Fluorescein as a label for non-radioactive in situ hybridization

Summary Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.     

Reprogramming homing endonuclease specificity through computational design and directed evolution

Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.     

Leg mass and lower body negative pressure tolerance in men and women

To explore the hypothesis that lower body musclemass correlates with orthostatic tolerance, 18 healthy volunteers (age18-48 yr; 10 men, 8 women) underwent a graded lower body negativepressure (LBNP) protocol consisting of six, 5-min stages of suction up to 60 mmHg in 10-mmHg increments. Forearm blood flow, heart rate, andblood pressure were measured, and forearm vascular resistance wascalculated. Leg muscle mass was assessed by dual-energy X-ray absorptiometry. All subjects received standard intravenous hydration for at least 8 h before the study. Six men and four women completed allstages of LBNP. Four men and four women developed presyncopal symptoms,including marked bradycardia and/or hypotension, at LBNP levelsof 30 mmHg (n = 2; 1 man, 1 woman), 40 mmHg (n = 2; 1 man, 1 woman), and 50 mmHg (n = 4; 2 men, 2 women). Thepresyncopal subjects had leg muscle masses ranging from 19.5 to 25.2 kgin men and from 11.7 to 16.6 kg in women. In subjects who completed allstages of LBNP, leg muscle mass ranged from 17.5 to 24.1 kg in men andfrom 10.4 to 18.0 kg in women. Leg muscle mass did not differ betweenpresyncopal subjects and those who completed the protocol. Furthermore,there were no differences in the hemodynamic responses to LBNP betweensubjects with low vs. high leg mass. These data suggest that leg musclemass is not a critical determinant of LBNP tolerance in otherwisehealthy men and women.

     

Endothelin--a new family of endothelium-derived peptides with widespread biological properties

Endothelin (ET) is a novel family of three isopeptides (ET-1, ET-2, ET-3) each containing twenty-one amino acids and two disulfide bonds. Initially isolated from the supernatant of cultured porcine aortic endothelial cells, ET is stored as a preproform and released through an unusual proteolytic cleavage. In general, ET-1, ET-2, ET-3 differ quantitatively but not qualitatively in their biologic activity. ET have potent contractile activity in a variety of isolated tissues including arteries veins, trachea, duodenum urinary bladder and uterus. In vivo, ET possesses potent vasodilator and vasoconstrictor properties. Although the mechanisms mediating the hemodynamic effects of ET are not entirely clarified, recent evidence indicates a role for endothelium-derived relaxant factor (EDRF), protein kinase C and extracellular calcium. Moreover, ET appears to produce inflammation and bronchoconstriction through the formation of arachidonic acid metabolites via the cyclooxygenase pathway. The presence of ET binding sites in blood vessels and in several organ systems suggests ET may have important regulatory functions, which remain to be determined.     

Human presence drives bobcat interactions among the U.S. carnivore guild

Biodiversity and Conservation - Mammalian carnivores are elusive and enigmatic species that often play keystone roles in ecosystems through direct and indirect effects. Growing evidence shows that...