TRIP8b Is Required for Maximal Expression of HCN1 in the Mouse Retina

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.     

Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters

“Super-blooms” of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species, were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystin-poisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine mammals due to cyanotoxins and confirms the existence of a novel class of marine “harmful algal bloom” in the Pacific coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from microcystin poisoning when consuming shellfish harvested at the land-sea interface.     

Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH

HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gram-negative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and colonize the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and partially unfolds. Studies with chemical reducing agents previously suggested that the intramolecular disulfide bond is important for maintaining residual structure in HdeA at low pH and may be responsible for positioning exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative cysteine to serine mutation of the disulfide. We found that, although residual structure is greatly diminished at pH 2 without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.     

Correlation of <Emphasis Type=Italic>trans</Emphasis>-Lycopene Measurements by the HPLC Method with the Optothermal and Photoacoustic Signals and the Color Readings of Fresh Tomato Homogenates

The trans-lycopene content of fresh tomato homogenates was assessed by means of the laser photoacoustic spectroscopy, the laser optothermal window, micro-Raman spectroscopy, and colorimetry; none of these methods require the extraction from the product matrix prior to the analysis. The wet chemistry method (high-performance liquid chromatography) was used as the absolute quantitative method. Analytical figures of merit for all methods were compared statistically; best linear correlation was achieved for the chromaticity index a* and chroma C*.     

Wet-dry cycling extends seed persistence by re-instating antioxidant capacity

Seeds in the field experience wet-dry cycling that is akin to the well-studied commercial process of seed priming in which seeds are hydrated and then re-dried to standardise their germination characteristics. To investigate whether the persistence (defined as in situ longevity) and antioxidant capacity of seeds are influenced by wet-dry cycling, seeds of the global agronomic weed Avena sterilis ssp. ludoviciana were subjected to (1) controlled ageing at 60% relative humidity and 53.5°C for 31 days, (2) controlled ageing then priming, or (3) ageing in the field in three soils for 21 months. Changes in seed viability (total germination), mean germination time, seedling vigour (mean seedling length), and the concentrations of the glutathione (GSH) / glutathione disulphide (GSSG) redox couple were recorded over time. As controlled-aged seeds lost viability, GSH levels declined and the relative proportion of GSSG contributing to total glutathione increased, indicative of a failing antioxidant capacity. Subjecting seeds that were aged under controlled conditions to a wet-dry cycle (to ?1 MPa) prevented viability loss and increased GSH levels. Field-aged seeds that underwent numerous wet-dry cycles due to natural rainfall maintained high viability and high GSH levels. Thus wet-dry cycles in the field may enhance seed longevity and persistence coincident with re-synthesis of protective compounds such as GSH.     

Allozyme diversity in Chinese,Seguin and American chestnut (Castanea spp.)

Allozyme genetic variability in three chestnut (Castanea) species was investigated using 19 loci from ten enzyme systems. G-tests of heterogeneity of isozymic allele distribution showed significant differences between the three species at 15 of the 19 loci, and between the 13 C. mollissima populations at 13 of the 19 loci examined. C. mollissima was found to possess a significantly-higher value of mean gene heterozygosity (H=0.3050±0.0419), the percentage of polymorphic loci (P=84.21%) and the average number of alleles per locus (A=2.05), than any other species in the Castanea section Eucastanon. When the genetic variability of populations of C. mollissima from four regions in China was investigated, the population from the Changjiang river region showed a markedly higher mean gene heterozygosity (H=0.3480±0.0436) than populations from the other regions. Genetic relationships among the four regions were assessed by Nei's genetic identity I and standard genetic distance D. An approximately-identical distance between the population from the Changjiang river region and populations from the three other regions was observed, while populations from the latter regions showed almost the same genetic distance from each other. These data, when considered with information existing prior to this study, contribute to an understanding of the possible origin and progenitor of the chestnut species.     

Lack of response to all-trans retinoic acid supplementation in adult dogs following left pneumonectomy.

We showed previously that removing 55-58% of the lung by right pneumonectomy (R-PNX) in adult dogs triggers compensatory growth of the remaining lung, but removing 42-45% of the lung by left PNX (L-PNX) does not. We also showed that, following R-PNX, supplemental all-trans retinoic acid (RA) selectively enhances alveolar capillary endothelial cell volume (Yan X, Bellotto DJ, Foster DJ, Johnson RL, Jr., Hagler HH, Estrera AS, and Hsia CC. J Appl Physiol 96: 1080-1089, 2004). We hypothesized that RA supplementation might enhance compensation following L-PNX and tested this hypothesis by administering RA (2 mg.kg(-1).day(-1), 4 days/wk) or placebo orally to litter-matched adult foxhounds for 4 mo following L-PNX. Resting lung function was measured under anesthesia. Air and tissue volumes of the remaining lung were assessed by high-resolution computed tomography scan and by detailed postmortem morphometric analysis of the fixed lung. There was no significant difference in resting lung function, lung volume, alveolar structure, or septal ultrastructure between RA and placebo treatment groups. We conclude that RA supplementation does not induce post-PNX compensatory lung growth in the absence of existing cellular growth activities initiated by other primary signals.