Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.     

Imatinib and Dasatinib Inhibit Hemangiosarcoma and Implicate PDGFR-β and Src in Tumor Growth

Hemangiosarcoma, a natural model of human angiosarcoma, is an aggressive vascular tumor diagnosed commonly in dogs. The documented expression of several receptor tyrosine kinases (RTKs) by these tumors makes them attractive targets for therapeutic intervention using tyrosine kinase inhibitors (TKIs). However, we possess limited knowledge of the effects of TKIs on hemangiosarcoma as well as other soft tissue sarcomas. We report here on the use of the TKIs imatinib and dasatinib in canine hemangiosarcoma and their effects on platelet-derived growth factor receptor β (PDGFR-β) and Src inhibition. Both TKIs reduced cell viability, but dasatinib was markedly more potent in this regard, mediating cytotoxic effects orders of magnitude greater than imatinib. Dasatinib also inhibited the phosphorylation of the shared PDGFR-β target at a concentration approximately 1000 times less than that needed by imatinib and effectively blocked Src phosphorylation. Both inhibitors augmented the response to doxorubicin, suggesting that clinical responses likely will be improved using both drugs in combination; however, dasatinib was significantly (P < .05) more effective in this context. Despite the higher concentrations needed in cell-based assays, imatinib significantly inhibited tumor growth (P < .05) in a tumor xenograft model, highlighting that disruption of PDGFR-β/PDGF signaling may be important in targeting the angiogenic nature of these tumors. Treatment of a dog with spontaneously occurring hemangiosarcoma established that clinically achievable doses of dasatinib may be realized in dogs and provides a means to investigate the effect of TKIs on soft tissue sarcomas in a large animal model.     

Opioid agonists have different efficacy profiles for G protein activation,rapid desensitization,and endocytosis of mu-opioid receptors

The differential ability of various mu-opioid receptor (MOP) agonists to induce rapid receptor desensitization and endocytosis of MOP could arise simply from differences in their efficacy to activate G proteins or, alternatively, be due to differential capacity for activation of other signaling processes. We used AtT20 cells stably expressing a low density of FLAG-tagged MOP to compare the efficacies of a range of agonists to 1) activate G proteins using inhibition of calcium channel currents (ICa) as a reporter before and after inactivation of a fraction of receptors by beta-chlornaltrexamine, 2) produce rapid, homologous desensitization of ICa inhibition, and 3) internalize receptors. Relative efficacies determined for G protein coupling were [Tyr-D-Ala-Gly-MePhe-Glyol]enkephalin (DAMGO) (1) > or = methadone (0.98) > morphine (0.58) > pentazocine (0.15). The same rank order of efficacies for rapid desensitization of MOP was observed, but greater concentrations of agonist were required than for G protein activation. By contrast, relative efficacies for promoting endocytosis of MOP were DAMGO (1) > methadone (0.59) > morphine (0.07) > or = pentazocine (0.03). These results indicate that the efficacy of opioids to produce activation of G proteins and rapid desensitization is distinct from their capacity to internalize mu-opioid receptors but that, contrary to some previous reports, morphine can produce rapid, homologous desensitization of MOP.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by β-adrenergic signals, leading to a rise in intracellular 3′,5′-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 ± 0.08. A change in assay temperature from a euthermic value (37 °C) to one typical of hibernating body temperature (5 °C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 °C to 8.7 at 5 °C, (b) K_m values at 37 °C for Mg.ATP (49.2±3.4 M) and for two phosphate acceptors, Kemptide (50.0±5.5 M) and Histone IIA (0.41 ± 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 °C, and (c) inhibition by KCl, NaCl and NH₄Cl was reduced. However, temperature change had little or no effect on K_m values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 °C and activation energies that were 29.1 ± 1.0 kJ/mol for temperatures >9 °C and 2.3-fold higher at 68.1 ± 2.1 kJ/mol for temperatures <9 °C, whereas the rabbit enzyme showed a breakpoint at 17 °C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 ± 0.18 at 37 °C to 2.19 ± 0.07 at 5 °C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with ³²P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 °C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor. Accepted: 10 July 1998     

When is a native species invasive? Incursion of a novel predatory marsupial detected using molecular and historical data

Aim

Range expansions facilitated by humans or in response to local biotic or abiotic stressors provide the opportunity for species to occupy novel environments. Classifying the status of newly expanded populations can be difficult, particularly when the timing and nature of the range expansion are unclear. Should native species in new habitats be considered invasive pests or actively conserved? Here, we present an analytical framework applied to an Australian marsupial, the sugar glider (Petaurus breviceps), a species that preys upon on an endangered parrot in Tasmania, and whose provenance was uncertain.

Location

Tasmania, Australia.

Methods

We conducted an extensive search of historical records for sugar glider occurrences in Tasmania. Source material included museum collection data, early European expedition logs, community observation records, and peer‐reviewed and grey literature. To determine the provenance of the Tasmanian population, we sequenced two mitochondrial genes and one nuclear gene in Tasmanian animals (n = 27) and in individuals across the species' native range. We then estimated divergence times between Tasmania and southern Australian populations using phylogenetic and Bayesian analyses.

