Segregation of infectious pancreatic necrosis resistance QTL in the early life cycle of Atlantic Salmon (Salmo salar)

In a previous study, three significant quantitative trait loci (QTL) associated with resistance to Infectious Pancreatic Necrosis (IPN) disease were identified by analysing challenge data from one sub-population of Landcatch Atlantic salmon (Salmo salar) smolt. While these QTL were shown to affect the resistance in seawater, their effect in freshwater was unknown. This study investigates the effect of these QTL on IPN resistance in salmon fry in freshwater. Twenty families with intermediate levels of IPN mortality were analysed from a freshwater challenge trial undertaken on a different sup-population of LNS salmon to that studied previously. Only the QTL from linkage group 21 (LG21) appeared to have a significant and large effect on resistance in freshwater; the same QTL was found to have the largest effect in seawater in the previous study. Variance component analysis showed a high heritability for the QTL: 0.45 ± 0.07 on the liability scale and 0.25 ± 0.05 on the observed scale. In a family where both parents were segregating for the QTL, there was a 0% vs. 100% mortality in homozygous offspring for resistant and susceptible QTL alleles. The finding that the same QTL has major effect in both freshwater and seawater has important practical implications, as this will allow the improvement of resistance in both phases through marker assisted selection by targeting this QTL. Moreover, the segregation of the LG21 QTL in a different sub-population gives further evidence of its association with IPN-resistance.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

Photoaffinity labeling of T4 bacteriophage 32 protein

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.     

Properties of bovine heart mitochondrial cytochrome b560

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

In vitro stimulation and inhibition of steroid hormone release from postovulatory follicles of the tilapia,Oreochromis mossambicus

Summary Postovulatory follicles of the tilapia, Oreochromis mossambicus, were incubated with graded doses of salmon gonadotropin to identify the steroid hormones released by this tissue. In addition, the effects of either cytochalasin B or colchicine on steroid hormone release were studied. After the incubation, the tissue was examined by electron microscopy. Postovulatory follicles released testosterone and estradiol-17B in a dose-dependent manner with gonadotropin. There was no detectable release of progesterone or 17a-OH-progesterone. When stimulated with high doses of gonadotropin, the steroidogenic cells showed an increase in smooth endoplasmic reticulum, Golgi complexes, and lipid droplets. Also, microfilaments became arranged in orderly bundles and were found close to the numerous secretory vesicles and lipid droplets. Upon incubation with gonadotropin and either colchicine or cytochalasin B, the cells still appeared steroidogenic, but the filaments were not organized nor associated with vesicles or lipid droplets. Release of steroid hormone decreased significantly. Also in these tissues, vesicles were no longer numerous in the apical region of the granulosa cells, but were located primarily near smooth endoplasmic reticulum and Golgi complexes. This suggests that disruption of the cytoskeleton results in reduced steroid hormone synthesis or release.     

Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.

Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half-maximal saturation of photoinsertion into the polypeptide hormone was at 8-12 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 microM enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse-phase high performance liquid chromatography (HPLC) revealed that the N-terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N-terminal region is suggestive of a physiological role for a glucagon-GTP complex in the mechanism of action of this hormone.