Multiview light-sheet microscope for rapid in toto imaging

We present a multiview selective-plane illumination microscope (MuVi-SPIM), comprising two detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of biological specimens with subcellular resolution. The fixed geometrical arrangement of the imaging branches enables multiview data fusion in real time. The high speed of MuVi-SPIM allows faithful tracking of nuclei and cell shape changes, which we demonstrate through in toto imaging of the embryonic development of Drosophila melanogaster.     

Thrombin inhibitors built on an azaphenylalanine scaffold

A series of azaphenylalanine derivatives were investigated as novel thrombin inhibitors based on the prodrug principle. By systematic structural modifications we have identified optimal groups for this series that led us to potent inhibitors of thrombin incorporating the benzamidine fragment at the P1 position, and their potentially orally active benzamidoxime prodrugs. The binding modes in the thrombin active site of two representative compounds were identified by X-ray crystallographic analysis.     

Delayed arteriolar relaxation after prolonged agonist exposure: functional remodeling involving tyrosine phosphorylation

Although arteriolar contraction is dependent on Ca2+-induced myosin phosphorylation, other mechanisms including Ca2+ sensitization and time-dependent phenomena such as cytoskeletal and cellular reorganization may contribute to contractile events. We hypothesized that if arteriolar smooth muscle exhibits time-dependent behavior this may be manifested in differences in relaxation after short- and long-term exposure to contractile agonists. Studies were conducted in isolated arterioles pressurized to 70 mmHg. In initial experiments (n = 10), rate of relaxation was measured after acute (5 min) or prolonged (4 h) exposure to 5 microM norepinephrine (NE). Prolonged exposure to NE resulted in significantly (P < 0.05) increased time for relaxation in physiological salt solution. Rapid relaxation of vessels exposed to NE for 4 h was observed after superfusion with 0 mM Ca2+ buffer, indicating that the alteration in relaxation was reversible and Ca2+ dependent. A similarly impaired dilation was not observed with 4-h exposure to KCl (75 mM). To determine mechanisms contributing to the effects of prolonged NE exposure, studies were performed in the presence of the microtubule depolymerizing agent demecolcine (10 microM) or a series of tyrosine phosphorylation inhibitors. Although demecolcine caused significant vasoconstriction (P < 0.05) and potentiated NE vasoconstriction, it did not prevent the effect of long-term NE exposure on relaxation. Genistein, although having no effect on acute NE-induced contraction, concentration-dependently inhibited prolonged NE constriction. Similarly, Src (PP1) and p42/44 MAP kinase (PD-98059) inhibitors prevented maintenance of long-term NE contraction. The data indicate that prolonged exposure to NE induces biochemical alterations that impair relaxation after removal of the agonist. The contractile effects are Ca2+ dependent and involve tyrosine phosphorylation but do not appear to involve the polymerization state of the microtubule network.     

Left ventricular end-diastolic pressure-volume relationship in septic rats with open thorax

To estimate changes in compliance, we evaluated the effects of sepsis on the end-diastolic pressure-volume relationship (EDPVR) in the left ventricle of rats that had undergone an open thorax procedure. Sepsis was induced in male Wistar Hannover rats (n = 7; 240 to 270 g) by intraperitoneal administration of a slurry of cecal contents; control rats (n = 7) were given 5% dextrose only. On the third day after induction of sepsis, left ventricular (LV) pressure and LV dimensions were recorded simultaneously in animals of both groups. Using a micromanometer and ultrasonic crystals, measurements were obtained at baseline and during the increase of afterload. Blood samples were taken for determination of complete blood count, white blood cell differential count, and lactate concentration, and for bacteriologic examination. Septic rats lost weight, and developed changes in body temperature, ascites, and abscesses in the abdominal and thoracic cavities, gram-negative bacteremia, and increase in heart rate. On the third day after induction of sepsis, LV EDPVR decreased, compared with that in the control rats (regression coefficients: control group, 8.41 to 23.95; sepsis group, 3.94 to 7.92). Myocardial compliance in the left ventricle increased on the third day of sepsis in the open-thorax rat model, as evidenced by the downward shift of LV EDPVR in rats with sepsis, compared with controls.     

Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence). Received: 27 August 1992 / Accepted: 5 December 1997     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

A holistic approach to marine eco-systems biology

The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.     

VizRank: finding informative data projections in functional genomics by machine learning

SUMMARY: VizRank is a tool that finds interesting two-dimensional projections of class-labeled data. When applied to multi-dimensional functional genomics datasets, VizRank can systematically find relevant biological patterns. AVAILABILITY: http://www.ailab.si/supp/bi-vizrank SUPPLEMENTARY INFORMATION: http://www.ailab.si/supp/bi-vizrank.