Separation of amino acids using ion-paired reversed-phase high-performance liquid chromatography with special reference to collagen hydrolysate

Summary The collagen study includes the analysis of its characteristic amino acids: proline, hydroxyproline, lysine, hydroxylysine. HPLC offers an interesting device if associated with on-line radiometric detection for the determination of radiolabelled amino acids in the case of metabolism studies. To avoid pre or post-column derivatization which may be poorly quantitative in the case of the hydrolysate of unpurified samples, we developed an ion-paired reversed-phase chromatography using a C8 column (econosphere C8 5µm, length: 250 mm, ID: 4.6 mm from Alltech Ass.) and an elution carried out with an acetonitrile gradient in heptane-sulfonate solution. A direct detection at 210 nm was used. Nineteen amino acids were separated within 40 min. Lag time was 7.3 min between hydroxyproline and proline, and 6.9 min between hydroxylysine and lysine. In the case of radiolabelled amino acid, there was a linear correlation (r = 0.92) between HPLC and ion-exchange chromatography.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Cell migration and proliferation are not discriminatory factors in the in vitro sociologic behavior of bronchial epithelial cell lines

A model of cellular cohesion has been developed, which permits the in vitro study of the spatial and temporal distribution of two human bronchial cell lines. The spatial distribution of cells in culture was characterized from videomicroscopic recordings and analyzed using an algorithmic program of cellular sociology based on the use of three geometrical models: Vorono?'s partition, Delaunay's graph, the and minimum spanning tree (MST). The results obtained suggested that the manner of cellular cohesiveness could be used to differentiate between the organizational behaviors of the cell lines: non-invasive 16HBE14o- cells rapidly formed clusters with a cohesive organization, whereas invasive BZR cells remained isolated and were characterized by a non-cohesive organization. Videomicroscopic and image analysis techniques also demonstrated that cell migration and proliferation are not discriminatory factors for explaining differences in the spatial organizations of the two cell lines. We concluded that the random nature of cell movement combined with the cell adhesion capacity are determinant factors in cell cluster formation.     

Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.     

Ecology of Testate Amoebae in an Amazonian Peatland and Development of a Transfer Function for Palaeohydrological Reconstruction

Tropical peatlands represent globally important carbon sinks with a unique biodiversity and are currently threatened by climate change and human activities. It is now imperative that proxy methods are developed to understand the ecohydrological dynamics of these systems and for testing peatland development models. Testate amoebae have been used as environmental indicators in ecological and palaeoecological studies of peatlands, primarily in ombrotrophic Sphagnum-dominated peatlands in the mid- and high-latitudes. We present the first ecological analysis of testate amoebae in a tropical peatland, a nutrient-poor domed bog in western (Peruvian) Amazonia. Litter samples were collected from different hydrological microforms (hummock to pool) along a transect from the edge to the interior of the peatland. We recorded 47 taxa from 21 genera. The most common taxa are Cryptodifflugia oviformis, Euglypha rotunda type, Phryganella acropodia, Pseudodifflugia fulva type and Trinema lineare. One species found only in the southern hemisphere, Argynnia spicata, is present. Arcella spp., Centropyxis aculeata and Lesqueresia spiralis are indicators of pools containing standing water. Canonical correspondence analysis and non-metric multidimensional scaling illustrate that water table depth is a significant control on the distribution of testate amoebae, similar to the results from mid- and high-latitude peatlands. A transfer function model for water table based on weighted averaging partial least-squares (WAPLS) regression is presented and performs well under cross-validation (r \(^{2}_{apparent} \,=\, 0.76, \text {RMSE} \,=\, 4.29; \mathrm {r}^{2}_{jack} \,=\, 0.68, \text {RMSEP} \,=\, 5.18\) ). The transfer function was applied to a 1-m peat core, and sample-specific reconstruction errors were generated using bootstrapping. The reconstruction generally suggests near-surface water tables over the last 3,000 years, with a shift to drier conditions at c. cal. 1218-1273 AD.     

Conservation physiology in practice: how physiological knowledge has improved our ability to sustainably manage Pacific salmon during up-river migration

Despite growing interest in conservation physiology, practical examples of how physiology has helped to understand or to solve conservation problems remain scarce. Over the past decade, an interdisciplinary research team has used a conservation physiology approach to address topical conservation concerns for Pacific salmon. Here, we review how novel applications of tools such as physiological telemetry, functional genomics and laboratory experiments on cardiorespiratory physiology have shed light on the effect of fisheries capture and release, disease and individual condition, and stock-specific consequences of warming river temperatures, respectively, and discuss how these findings have or have not benefited Pacific salmon management. Overall, physiological tools have provided remarkable insights into the effects of fisheries capture and have helped to enhance techniques for facilitating recovery from fisheries capture. Stock-specific cardiorespiratory thresholds for thermal tolerances have been identified for sockeye salmon and can be used by managers to better predict migration success, representing a rare example that links a physiological scope to fitness in the wild population. Functional genomics approaches have identified physiological signatures predictive of individual migration mortality. Although fisheries managers are primarily concerned with population-level processes, understanding the causes of en route mortality provides a mechanistic explanation and can be used to refine management models. We discuss the challenges that we have overcome, as well as those that we continue to face, in making conservation physiology relevant to managers of Pacific salmon.     

