Differences in Mate Pairings of Hatchery- and Natural-Origin Coho Salmon Inferred from Offspring Genotypes.

Captive breeding can affect how sexual selection acts on subsequent generations. One context where this is important is in fish hatcheries. In many salmon hatcheries, spawning is controlled artificially and offspring are reared in captivity before release into the wild. While previous studies have suggested that hatchery- and natural-origin fish may make different mate choice decisions, it remains to be determined how hatchery fish may be making different mate choice decisions compared with natural-origin fish at a genetic level. Using genotyping-by-sequencing, we identify single-nucleotide polymorphisms (SNPs) associated with variation in mate pairings from a natural context involving hatchery- and natural-origin coho salmon (Oncorhynchus kisutch). In both natural-origin and hatchery mate pairs, we observed more SNPs with negative assortment than positive assortment. However, only 3% of the negative assortment SNPs were shared between the two mating groups, and 1% of the positive assortment SNPs were shared between the two mating groups, indicating divergence in mating cues between wild and hatchery-raised salmon. These findings shed light on mate choice in general and may have important implications in the conservation management of species as well as for improving other captive breeding scenarios. There remains much to discover about mate choice in salmon and research described here reflects our intent to test the potential of ongoing advances in population genomics to develop new hatchery practices that may improve the performance of hatchery offspring, lessening the differences and thus potential impacts upon wild stocks.

Influence of habitat discontinuity, geographical distance, and oceanography on fine-scale population genetic structure of copper rockfish (Sebastes caurinus).

The copper rockfish is a benthic, nonmigratory, temperate rocky reef marine species with pelagic larvae and juveniles. A previous range-wide study of the population-genetic structure of copper rockfish revealed a pattern consistent with isolation-by-distance. This could arise from an intrinsically limited dispersal capability in the species or from regularly-spaced extrinsic barriers that restrict gene flow (offshore jets that advect larvae offshore and/or habitat patchiness). Tissue samples were collected along the West Coast of the contiguous USA between Neah Bay, WA and San Diego, CA, with dense sampling along Oregon. At the whole-coast scale (approximately 2200 km), significant population subdivision (F(ST) = 0.0042), and a significant correlation between genetic and geographical distance were observed based on 11 microsatellite DNA loci. Population divergence was also significant among Oregon collections (approximately 450 km, F(ST) = 0.001). Hierarchical amova identified a weak but significant 130-km habitat break as a possible barrier to gene flow within Oregon, across which we estimated that dispersal (N(e)m) is half that of the coast-wide average. However, individual-based Bayesian analyses failed to identify more than a single population along the Oregon coast. In addition, no correlation between pairwise population genetic and geographical distances was detected at this scale. The offshore jet at Cape Blanco was not a significant barrier to gene flow in this species. These findings are consistent with low larval dispersal distances calculated in previous studies on this species, support a mesoscale dispersal model, and highlight the importance of continuity of habitat and adult population size in maintaining gene flow.

Duplicated Clock genes with unique polyglutamine domains provide evidence for nonhomologous recombination in Chinook salmon (Oncorhynchus tshawytscha).

Circadian rhythms underlie diverse life functions ranging from cellular activities to behavior. Multiple clock genes play a central role in the generation of these rhythms. We partially characterized two copies of the Clock gene from Chinook salmon (Oncorhynchus tshawytscha), OtsClock1a and OtsClock1b. The 6,460 bp OtsClock1a sequence contains 16 exons, 15 introns and encompasses three highly conserved domains indicating it is a novel member of the bHLH-PAS superfamily of transcription factors. The second copy, OtsClock1b, consists of five exons and five introns spanning 1,945 bp. A polyglutamine repeat motif (PolyQ), characteristic of a majority of CLOCK proteins, is present in both OTSCLOCK1a and OTSCLOCK1b. However, the Chinook PolyQ domains are uniquely positioned inside the gene. Interestingly, a 1,200 bp non-coding segment located downstream of the OtsClock1a PolyQ domain is absent from OtsClock1b. This insertion/deletion is 91% similar to the Salmo salar Transferrin gene. A phylogenetic analysis of 11 CLOCK proteins shows that OtsClock1a and OtsClock1b are paralogs which likely arose subsequent to the salmonid genome-wide duplication event. Ultimately, the Chinook salmon Clock genes are key components to our understanding the genetic mechanisms underlying temporally regulated life history traits in Pacific salmonids.

Bone marrow cells produce soluble factors that inhibit osteoclast activity.

