Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1′ pocket.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Rapid morphological divergence of a stream fish in response to changes in water flow

Recent evidence indicates that evolution can occur on a contemporary time scale. However, the precise timing and patterns of phenotypic change are not well known. Reservoir construction severely alters selective regimes in aquatic habitats due to abrupt cessation of water flow. We examined the spatial and temporal patterns of evolution of a widespread North American stream fish (Pimephales vigilax) in response to stream impoundment. Gross morphological changes occurred in P. vigilax populations following dam construction in each of seven different rivers. Significant changes in body depth, head shape and fin placement were observed relative to fish populations that occupied the rivers prior to dam construction. These changes occurred over a very small number of generations and independent populations exhibited common responses to similar selective pressures. The magnitude of change was observed to be greatest in the first 15 generations post-impoundment, followed by continued but more gradual change thereafter. This pattern suggests early directional selection facilitated by phenotypic plasticity in the first 10–20 years, followed by potential stabilizing selection as populations reached a new adaptive peak (or variation became exhausted). This study provides evidence for rapid, apparently adaptive, phenotypic divergence of natural populations due to major environmental perturbations in a changing world.     

A laboratory study on the thermal tolerance of four southeastern stream insect species (Trichoptera,Ephemeroptera)

The acute thermal tolerances of four southeastern stream insect species, Ephemerella invaria (Walker), Stenonema ithaca (Clemens and Leonard), Symphitopsyche morosa (Hagun), and Brachycentrus lateralis (Say) were determined using an artificial stream enclosure. All species were acclimated at 10°C for 72 hours prior to instantaneous immersion into heated water for 96 hours. Percent mortality was recorded and the temperature at which 50% mortality occurred determined (LT5o). Data were subjected to standard statistical analysis.Thermal tolerance values were compared between species tested and to results from previous investigations using similar methodologies. The evolution and life histories of these species were also discussed in relation to their thermal tolerance values.     

Multicolor FISH analysis of chromosomal breaks, duplications, deletions, and numerical abnormalities in the sperm of healthy men

Transmitted de novo structural chromosomal abnormalities, the majority of which are paternally derived, can lead to abnormal reproductive outcomes as well as genetic diseases in offspring. We developed and validated a new multicolor FISH procedure (sperm ACM, which utilizes DNA probes specific for the alpha [1cen], classical, [1q12], and midi [1p36.3] satellites of chromosome 1) which utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical abnormalities plus two categories of structural aberrations: (1) duplications and deletions of 1pter and 1cen, and (2) chromosomal breaks within the 1cen-1q12 region. In healthy men, the average frequencies of sperm with duplications and deletions were (a) 4.5 +/- 0.5 and 4.1 +/- 1.3 per 10(4) involving 1pter and (b) 0.9 +/- 0.4 and 0.8 +/- 0.3 per 10(4) involving 1cen, respectively. The frequency of sperm exhibiting breaks within the 1cen-1q12 region was 14.1 +/- 1.2 per 10(4). Structural aberrations accounted for 71% of the abnormalities detected by sperm ACM, which was significantly higher than numerical abnormalities (P=2x10-8). Our findings also suggest that, for healthy men, (a) sperm carrying postmeiotic chromosomal breaks appear to be more prevalent than those carrying products of premeiotic or meiotic breakage or rearrangements, (b) the high frequency of chromosome breaks measured after "fertilization" by the hamster-egg cytogenetic method already appear to be present and detectable within human sperm by FISH, and (c) there are nonrandom and donor-specific distributions of breakpoint locations within 1q12 in sperm. FISH facilitates the analysis of much larger numbers of sperm than was possible when the hamster-egg method was used. Therefore, FISH-based procedures for simultaneously detecting chromosomal breaks, rearrangements, and numerical abnormalities in sperm may have widespread applications in human genetics, genetic toxicology, and reproductive medicine.     

Phylogenetic relationships in the iguanid lizard genus Liolaemus: multiple origins of viviparous reproduction and evidence for recurring Andean vicariance and dispersal

Phylogenetic relationships within the iguanid lizard genus Liolaemus are investigated using 1710 aligned base positions (785 phylogenetically informative) of mitochondrial DNA sequences, representing coding regions for eight tRNAs, ND2, and portions of ND1 and COL Sixty new sequences ranging in length from 1736 to 1754 bases are compared with four previously reported sequences. Liolaemus species form two well-supported monophyletic groups of subgeneric status, Liolaemus and Eulaemus. These subgenera appear to have separated at least 12.6 million years ago based on the amount of molecular evolutionary divergence between them. Hypotheses that species occurring in the Andes, west of the Andes, and east of the Andes, each comprise distinct monophyletic groups are independently rejected statistically. The shortest estimate of phylogeny suggests mat Liolaemus originated either in the Andes or the eastern lowlands. Numerous evolutionary shifts have occurred between the Andes, and the eastern and western lowlands, suggesting recurring vicariance and dispersal. Species occurring at high elevations or high latitudes usually have viviparous reproduction. Depending on whether parity mode is considered reversible in Liolaemus , the most parsimonious reconstruction supports at least six independent origins of viviparity or at least three gains followed by three losses of viviparity among die 60 Liolaemus lineages examined.     

An automated climbing apparatus to measure chemotherapy-induced neurotoxicity in Drosophila melanogaster

We have developed a novel model system in Drosophila melanogaster to study chemotherapy-induced neurotoxicity in adult flies. Neurological deficits were measured using a manual geotactic climbing assay. The manual assay is commonly used; however, it is laborious, time-consuming, subject to human error and limited to observing one sample at a time. We have designed and built a new automated fly-counting apparatus that uses a “video capture-particle counting technology” to automatically measure 10 samples at a time, with 20 flies per sample. Climbing behavior was assessed manually, as in our previous studies, and with the automated apparatus within the same experiment yielding statistically similar results. Both climbing endpoints as well as the climbing rate can be measured in the apparatus, giving the assay more versatility than the manual assay. Automation of our climbing assay reduces variability, increases productivity and enables high throughput drug screens for neurotoxicity.