New backbone cyclic substance P analogs

Summary Structure-activity relationships of cyclic substance P (SP) analogs were extensively studied in our laboratories utilizing the new concept of backbone cyclization. Employing the C-terminal hexapeptide SP_6–11, we examined the influence of chemical changes in peptides containing backbone-to-amino-end cyclization. These changes in the ring have significant influence on activity, and should be carefully designed in order to optimize pharmacological feature.     

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor. By using a cDNA library from an Loxosceles intermedia venom gland, we cloned and expressed a 900 bp cDNA, which encoded a signal peptide and a propeptide, which corresponded to a 30 kDa metalloprotease, now named LALP (Loxosceles astacin-like protease). Recombinant LALP was refolded and used to produce a polyclonal antiserum, which showed cross-reactivity with a 29 kDa native venom protein. CD analysis provided evidence that the recombinant LALP toxin was folded correctly, was still in a native conformation and had not aggregated. LALP addition to endothelial cell cultures resulted in de-adhesion of the cells, and also in the degradation of fibronectin and fibrinogen (this could be inhibited by the presence of the bivalent chelator 1,10-phenanthroline) and of gelatin in vitro. Sequence comparison (nucleotide and deduced amino acid), phylogenetic analysis and analysis of the functional recombinant toxin revealed that LALP is related in both structure and function to the astacin family of metalloproteases. This suggests that an astacin-like toxin is present in a animal venom secretion and indicates that recombinant LALP will be a useful tool for future structural and functional studies on venom and the astacin family.     

Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 +/- 9, 18.6 +/- 6.9, and 58.4 +/- 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice (n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.     

B cells, BAFF/zTNF4, TACI, and systemic lupus erythematosus

B cells and B-cell/T-cell collaborations are instrumental in the pathophysiology of systemic lupus erythematosus (SLE). This commentary highlights in particular the newly discovered role of B-cell-activating factor (BAFF; also known as TALL-1, THANK, BlyS, and zTNF4) as a positive regulator of B-cell functions, such as B-cell activation and differentiation. Two members of the tumor necrosis factor(TNF)-receptor superfamily were recently identified as receptors for BAFF on B cells. The interaction between BAFF and its receptors may be important in the pathogenesis of lupus. Advances in our understanding of abnormalities in immune regulation in lupus might provide the opportunity to improve our current therapeutic approaches to this disorder.     

Alcohol sales and risk of serious assault

Background

Alcohol is a contributing cause of unintentional injuries, such as motor vehicle crashes. Prior research on the association between alcohol use and violent injury was limited to survey-based data, and the inclusion of cases from a single trauma centre, without adequate controls. Beyond these limitations was the inability of prior researchers to comprehensively capture most alcohol sales. In Ontario, most alcohol is sold through retail outlets run by the provincial government, and hospitals are financed under a provincial health care system. We assessed the risk of being hospitalized due to assault in association with retail alcohol sales across Ontario.

Methods and Findings

We performed a population-based case-crossover analysis of all persons aged 13 years and older hospitalized for assault in Ontario from 1 April 2002 to 1 December 2004. On the day prior to each assault case''s hospitalization, the volume of alcohol sold at the store in closest proximity to the victim''s home was compared to the volume of alcohol sold at the same store 7 d earlier. Conditional logistic regression analysis was used to determine the associated relative risk (RR) of assault per 1,000 l higher daily sales of alcohol. Of the 3,212 persons admitted to hospital for assault, nearly 25% were between the ages of 13 and 20 y, and 83% were male. A total of 1,150 assaults (36%) involved the use of a sharp or blunt weapon, and 1,532 (48%) arose during an unarmed brawl or fight. For every 1,000 l more of alcohol sold per store per day, the relative risk of being hospitalized for assault was 1.13 (95% confidence interval [CI] 1.02–1.26). The risk was accentuated for males (1.18, 95% CI 1.05–1.33), youth aged 13 to 20 y (1.21, 95% CI 0.99–1.46), and those in urban areas (1.19, 95% CI 1.06–1.35).

Conclusions

The risk of being a victim of serious assault increases with alcohol sales, especially among young urban men. Akin to reducing the risk of driving while impaired, consideration should be given to novel methods of preventing alcohol-related violence.     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function

Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 A resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 5'-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az.     

Structural insights into the binding and catalytic mechanisms of the Listeria monocytogenes bacteriophage glycosyl hydrolase PlyP40

Endolysins are bacteriophage‐encoded peptidoglycan hydrolases that specifically degrade the bacterial cell wall at the end of the phage lytic cycle. They feature a distinct modular architecture, consisting of enzymatically active domains (EADs) and cell wall‐binding domains (CBDs). Structural analysis of the complete enzymes or individual domains is required for better understanding the mechanisms of peptidoglycan degradation and provides guidelines for the rational design of chimeric enzymes. We here report the crystal structure of the EAD of PlyP40, a member of the GH‐25 family of glycosyl hydrolases, and the first muramidase reported for Listeria phages. Site‐directed mutagenesis confirmed key amino acids (Glu98 and Trp10) involved in catalysis and substrate stabilization. In addition, we found that PlyP40 contains two heterogeneous CBD modules with homology to SH3 and LysM domains. Truncation analysis revealed that both domains are required for full activity but contribute to cell wall recognition and lysis differently. Replacement of CBDP40 with a corresponding domain from a different Listeria phage endolysin yielded an enzyme with a significant shift in pH optimum. Finally, domain swapping between PlyP40 and the streptococcal endolysin Cpl‐1 produced an intergeneric chimera with activity against Listeria cells, indicating that structural similarity of individual domains determines enzyme function.