Astaxanthin production in transgenicArabidopsis with chyB gene encoding β;-carotene Hydroxylase

Oxycarotenoids, produced through the oxidation of carotenoids, play critical roles in plants. This reaction is mediated by a specific enzyme, β;-carotene hydroxylase, which adds hydroxyl groups to the β;-rings of carotenes. To investigate the effect of the β;-carotene hydroxylase gene (Chyb) on oxycarotenoid biosynthesis, we generated transgenicArabidopsis plants that over-expressedChyb under the control of a 35S promoter. Their levels of zeaxanthin and neoxanthin were two- to three-fold greater relative to the WT, while that of violaxanthin, a final product in the xanlthophyll pathway, was 1.3-fold higher than the control. In contrast, the amount of β;-carotene declined as much as 2.4-fold, depending on the particular transgenic line. Interestingly, astaxanthin was produced in the transgenics, but not in the WT. These data suggest that, with the aid of unknown factors in the host, carotenoids could be converted into metabolites in the astaxanthin biosynthetic pathway. Microarray analysis was used lo identify several genes that were consistently up-or down-regulated in transgenic chyB leaves compared with the controls. Here, we also discuss possible modifications in leaf carotenoids, and the importance of these data from a nutritional standpoint. These authors contributed equally to this work.     

NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.     

Soil type does not affect seed ageing when soil water potential and temperature are controlled

To investigate the effects of soil type on seed persistence in a manner that controlled for location and climate variables, three weed species—Gomphocarpus physocarpus (swan plant), Avena sterilis ssp. ludoviciana (wild oat) and Ligustrum lucidum (broadleaf privet)—were buried for 21 months in three contrasting soils at a single location. Soil type had a significant effect on seed persistence and seedling vigour, but soil water content and temperature varied between soils due to differences in physical and chemical properties. Warmer, wetter conditions favoured shorter persistence. A laboratory-based test was developed to accelerate the rate of seed ageing within soils, using controlled superoptimal temperature and moisture conditions (the soil-specific accelerated ageing test, SSAAT). The SSAAT demonstrated that soil type per se did not influence seed longevity. Moreover, the order in which seeds aged was the same whether aged in the field or SSAAT, with L. lucidum being shortest-lived and A. sterilis being longest-lived of the three species.     

Trabeculae carneae as models of the ventricular walls: implications for the delivery of oxygen

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure (PO₂) in the coronary arterioles is identical in specimens from both ventricles, whereas that of ventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a “natural laboratory” in which to examine the influence of “extravascular” PO₂ on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated segmentation techniques to distinguish capillaries from myocytes. We quantified the capillary-to-myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.     

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.     

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.     

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.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.