi occupancies. The results of a regression analysis model has identified three principal factors which explain 80% of the variability in the average yield of the principal dry recyclate services: weekly residual waste capacity, number of materials collected and the weekly recycling capacity. The use of the model has been evaluated and recommendations made on ongoing methodological development and the use of the results in informing the design of kerbside recycling programmes. The authors hope that the research can provide insights for the ongoing development of methods to optimise the design and operation of kerbside recycling programmes.

Molecular evolution of proadrenomedullin N-terminal 20 peptide (PAMP):evidence for gene co-option

Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP's better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.

The C-Terminal Variable Region Specifies the Dynamic Properties of Arabidopsis Microtubule-Associated Protein MAP65 Isotypes

The microtubule-associated protein, MAP65, is a member of a family of divergent microtubule-associated proteins from different organisms generally involved in maintaining the integrity of the central spindle in mitosis. The dicotyledon Arabidopsis thaliana and the monocotyledon rice (Oryza sativa) genomes contain 9 and 11 MAP65 genes, respectively. In this work, we show that the majority of these proteins fall into five phylogenetic clades, with the greatest variation between clades being in the C-terminal random coil domain. At least one Arabidopsis and one rice isotype is within each clade, indicating a functional specification for the C terminus. In At MAP65-1, the C-terminal domain is a microtubule binding region (MTB2) harboring the phosphorylation sites that control its activity. The At MAP65 isotypes show differential localization to microtubule arrays and promote microtubule polymerization with variable efficiency in a MTB2-dependent manner. In vivo studies demonstrate that the dynamics of the association and dissociation of different MAP65 isotypes with microtubules can vary up to 10-fold and that this correlates with their ability to promote microtubule polymerization. Our data demonstrate that the C-terminal variable region, MTB2, determines the dynamic properties of individual isotypes and suggest that slower turnover is conditional for more efficient microtubule polymerization.