Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle.

A potent (Na,K)-ATPase inhibitor purified from "Sigma Grade* ATP has been identified as vanadium using electron probe microanalysis and confirmed by microwave-induced emission spectroscopy and electron paramagnetic resonance spectroscopy. Sodium orthovanadate (Na3 VO4) is identical with the purified inhibitor with respect to ultraviolet absorbance, migration on thin layer chromatography, and inhibition of (Na,K)-ATPase. The (Na,K)-ATPase is in-inhibited 50% by 40 nM Na3 VO4 under optimal conditions (28 mM Mg2+) and the inhibition is 100% reversible by millimolar concentrations of norepinephrine. The physiological significance of this inhibition is discussed in relation to vanadium concentrations in vivo.     

Fate of a bioactive fluorescent wortmannin derivative in cells

Here, we report on NBD-Wm, a fluorescent wortmannin (Wm) probe that maintains the bioactivity of Wm as an inhibitor of PI3 kinase and as an antiproliferative agent. The attachment of the NBD fluorochrome permits NBD-Wm in cells to be monitored by NBD fluorescence-based methods such as FACS or fluorescence microscopy or with an anti-NBD antibody. The fluorescence of NBD-Wm treated cells reached a peak at 1.5 h and then decreased because of the extrusion of a fluorescent compound into the culture media. Cells accumulated NBD-Wm to levels about 30-fold higher than those in the media. NBD-Wm modified five major proteins, with the modification of the catalytic subunit of PI3 kinase being a minor band. The bioactivity of NBD-Wm, coupled with a variety of techniques available for determining its disposition, suggest that NBD-Wm can be a useful tool in understanding the mechanism of action of viridins.     

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G₁/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.Key words: cyclin A, cyclin E, Cdk2, centrosome, CLS, PSTAIRE, DNA synthesis     

Spatial and seasonal distribution of American whaling and whales in the age of sail

American whalemen sailed out of ports on the east coast of the United States and in California from the 18(th) to early 20(th) centuries, searching for whales throughout the world's oceans. From an initial focus on sperm whales (Physeter macrocephalus) and right whales (Eubalaena spp.), the array of targeted whales expanded to include bowhead whales (Balaena mysticetus), humpback whales (Megaptera novaeangliae), and gray whales (Eschrichtius robustus). Extensive records of American whaling in the form of daily entries in whaling voyage logbooks contain a great deal of information about where and when the whalemen found whales. We plotted daily locations where the several species of whales were observed, both those caught and those sighted but not caught, on world maps to illustrate the spatial and temporal distribution of both American whaling activity and the whales. The patterns shown on the maps provide the basis for various inferences concerning the historical distribution of the target whales prior to and during this episode of global whaling.     

Noncompetitive antibody neutralization of IL-10 revealed by protein engineering and x-ray crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.     

Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles

Superparamagnetic nanoparticles have a number of important biomedical applications, serving as MR contrast agents for imaging specific molecular targets, as reagents for cell labeling and cell tracking, and for the isolation of specific classes of cells. We have determined the physical and biological properties of MION-47 and amino-CLIO, nanoparticles which serve as precursors for the synthesis of targeted MR contrast agents, and Tat-CLIO, a nanoparticle used as a cell labeling reagent. Blood half-lives for MION-47 and amino-CLIO were 682 +/- 34 and 655 +/- 37 min, respectively. The attachment of 9.7 tat peptides per crystal to amino-CLIO resulted in a reduction in blood half-life to 47 +/- 6 min. MION-47, amino-CLIO, and Tat-CLIO were present in highest concentrations in liver and spleen and lymph nodes, where concentrations for all three nanoparticles ranged from 8.80 to 6.11% of injected dose per gram. Twenty-four hours after the intravenous injection of amino-CLIO, the nanoparticle was concentrated in cells surrounding hepatic blood vessels (endothelial and Kupffer cells), in a fashion similar to that obtained with other nanoparticle preparations. In contrast, Tat-CLIO was present as numerous discrete foci of intense fluorescence throughout the parenchyma. Using the peptide as a component of future nanoparticles, it might be possible to design sensors for the detection of macromolecules present in intracellular compartments.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.