Cytokines that stimulate bone resorption are produced by cells found in bone marrow. However, marrow cells produce multiple factors, some of which may be inhibitors of osteoclast differentiation or activity. Thus, it is not possible to predict a priori whether the mixture of factors produced by marrow cells will have a net stimulatory or inhibitory effect on bone resorption. In this study, we showed that the net effect of whole marrow is to inhibit osteoclast activity induced by parathyroid hormone. Fractionation of the marrow revealed that the inhibitory activity was in the marrow fluid. However, conditioned media obtained from marrow cell cultures also inhibited osteoclast activity. Thus, it is likely that the inhibitory factors are produced in vivo by cells residing in the marrow. These inhibitory factors may represent a physiological regulatory process that plays an important role in maintaining the balance between bone resorption and formation. Because we have previously shown that interleukin-6 is one of the cytokines that parathyroid hormone induces in osteoblastic cells to stimulate osteoclast activity, one potential mechanism by which the marrow-derived inhibitory factors might act is by preventing this production of interleukin-6. However, we found that the marrow cell-conditioned media do not inhibit the production or activity of interleukin-6. Thus, the inhibitory factors appear to block osteoclast activity through a mechanism that does not involve interleukin-6. Taken together, these results demonstrate the importance of factors that inhibit bone resorption and emphasize that the presence of cytokines that stimulate bone resorption in conditions such as osteoporosis and orthopaedic implant loosening should be interpreted with caution unless evidence exists demonstrating their functional importance.

Osteoclast differentiation requires ascorbic acid.

Osteoclast differentiation assays are usually conducted in alpha minimal essential medium (alpha-MEM). We reasoned that determining which components of this media are critical for osteoclast differentiation might provide insight into the mechanisms that regulate osteoclast differentiation. This study demonstrates that ascorbic acid is the crucial component of alpha-MEM that stimulates differentiation of murine osteoclasts in cocultures with murine mesenchymal support cells. Thus, supplementation with ascorbic acid allows osteoclast differentiation to occur in basal MEM media as well as in RPMI-1640 and basal media Eagle (BME) media. The conclusion that osteoclast differentiation is stimulated by ascorbic acid was obtained whether osteoclast differentiation was induced by 1,25-dihydroxyvitamin D3 or parathyroid hormone, whether ST2 or CIMC-2 cells were used as mesenchymal support cells, and whether osteoclast precursors were obtained from spleen or bone marrow. Time course studies revealed that although ascorbic acid only modestly increases the rate at which osteoclast precursors begin to express tartrate-resistant acid phosphatase, it strongly increases the rate at which precursors fuse into mature, multinucleated cells. Moreover, ascorbic acid strongly increases the life span of both osteoclasts and their precursors. The increases in precursor formation, fusion, and life span induced by ascorbic acid are together responsible for the stimulation of osteoclast differentiation by ascorbic acid. Given the known effects of ascorbic acid on differentiation of mesenchymal cells, it may stimulate osteoclast differentiation indirectly by regulating the differentiation state of the mesenchymal cells that support osteoclast differentiation.

Vitamin E levels and susceptibility to lipid peroxidation increase with aging in heart plasma membrane from miniature swine.

Age-related changes in heart plasma membrane fatty acid composition, vitamin E content, membrane fluidity, susceptibility to lipid peroxidation, and the subcellular distribution of vitamin E were observed in male and female Hormel-Hanford miniswine over a wide range of ages: prepubertal, < 0.5 years; young, 0.5-2.5 years; middle-aged, 5.9-10 years; and old, 11.5-13.9 years. Pigs were continuously fed the same low-fat, cholesterol-free, vitamin E-adequate stock diet at restricted maintenance levels. Membrane lipid peroxidation tended to increase in middle-aged and elderly pigs, but not significantly, perhaps being somewhat ameliorated by the significantly increased membrane vitamin E in middle-aged and old pigs. Mid-bilayer membrane fluidity was significantly increased in old pigs, but fluidity of the polar headgroup domains decreased with age. Thus, lipid peroxidation tended to increase over the long life span of miniswine even when they are food restricted.

Adenyl cyclase and interleukin 6 are downstream effectors of parathyroid hormone resulting in stimulation of bone resorption.

Parathyroid hormone and other bone resorptive agents function, at least in part, by inducing osteoblasts to secrete cytokines that stimulate both differentiation and resorptive activity of osteoclasts. We previously identified two potentially important cytokines by demonstrating that parathyroid hormone induces expression by osteoblasts of IL-6 and leukemia inhibitory factor without affecting levels of 14 other cytokines. Although parathyroid hormone activates multiple signal transduction pathways, induction of IL-6 and leukemia inhibitory factor is dependent on activation of adenyl cyclase. This study demonstrates that adenyl cyclase is also required for stimulation of osteoclast activity in cultures containing osteoclasts from rat long bones and UMR106-01 rat osteoblast-like osteosarcoma cells. Since the stimulation by parathyroid hormone of both cytokine production and bone resorption depends on the same signal transduction pathway, we hypothesized that IL-6 might be a downstream effector of parathyroid hormone. We found that addition of exogenous IL-6 mimics the ability of parathyroid hormone to stimulate bone resorption. More importantly, an antibody directed against the IL-6 receptor blocks moderate stimulation of osteoclast activity induced by the hormone. Interestingly, strong stimulation of resorption overcomes this dependence on IL-6. Thus, parathyroid hormone likely induces multiple, redundant cytokines that can overcome the IL-6 requirement associated with moderate stimulation. Taken together with studies showing that many other bone resorptive agents also stimulate IL-6 production, our results suggest that IL-6 may be a downstream effector of these agents as well as of parathyroid hormone.