Polymorphisms in IL12A and cockroach allergy in children with asthma

Hereditary angioedema is a serious medical condition caused by a deficiency of C1-inhibitor. The condition is the result of a defect in the gene controlling the synthesis of C1-inhibitor, which regulates the activity of a number of plasma cascade systems. Although the prevalence of hereditary angioedema is low – between 1:10,000 to 1:50,000 – the condition can result in considerable pain, debilitation, reduced quality of life, and even death in those afflicted. Hereditary angioedema presents clinically as cutaneous swelling of the extremities, face, genitals, and trunk, or painful swelling of the gastrointestinal mucosa. Angioedema of the upper airways is extremely serious and has resulted in death by asphyxiation. Subnormal levels of C1-inhibitor are associated with the inappropriate activation of a number of pathways – including, in particular, the complement and contact systems, and to some extent, the fibrinolysis and coagulation systems. Current findings indicate bradykinin, a product of contact system activation, as the primary mediator of angioedema in patients with C1-inhibitor deficiency. However, other systems may play a role in bradykinin's rapid and excessive generation by depleting available levels of C1-inhibitor. There are currently no effective therapies in the United States to treat acute attacks of hereditary angioedema, and currently available agents used to treat hereditary angioedema prophylactically are suboptimal. Five new agents are, however, in Phase III development. Three of these agents replace C1-inhibitor, directly addressing the underlying cause of hereditary angioedema and re-establishing regulatory control of all pathways and proteases involved in its pathogenesis. These agents include a nano-filtered C1-inhibitor replacement therapy, a pasteurized C1-inhibitor, and a recombinant C1-inhibitor isolated from the milk of transgenic rabbits. All C1-inhibitors are being investigated for acute angioedema attacks; the nano-filtered C1-inhibitor is also being investigated for prophylaxis of attacks. The other two agents, a kallikrein inhibitor and a bradykinin receptor-2 antagonist, target contact system components that are mediators of vascular permeability. These mediators are formed by contact system activation as a result of C1-inhibitor consumption.     

A field test of the use of pop‐off data storage tags in freshwater fishes

In the present study, pop‐off data storage tags (pDST) without any transmitting capabilities were attached to 118 adult salmonids in a 19 000 km² freshwater system (Lake Ontario). The 9·3 cm long cylindrical tags were externally attached to fishes using a backpack‐style harness, set to record pressure (dBar ≈ depth in m) and temperature every 70 s (and at some key times, every 5 s) and programmed to release from the harness and float to the surface after c. 1 year. Recapture of the bright‐orange tags for data retrieval relied on members of the public finding tags on shore, or on anglers capturing fishes with tags attached and using the contact information displayed on each tag to mail tags to the research team in exchange for a monetary reward. Thirty‐seven tags were found and returned from the 118 released (31%), while 26 of the 118 tags (22%) remained scheduled to pop‐off in summer 2017. Of the 37 tags returned, 23 were from wild‐caught fishes (out of 88 wild‐caught and tagged fishes; 26%) and yielded useful data whereas 14 were from hatchery‐reared fishes that were opportunistically tagged and appear to have been unable to acclimate to life in the wild and died days to weeks after release. The field study described here thus demonstrated that pDSTs can be a viable option for collecting large amounts of high‐resolution depth and temperature data for salmonids in freshwater systems. Technical challenges, limitations and unknowns related to the use of pDSTs with freshwater fishes are discussed. In addition, pDSTs are compared with alternate electronic tagging technologies and assessed for their potential as a more widespread tool in research on freshwater fishes.     

Effects of intracoelomic transmitter implantation on metabolic rate,swimming performance,growth and survival in juveniles of two salmonids

In this study, we investigated the effects of acoustic tag implantation on standard and routine metabolic rate (SMR and RMR, estimated via oxygen consumption), critical swimming speed (U_crit), survival and growth in juveniles of rainbow trout Oncorhynchus mykiss and lake trout Salvelinus namaycush. Tag burdens ranged from 1.8% to 7.5% across the two species. Growth rates in acoustic-tagged fish were equal to or higher than those in other treatments. Acoustic-tagged S. namaycush had a marginally lower U_crit than controls but that effect was not replicated in the O. mykiss experiment. Tagging did not have clear effects on metabolic rate but there was an interaction whereby SMR and RMR tended to increase with time since surgery in tagged O. mykiss but not in other treatments (the same trend did not occur in S. namaycush). Survival was high across treatments (mean 98% survival among O. mykiss, 97.5% among S. namaycush). There were no statistically significant effects of tag burden (percentage of body mass) except for a weak negative relationship with growth rate (across species) and a weak positive relationship with U_crit but only in the O. mykiss. Collectively, our findings suggest there were minor, context-dependent effects of acoustic tagging in juvenile S. namaycush and O. mykiss during an eight-week laboratory experiment. Further research will be required to assess whether tagging can cause meaningful behavioural effects in these species in captivity or in the wild and whether there is a tag burden threshold above which deleterious effects consistently occur.