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.     

In vivo calibration of a femoral fixation device transducer for measuring anterior cruciate ligament graft tension: a study in an ovine model

Toward developing a transducer for measuring in vivo tension in anterior cruciate ligament grafts in humans, the objectives of this study were to determine the following: (1) whether the calibration of a previously reported femoral fixation device transducer (FDT) (Ventura et al., 1998) is affected by the presence of the graft when implanted in the tibial metaphysis of an ovine model, (2) whether the FDT remains calibrated at 4 weeks postoperatively, and (3) whether the biological incorporation of the graft occurs prior to a change in the FDT calibration. The FDT was implanted in the hind limb of five sheep using an extra-articular procedure. Both the proximal common digital extensor tendon (i.e., graft) and a Teflon-coated wire were looped around the FDT inside a tunnel in the tibial metaphysis. The FDT was calibrated on three occasions using the loop of wire: once intraoperatively before graft insertion, once intraoperatively after graft insertion, and once postoperatively after the animals had been sacrificed at 4 weeks. Following sacrifice, the load transmitted to the FDT by the graft was also determined. The FDT exhibited linear calibration intraoperatively both before and after graft insertion with an average error relative to the calibration before insertion of the graft of -4.6 percent of full-scale load (150 N) and this average relative error was not significantly different from zero (p = 0.183). After 4 weeks of implantation, the average relative percent error was -5.0 percent and was not significantly different from zero (p = 0.434) indicating that the FDT remained calibrated in the in vivo environment. Because only 15 percent of the graft tension was transmitted to the FDT after 4 weeks, biological incorporation of the graft preceded the loss of calibration. In light of these findings, the FDT offers the capability of measuring the intra-articular ACL graft tension in vivo in animal models and possibly humans before the biological bond develops and also of monitoring the formation and maturation of the biological bond between a graft and bone tunnel.     

Hierarchical phenotypic and epigenetic variation in cloned swine

Cloning by somatic cell nuclear transfer can result in the birth of animals with phenotypic and gene expression abnormalities. We compared adult cloned pigs and adult pigs from naturally bred control females using a series of physiological and genetic parameters, including detailed methylation profiles of selected genomic regions. Phenotypic and genetic analyses indicated that there are two classes of traits, one in which the cloned pigs have less variation than controls and another characterized by variation that is equally high in cloned and control pigs. Although cloning creates animals within the normal phenotypic range, it increases the variability associated with some traits. This finding is contrary to the expectation that cloning can be used to reduce the size of groups involved in animal experimentation and to reproduce an animal, including a pet, with a homogenous set of desired traits.     

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.      

Atypical endogenous opioid systems in mice in relation to an auto-addiction opioid model of anorexia nervosa

We have proposed that the atypical opioid system in the mouse may be representative of that in the anorexia nervosa patient and may account for a biological predisposition to the disorder. This is in the context of our auto-addiction model of anorexia nervosa in which endogenous opioids play a critical role in its etiology. Morphine activation of the endogenous opioid systems increases food intake and causes sedation in most species, including normal humans and rats. In contrast in BALB/C mice, morphine causes anorexia and hyperactivity, which we suggest may be true in the anorexia nervosa patient. A variety of atypical opioid systems have been demonstrated in different mouse strains, based on other responses. The present study examines these strains with reference to the responses relevant to our anorexia nervosa model. Three patterns are described--anorexia with hyperactivity (BALB/C and C57BL/6J mice), anorexia without hyperactivity (DBA/J mice), and a biphasic curve with hyperphagia at low doses and anorexia and hyperactivity at higher doses (CF-1 mice). Only female mice were used. These atypical opioid systems may reflect a spectrum of biological predispositions to the disorder. These strain differences may also provide useful correlations of the genetic determinants of various opiate responses and provide useful comparisons in characterizing the essential features responsible for the atypical responses.     

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.     

Discovery of renin inhibitors containing a simple aspartate binding moiety that imparts reduced P450 inhibition

Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.     

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.