Home infusion of intravenous immunoglobulin.

Intravenous immunoglobulin (IGIV) is being used extensively in the home-care setting to treat patients with a wide variety of disorders. This therapy can be managed more effectively when the patient and/or caregiver have been educated regarding the therapy. In this article, a therapy education plan, policies, and procedures that address the key components of providing IGIV treatments in the home-care setting will be presented. Instructional information is presented in "patient-friendly terms" that can be readily applied to the nursing care plan. This plan will be applicable for patients who will become independent with the administration of their IGIV as well as for those who will have their IGIV administered by the home-care nurse.

Heart and liver fatty acid composition and vitamin E content in miniature swine fed diets containing corn and menhaden oils.

Female miniature swine, 4-11 yr, were fed 15% fat diets containing n-3 and/or n-6 polyunsaturated fat for 6 months, at 1.95 g fat/kg body weight. Liver lipids from menhaden oil-fed minipigs were elevated in the n-3 fatty acids: 20:5, 22:5 and 22:6, but heart lipids only in 20:5 and 22:6. Liver cell plasma membrane was elevated in 20:5, 22:5 and 22:6 and lowered in the n-6 acids 18:2 and 20:4 in menhaden oil-fed animals, to a greater extent than in the total tissue lipids. Liver alpha-tocopherol tended to decrease upon feeding menhaden oil, but heart alpha-tocopherol concentrations were not affected.

Robinson anterior cervical fusion comparison of the standard and modified techniques.

In 1990 the authors modified the Robinson anterior cervical interbody fusion technique by burring the endplates to expose subchondral bone. The authors compared 31 patients having the standard technique and 29 patients having the modified technique to evaluate 1) setting of the bone graft, 2) kyphotic angulation, 3) pseudarthrosis rate, and 4) pain outcome. In the standard Robinson fusion technique, the average loss of height across the fused segments was 0.8 mm and the average increase in kyphosis 4.9 degrees. Values for the modified technique were 1.9 mm and 3.1 degrees, respectively. The change in height was statistically significant (P = .01), as was the difference in angulation (P = .028), though the latter was in the opposite direction predicted. The pseudarthrosis rate using the modified technique decreased to 4.4% per level. Pain outcome for the two groups was equivalent. Burring of the endplates for anterior cervical interbody arthrodesis results in a detectable but not clinically important amount of graft settling with a higher success rate for arthrodesis.

Mice, hamsters and guinea pigs differ in efficiency of pyridoxine-5'-beta-D-glucoside utilization.

Mice, hamsters and guinea pigs were studied to assess species variation in utilization of pyridoxine-5'-D-glucoside (PN-glucoside), a form of vitamin B-6 found in plants. Animals fed vitamin B-6-deficient or marginally supplemented diets [1 mg pyridoxine (PN)/kg] were given an oral dose of [3H]PN-glucoside plus [14C]PN. Urinary and fecal isotopic excretion was measured over 24 h and the distribution of B-6 vitamins in liver determined at the end of the 24-h period. Intestinal absorption was nearly complete, as very little (< 6%) of each isotope was excreted in the feces. In mice, hamsters and guinea pigs, 31.3, 31.5 and 9.5%, respectively, of urinary 3H was present as intact PN-glucoside. Incorporation into liver was reflected by 3H/14C ratios of hepatic vitamin B-6 as follows: mice, 0.39; hamsters, 0.73; guinea pigs, 1.49 (means for both diets). The intake of dietary vitamin B-6 had little effect on [3H]PN-glucoside metabolism. Guinea pigs displayed greater utilization of PN-glucoside than did mice, hamsters or rats (seen previously), although they may not be the best animal model for the study of PN-glucoside metabolism. Because the bioavailability of PN-glucoside in humans has been estimated to be 58% relative to PN, mice or hamsters, rather than guinea pigs or rats, would be better species for quantitative studies of PN-glucoside bioavailability and associated enzymatic processes.

Discrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large subunit rRNA.

