Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Vortexlet models of flapping flexible wings show tuning for force production and control

Insect wings are compliant structures that experience deformations during flight. Such deformations have recently been shown to substantially affect induced flows, with appreciable consequences to flight forces. However, there are open questions related to the aerodynamic mechanisms underlying the performance benefits of wing deformation, as well as the extent to which such deformations are determined by the boundary conditions governing wing actuation together with mechanical properties of the wing itself. Here we explore aerodynamic performance parameters of compliant wings under periodic oscillations, subject to changes in phase between wing elevation and pitch, and magnitude and spatial pattern of wing flexural stiffness. We use a combination of computational structural mechanics models and a 2D computational fluid dynamics approach to ask how aerodynamic force production and control potential are affected by pitch/elevation phase and variations in wing flexural stiffness. Our results show that lift and thrust forces are highly sensitive to flexural stiffness distributions, with performance optima that lie in different phase regions. These results suggest a control strategy for both flying animals and engineering applications of micro-air vehicles.     

Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain.The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.     

BEEtag: A Low-Cost,Image-Based Tracking System for the Study of Animal Behavior and Locomotion

A fundamental challenge common to studies of animal movement, behavior, and ecology is the collection of high-quality datasets on spatial positions of animals as they change through space and time. Recent innovations in tracking technology have allowed researchers to collect large and highly accurate datasets on animal spatiotemporal position while vastly decreasing the time and cost of collecting such data. One technique that is of particular relevance to the study of behavioral ecology involves tracking visual tags that can be uniquely identified in separate images or movie frames. These tags can be located within images that are visually complex, making them particularly well suited for longitudinal studies of animal behavior and movement in naturalistic environments. While several software packages have been developed that use computer vision to identify visual tags, these software packages are either (a) not optimized for identification of single tags, which is generally of the most interest for biologists, or (b) suffer from licensing issues, and therefore their use in the study of animal behavior has been limited. Here, we present BEEtag, an open-source, image-based tracking system in Matlab that allows for unique identification of individual animals or anatomical markers. The primary advantages of this system are that it (a) independently identifies animals or marked points in each frame of a video, limiting error propagation, (b) performs well in images with complex backgrounds, and (c) is low-cost. To validate the use of this tracking system in animal behavior, we mark and track individual bumblebees (Bombus impatiens) and recover individual patterns of space use and activity within the nest. Finally, we discuss the advantages and limitations of this software package and its application to the study of animal movement, behavior, and ecology.     

A calorimetric study of the thermotropic behavior of aqueous dispersions of natural and synthetic sphingomyelins.

A recently developed differential scanning calorimeter has been used to characterize the thermotropic behavior of aqueous dispersions of liposomes containing sphingomyelin. Liposomes derived from sheep brain sphingomyelin exhibit a broad gel-liquid crystalline phase transition in the temperature range of 20-45 degrees C. The transition is characterized by maxima in the heat capacity function at 31.2 and 37.1 degrees C and a total enthalpy change of 7.2 +/-0.4 kcal/mol. Beef brain sphingomyelin liposomes behave similarly but exhibit heat capacity maxima at 30, 32, and 38 degrees C and a total enthalpy change of 6.9 kcal/mol. The thermotropic behavior of four pure synthetic sphingomyelins is reminiscent of multilamellar lecithin liposomes in that a single, sharp, main transition is observed. Results obtained for liposomes containing mixtures of different sphingomyelins are complex. A colyophilized mixture of N-palmitoylsphingosinephosphorylcholine, N-stearoylsphingosinephosphorylcholine, and N-lignocerylsphingosinephosphorylcholine in a 1 : 1 : 1 mol ratio exhibits a single transition with a Tm below that observed for the individual components. On the other hand a 1 : 1 mixture of N-stearoylsphingosinephosphorylcholine and 1-palmitoyl-2-oleylphosphatidylcholine exhibits three maxima in the heat capacity function. It is clear from these results that the thermotropic behavior of sphingomyelin-containing liposomes is a complex function of the exact composition. Furthermore, it appears that the behavior of the liposomes derived from natural sphingomyelins cannot be explained in terms of phase separation of the individual components.     

Nucleocapsid Protein Subunits of Simian Virus 5, Newcastle Disease Virus, and Sendai Virus

Helical nucleocapsids of each of the paramyxoviruses simian virus 5 (SV5), Newcastle disease virus (NDV), and Sendai virus have been isolated in two different forms. One form contains larger protein subunits and is obtained from mature virions or infected cells dispersed by ethylenediaminetetraacetic acid. The other form possesses smaller subunits and is obtained from infected cells dispersed by trypsin. The estimated molecular weights of the larger subunits in the three viruses are similar: SV5, 61,000; Sendai virus, 60,000; NDV, 56,000. The smaller nucleocapsid subunits are also very similar: SV5, 43,000; Sendai virus, 46,000; NDV, 47,000. The helical nucleocapsid composed of the smaller subunit appears to be less flexible and more stable than that formed by the larger subunit. There is suggestive evidence that conversion of the larger subunit to the smaller by proteolytic cleavage may occur intracellularly. The possibility that such a mechanism could be involved in the accumulation of nucleocapsid in cells persistently infected with paramyxoviruses is discussed.     

A common polygenic basis for quinine and PROP avoidance in mice

Inbred strains of mice (Mus musculus) differ greatly in ability to taste various bitter compounds. For some compounds, the differences result from allelic variation at a single locus. However, segregation patterns incompatible with monogenic inheritance have been found for quinine avoidance. The Soa bitter sensitivity locus exerts some influence on this phenotype, but an unknown number of other loci also contribute. Relative avoidance patterns for quinine sulfate in panels of naive inbred strains resembled avoidance patterns for 6-n-propyl-2- thiouracil (PROP), suggesting a common genetic basis. In particular, C57BL/6J mice strongly avoided both 0.1 mM quinine sulfate and 1 mM PROP in two-bottle preference tests, whereas C3H/HeJ mice were indifferent to both. Therefore, 12 BXH/Ty recombinant inbred strains, derived from these strains, were tested with both solutions to begin identification of the unknown bitter loci. Naive mice were tested for four consecutive days with each compound (order counterbalanced). Some BXH/Ty strain means resembled those of the parent strains, but others were intermediate. This indicated recombination among loci affecting avoidance, and therefore polygenic inheritance. The strain means were highly correlated across compounds (r = 0.98), suggesting that the same polygenes controlled both phenotypes. The BXH/Ty means for both compounds were then compared with the strain genotypes at 212 chromosome position markers distributed throughout the genome. Eight markers on five chromosomes (3, 6, 7, 8 and 9) yielded significant correlations. Six of the markers were correlated with both phenotypes, again suggesting common polygenic inheritance. The marker with the highest correlation was Prp, tightly linked to Soa on chromosome 6. The correlated marker regions likely contain quantitative trait loci affecting bitter avoidance. The phenotypic similarity of PROP to quinine, rather than to phenylthiourea, apparently stemming from a common polygenic basis, indicates a difference between mice and humans in gustatory organization related to bitters.      

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

A phylogenetic approach to the identification of phosphoglucomutase genes

The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes. We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM). Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms. In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3. Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database. Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium-- and sugar-binding sites, were conserved in all 47 sequences. The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified. Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2. Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2. A third lineage may contain homologs to human PGM3. On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.      

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.