Results

We found no historical evidence of sugar gliders occurring in Tasmania prior to 1835. All Tasmanian individuals (n = 27) were genetically identical at the three genes surveyed here with those individuals being 0.125% divergent from individuals from a population in Victoria. Bayesian analysis of divergence between Tasmanian individuals and southern Australian individuals suggested a recent introduction of sugar gliders into Tasmania from southern Australia.

Main conclusions

Molecular and historical data demonstrate that Tasmanian sugar gliders are a recent, post‐European, anthropogenic introduction from mainland Victoria. This result has implications for the management of the species in relation to their impact on an endangered parrot. The analytical framework outlined here can assist environmental managers with the complex task of assessing the status of recently expanded or introduced native species.

     

Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis

The initial steps of Venezuelan equine encephalitis virus (VEE) spread from inoculation in the skin to the draining lymph node have been characterized. By using green fluorescent protein and immunocytochemistry, dendritic cells in the draining lymph node were determined to be the primary target of VEE infection in the first 48 h following inoculation. VEE viral replicon particles, which can undergo only one round of infection, identified Langerhans cells to be the initial set of cells infected by VEE directly following inoculation. These cells are resident dendritic cells in the skin, which migrate to the draining lymph node following activation. A point mutation in the E2 glycoprotein gene of VEE that renders the virus avirulent and compromises its ability to spread beyond the draining lymph blocked the appearance of virally infected dendritic cells in the lymph node in vivo. A second-site suppressor mutation that restores viral spread to lymphoid tissues and partially restore virulence likewise restored the ability of VEE to infect dendritic cells in vivo.     

Testing a ‘genes‐to‐ecosystems’ approach to understanding aquatic–terrestrial linkages

A ‘genes‐to‐ecosystems’ approach has been proposed as a novel avenue for integrating the consequences of intraspecific genetic variation with the underlying genetic architecture of a species to shed light on the relationships among hierarchies of ecological organization (genes → individuals → communities → ecosystems). However, attempts to identify genes with major effect on the structure of communities and/or ecosystem processes have been limited and a comprehensive test of this approach has yet to emerge. Here, we present an interdisciplinary field study that integrated a common garden containing different genotypes of a dominant, riparian tree, Populus trichocarpa, and aquatic mesocosms to determine how intraspecific variation in leaf litter alters both terrestrial and aquatic communities and ecosystem functioning. Moreover, we incorporate data from extensive trait screening and genome‐wide association studies estimating the heritability and genes associated with litter characteristics. We found that tree genotypes varied considerably in the quality and production of leaf litter, which contributed to variation in phytoplankton abundances, as well as nutrient dynamics and light availability in aquatic mesocosms. These ‘after‐life’ effects of litter from different genotypes were comparable to the responses of terrestrial communities associated with the living foliage. We found that multiple litter traits corresponding with aquatic community and ecosystem responses differed in their heritability. Moreover, the underlying genetic architecture of these traits was complex, and many genes contributed only a small proportion to phenotypic variation. Our results provide further evidence that genetic variation is a key component of aquatic–terrestrial linkages, but challenge the ability to predict community or ecosystem responses based on the actions of one or a few genes.     

Diaspirin cross-linked Hb and norepinephrine prevent the sepsis-induced increase in critical O(2) delivery

We hypothesized that support of arterial perfusion pressure with diaspirin cross-linked Hb (DCLHb) would prevent the sepsis-induced attenuation in the systemic O(2) delivery-O(2) uptake relationship. Awake septic rats were treated with a chronic infusion of DCLHb or a reference treatment [norepinephrine (NE)] to increase mean arterial pressure by 10-20% over 18 h. Septic and sham control groups received normal saline. Isovolemic hemodilution to create anemic hypoxia was then performed in a metabolic box during continuous measurement of systemic O(2) uptake. O(2) delivery was calculated from hemodynamic variables, and the critical point of O(2) delivery (DO(2 crit)) was determined using piecewise regression analysis of the O(2) delivery-O(2) uptake relationship. Sepsis increased DO(2 crit) from 4.99 +/- 0.17 to 6.69 +/- 0.42 ml x min(-1) x 100 g(-1) (P < 0.01), while O(2) extraction capacity was decreased (P < 0.05). DCLHb and NE infusion prevented the sepsis-induced increase in DO(2 crit) [4.56 +/- 0.42 ml x min(-1) x 100 g(-1) (P < 0.01) and 5.04 +/- 0.56 ml x min(-1) x 100 g(-1) (P < 0.05), respectively]. This was explained by a 59% increase in O(2) extraction capacity in the DCLHb group compared with septic controls (P < 0.05), whereas NE treatment decreased systemic O(2) uptake in anemic hypoxia (1.51 +/- 0.08 vs. 1.87 +/- 0.1 ml x min(-1) x 100 g(-1) in septic controls, P < 0.05). We conclude that DCLHb ameliorated O(2) extraction capacity in the septic microcirculation, whereas NE decreased the metabolic demands of the tissues.     

Phospholipid exchange between bilayer membrane vesicles.

The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.