Angiotensinase activity and angiotensin receptor binding in guinea pig aorta.

The angiotensinase (EC 3.4.99.3) activity of the subcellular fractions of guinea pig aorta has been studied in relation to their [14C]angiotensin binding capacity. The enzyme activity occurs in the following decreasing order: supernatant greater than plasma membrane fraction greater than 105 000 X g pellet greater than mitochondrial fraction. The specific binding of [14C]angiotensin to these fractions follows the same pattern. Pretreatment of the subcellular fractions at 47 degrees C for 20 min was performed in an attempt to differentiate binding of angiotensin to the pharmacological receptor from binding to the destroying enzymes. This procedure decreased the angiotensinase activity in the plasma membrane fraction only whereas the specific binding of [14C]angiotensin to this fraction was not significantly decreased, suggesting that the plasma membrane angiotensinase is a thermolabile enzyme.     

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

Diurnal variation in Enterococcus species composition in polluted ocean water and a potential role for the enterococcal carotenoid in protection against photoinactivation

Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa Catalina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcus faecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Enterococcus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented E. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing photoinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

Structural characterization of (1-->3)-beta-D-glucans isolated from blastospore and hyphal forms of Candida albicans

Glucans are (1-->3)-beta-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.     

The effect of visitors on the behavior of zoo-housed western lowland gorillas (Gorilla gorilla gorilla)

Primates, especially apes, are popular with the public, often attracting large crowds. These crowds could cause behavioral change in captive primates, whether positive, neutral, or negative. We examined the impact of visitors on the behavior of six western lowland gorillas (Gorilla gorilla gorilla), observing the troop over 6 weeks during high season (4.5 hr/day, 35 days, May–July 2016). We used focal scan sampling to determine activity budget and enclosure usage, and focal continuous sampling to identify bouts of anxiety-related behavior (visitor-directed vigilance, self-scratching, and aggression). Both daily zoo-entry numbers (V_GATE) and instantaneous crowds at the exhibit (V_DENSITY) were measured. Overall, V_GATE had little effect across behaviors. However, consistent with the more acute time frame of measurement, V_DENSITY was a better predictor of behavior; at high crowd volumes, we observed significant group-level changes in activity budget (increased inactivity, increased locomotion, and decreased environment-related behaviors), increase in some anxiety-related behaviors, and decreased enclosure usage. Although contributing similar effects, it could not be determined if crowd numbers, composition, or noise most affected the troop, nor any chronic effects of exposure to large crowds. Nevertheless, our findings suggest that measures to minimize the impacts of large crowds at the exhibit would be beneficial. Furthermore, we highlight potential discrepancies between common methods for measuring visitor numbers: V_GATE is less sensitive to detecting visitor effects on behavioral indices than V_DENSITY. Future studies should appropriately match the biological time frame of welfare indicators and visitor measures used to ensure the reliability of findings.     

Structural characterization of NETNES, a novel glycoconjugate in Trypanosoma cruzi epimastigotes

The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucinlike molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligomannose glycans, two linear alpha-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn; the phosphate-linked glycans are a mixture of (Manalpha1-2)0-3Man1-P-Ser; and the GPI anchor has the structure Manalpha1-2(ethanolamine phosphate)Manalpha1-2Manalpha1-6Manalpha1-4(2-aminoethylphosphonate-6)GlcNalpha1-6-myo-inositol-1-P-3(sn-1-O-(C16:0)alkyl-2-O-(C16:0)acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid N-terminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.     

Y25S variant of Paracoccus pantotrophus cytochrome cd1 provides insight into anion binding by d1 heme and a rare example of a critical difference between solution and crystal structures

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.