Mitochondrial DNA sequence variation was characterized for the large subunit rRNA-coding gene (16SrDNA) in two closely related Pacific oyster species (Crassostrea gigas and C. sikamea) and an out group, the Olympia oyster (Ostrea lurida). Although each species was shown to have a single, fixed haplotype for the DNA sequence under study, 7 nucleotide differences were found between C. gigas and C. sikamea, and these two species differed from the O. lurida haplotype at 62 and 60 nucleotide sites, respectively. Nucleotide differences for the two Crassostrea species showed a notable transition bias (85.7%) in contrast to the marginal transversion bias (54.5%) in nucleotide differences between Crassostrea haplotypes and the more distantly related O. lurida. Conservation of primary sequence in all three oyster species as well as other published 16SrDNA sequences was noted for regions with apparent functional significance. We developed DNA sequence-specific discrimination techniques and employed sequence-specific PCR primers, dot-blot hybridization, and restriction digests as alternate techniques for rapid diagnosis of Crassostrea oyster larvae.

Taurine content of isolated rat alveolar type I cells.

1. Rat alveolar type I cells were isolated by enzymatic digestion and purified by centrifugal elutriation and specific surface adsorption. 2. The identity of the harvested cells was confirmed using electronic cell sizing and transmission electron microscopy. 3. Purified cell preparations contained 4.6 +/- 2.3 x 10(6) type I cells/rat lung with a purity of 79 +/- 3%. 4. Isolated type I cells exhibited the following characteristics: mean cell volume = 716 +/- 48 microns 3; diameter = 11.1 +/- 0.7 microns; and cell water content = 0.50 +/- 0.03 microliter/10(6) cells. 5. Taurine content of these alveolar type I cells was measured by HPLC. 6. The intracellular taurine concentration of type I cells was 0.14 +/- 0.07 mM, a value close to that of plasma (0.1 mM).

Hydrolysis of pyridoxine-5'-beta-D-glucoside by a broad-specificity beta-glucosidase from mammalian tissues.

Research was conducted to evaluate the ability of a broad-specificity beta-glucosidase in mammalian tissues to catalyze the hydrolytic release of free pyridoxine from pyridoxine-5'-beta-D-glucoside, a naturally occurring form of vitamin B6 in plant-derived foods. Activity was detected in liver and intestinal mucosa using tritiated pyridoxine glucoside as a substrate. In the rat and guinea pig, enzyme activity was greater in intestine than in liver or kidney while even greater activity was detected in human intestinal tissue. Reaction rates were, however, low in all tissues. Hydrolysis of the synthetic substrate 4-methylumbelliferyl-beta-D-glucoside was also greatest in intestinal tissue. The characteristics of the enzymatic hydrolysis of pyridoxine glucoside to pyridoxine included: (i) most activity in the soluble tissue fraction, (ii) a pH optimum of approximately 6.0, and (iii) inhibition caused by the addition of sodium taurocholate. These characteristics are very similar to those of the broad-specificity beta-glucosidase in mammalian tissues with respect to the hydrolysis of a variety of naturally occurring and synthetic substrates. The apparent Km was greater than 2 mM for pyridoxine glucoside hydrolysis by intestinal preparations of each species, which is much greater than expected intestinal concentrations derived from dietary sources. In vivo studies have indicated that the intestine is involved in the metabolic utilization of dietary pyridoxine glucoside. The results observed here suggest that an alternate process, possibly involving intestinal microorganisms, may also be involved in the in vivo hydrolysis of pyridoxine glucoside.

Effects of in vitro ozone exposure on peroxidative damage, membrane leakage, and taurine content of rat alveolar macrophages.

Rat alveolar macrophages (AM) were isolated by pulmonary lavage, allowed to adhere to a tissue culture flask, and then exposed to 0.45 +/- 0.05 ppm ozone. After exposures ranging from 0 to 60 min, the medium was decanted and cells were harvested. Cells were assayed for oxidant damage and media analyzed for leakage of intracellular components. Increasing length of exposure to ozone resulted in a decreased number of adherent AM and decreased cell viability. Resting and zymosan-stimulated chemiluminescence increased immediately after ozone exposure and reached a maximum at 15-30 min, then declined to initial levels after 60 min of ozone exposure. Lipid peroxidation and leakage of protein and K+ ions increased with increasing length of exposure to ozone, while leakage of reduced and oxidized glutathione increased through 30 min, then declined (reduced) or leveled off (oxidized). Activity of the Na+/K+ ATPase decreased with time while intracellular taurine concentration exhibited an initial rise, peaked at 30 min, and then returned to the untreated level. Leakage of taurine into the medium increased with time of exposure, suggesting that exposure of AM to ozone results in a shift from bound to free intracellular taurine. These data indicate that in vitro exposure of AM to ozone results in a time-dependent alteration of cell function, membrane integrity, and viability.