The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin

Angiogenesis inhibitors have gained much public attention recently as anti-cancer agents and several are currently in clinical trials, including angiostatin (Phase I, Thomas Jefferson University Hospital, Philadelphia, PA). We report here the bowl-shaped structure of angiostatin kringles 1-3, the first multi-kringle structure to be determined. All three kringle lysine-binding sites contain a bound bicine molecule of crystallization while the former of kringle 2 and kringle 3 are cofacial. Moreover, the separation of the kringle 2 and kringle 3 lysiner binding sites is sufficient to accommodate the alpha-helix of the 30 residue peptide VEK-30 found in the kringle 2/VEK-30 complex. Together the three kringles produce a central cavity suggestive of a unique domain where they may function in concert.     

Isolation, characterization and biological activity of acidic phospholipase A2 isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIB diffracted beyond 1.8 A resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 A, b = 54.2 A and c = 90.7 A. The crystal structure has been refined to a crystallographic residual of 16.1% (R(free) = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities.     

Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2

Congenital cataracts are a common major abnormality of the eye that frequently cause blindness in infants. At least one-third of all cases are familial; autosomal-dominant congenital cataract appears to be the most-common familial form in the Western world. Elsewhere, in family ADCC-3, we mapped an autosomal-dominant cataract gene to chromosome 3q21-q22, near the gene that encodes a lens-specific beaded filament protein gene, BFSP2. By sequencing the coding regions of BFSP2, we found that a deletion mutation, DeltaE233, is associated with cataracts in this family. This is the first report of an inherited cataract that is caused by a mutation in a cytoskeletal protein.     

Reversible decreases in ATP and PCr concentrations in anoxic turtle brain

A hallmark of anoxia tolerance in western painted turtles is relative constancy of tissue adenylate concentrations during periods of oxygen limitation. During anoxia heart and brain intracellular compartments become more acidic and cellular energy demands are met by anaerobic glycolysis. Because changes in adenylates and pH during anoxic stress could represent important signals triggering metabolic and ion channel down-regulation we measured PCr, ATP and intracellular pH in turtle brain sheets throughout a 3-h anoxic-re-oxygenation transition with ³¹P NMR. Within 30 min of anoxia, PCr levels decrease 40% and remain at this level during anoxia. A different profile is observed for ATP, with a statistically significant decrease of 23% occurring gradually during 110 min of anoxic perfusion. Intracellular pH decreases significantly with the onset of anoxia, from 7.2 to 6.6 within 50 min. Upon re-oxygenation PCr, ATP and intracellular pH recover to pre-anoxic levels within 60 min. This is the first demonstration of a sustained reversible decrease in ATP levels with anoxia in turtle brain. The observed changes in pH and adenylates, and a probable concomitant increase in adenosine, may represent important metabolic signals during anoxia.     

A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.     

New-onset atrial fibrillation in sepsis is associated with increased morbidity and mortality

BackgroundThe development of new-onset atrial fibrillation in sepsis has been associated with adverse outcomes.MethodsA systematic literature search was conducted to retrieve articles that investigated the association of new-onset atrial fibrillation in patients diagnosed with sepsis. The primary outcome of interest was the pooled risk ratio (RR) of in-hospital mortality in patients with new-onset atrial fibrillation and sepsis.ResultsSix studies included 3100 patients with new-onset atrial fibrillation in sepsis and 36,900 patients without new-onset atrial fibrillation in sepsis. The pooled RR for in-hospital mortality was 1.45 (95 % CI 1.32–1.60, p < 0.00001, I^2 =24 %). New-onset atrial fibrillation was also associated with increased ICU mortality, ICU and in-hospital length of stay and stroke. New-onset atrial fibrillation occurred more in the elderly, those with a prior history of cardiovascular and respiratory disease, and those with increased severity of illness.ConclusionProspective randomised trials are needed to clarify the significance of new-onset atrial fibrillation in sepsis, optimal treatment strategies for these patients, and the benefit of systemic anticoagulation. Physicians should be aware that new-onset atrial fibrillation in sepsis is not merely an observed temporary arrhythmia but a marker of poor prognosis and should be managed accordingly.     

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.     

The structure of a native l-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 ? resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers. The binding of four Zn(2+) ions stabilizes the enzymatically active quaternary structure and is considered important for the biological activity of LAAO and other flavoproteins. Each monomer consists of three domains with a cofactor bound between the FAD and substrate binding domains, and a solvent exposed glycosylation site which is considered crucial for the toxicity. Comparison of LAAO structures in the absence and presence of a substrate indicates conformational changes in the dynamic active site. The active site H-bond network involving the triad Lys326-Water-N5 of FAD is formed only upon substrate binding, and results in the increased mobility of the isoalloxazine system. Details of the catalytic transformation of amino acid substrates are discussed.     

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.     

Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene.

The gene encoding the dopamine D2 receptor (DRD2) has been suggested as a candidate gene for several mental disorders. We previously described the cloning and chromosomal mapping (to 11q22-q23) of a human DRD2 gene as well as its use for the detection of a two-allele TaqI RFLP with a minor allele frequency of 0.24, corresponding to a PIC of 0.30. Family linkage utilizing DRD2 would be facilitated if the PIC of the DRD2 locus were increased. To this end, we have used additional phage and cosmid clones in the vicinity of DRD2 to identify a new two-allele TaqI RFLP as well as a TG microsatellite polymorphism with a PIC of 0.62. We report localizations of the three polymorphisms on the restriction map of the DRD2 locus. The TaqI RFLPs are in apparent linkage equilibrium with the microsatellite, yielding a highly informative compound marker locus with a PIC of 0.76.