Fixed and suspended coral nurseries in the Philippines: Establishing the first step in the “gardening concept” of reef restoration

The worldwide degradation of reef ecosystems has promoted the advocators of restoration acts to the foreground. Here, we describe the results of the first step of large-scale restoration based on the “gardening with corals” concept. During June-September 2005, two coral nurseries were established in Bolinao, the Philippines, in front of Silaqui Island, in a shallow (2 m depth) sandy lagoon. Two types of nurseries were employed: (1) suspended nursery; (2) leg-fixed nursery. The nursery held a total number of 6824 ramets, from seven coral species representing different growth forms (branching, leaf-like and sub-massive forms) and different growth rates (fast and slow growing species). Each species was represented by several genotypes. During one year, we analyzed and compared survivorship, bleaching and growth rates of fragments between the different nurseries, species and genotypes. Survivorship, which was high in both nurseries, > 85%, fluctuated between the different species indicating that different species require different rearing methodologies. Mortality and detachment was subjected to environmental conditions, especially affected by the typhoons prevailing in this part of the world. The one-year nursery phase produced sizeable colonies, especially of branching forms, suitable for transplantation.     

The Inhibition of Macrophage Foam Cell Formation by 9-Cis β-Carotene Is Driven by BCMO1 Activity

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.     

Employing a highly fragmented,weedy coral species in reef restoration

A major determinant in reef restoration programs is the choice of species employed. In this paper, we concentrate on the potential use of Montipora digitata (Dana, 1846), a highly fragmented and weedy coral species, for reef restoration and for recreation of reef physiognomy in Bolinao, The Philippines, under the ‘gardening concept’. Coral ramets (n = 1960) were reared in a shallow lagoon nursery for 1 year and then, the resulting nursery-grown colonies were transplanted onto denuded reef knolls at two different sites and according to three transplantation designs (grid design with high or low density and patch design). The nursery grown ramets, which had initially exhibited a 99% survivorship, were reduced to 87% by a super typhoon. Low detachment rates, low mortality (<1% both) and low bleaching (<3% over most months), have been indicating good nursery conditions for corals. Monitoring transplanted colonies for over 15 months showed no significant differences between the sites or among the three different transplantation designs. The major events of mortality and detachment were documented during the first 3 months post-transplantation and in the wake of a bleaching event. M. digitata transplants exhibited rapid growth (a 384% increase in ecological volume) and frequent fragmentation. Some fragments remained by their colonies of origin entangled in the attached branches, while other fragments fell onto the surrounding sandy substrate, forming M. digitata ‘beds’ around the knolls. In both, nursery and transplanted corals, creation of complex 3D structures was followed by recruitment of juvenile fishes into aggregations. Above results reveal that employing fast growing, weedy and easily fragmenting branching species, which are also considered as ‘modifier species’, in reef restoration may result in a fast regeneration of reef physiognomy. Instead of ‘copying’ natural processes, reef managers should therefore concentrate on recovering the reef's ability for self-maintenance.     

Reduced atherosclerosis and inflammatory cytokines in apolipoprotein-E-deficient mice lacking bone marrow-derived interleukin-1α

Objective

Interleukin (IL)-1α and IL-1β are products of macrophages, endothelial cells and vascular smooth muscle cells; moreover, each of these cell types is affected by the pro-inflammatory properties of both IL-1’s. Whereas several studies demonstrate the proatherogenic properties of IL-1β, the role of IL-1α in atherogenesis remains unclear. We assessed whether IL-1α and IL-1β from tissue resident vascular cells or emigrating bone marrow-derived cells promote the development of atherosclerosis in apoE−/− mice and determined the effect of selective macrophage IL-1α or IL-1β deficiency on degradation of LDL and cytokine production.

Methods

We generated strains of double knock-out (KO) mice (apoE−/−/IL-1α−/− and apoE−/−/IL-1β−/−) and created chimeras consisting of apoE−/− mice reconstituted with bone marrow-derived cells from apoE−/−/IL-1+/+, apoE−/−/IL-1α−/− and apoE−/−/IL-1β−/−.

Results

The areas of aortic sinus lesions were lower in either double KO mice compared to solely apoE−/− mice, despite higher non-HDL cholesterol levels. Importantly, selective deficiency of IL-1α or IL-1β in bone marrow-derived cells inhibited atherogenesis to the same extent as in double KO mice without affecting plasma lipids. Aortic sinus lesions in apoE−/− mice transplanted with IL-1β−/− or IL-1α−/− cells were 32% and 52% lower, respectively, than in IL-1+/+ transplanted mice. Ex vivo, isolated IL-1α−/− macrophages from atherosclerotic mice degraded LDL and secreted IL-6, TNFα and IL-12 similarly to IL-1+/+ macrophages; however, IL-1α deficient macrophages secreted reduced levels of IL-1β (−50%) and 2–3-fold higher levels of the anti-inflammatory cytokine IL-10.

Conclusion

We show for the first time that it is IL-1α from bone marrow-derived cells that accelerates atherogenesis in apoE-deficient mice rather than constitutive IL-1α in vascular cells, possibly by increasing the inflammatory cytokine profile of macrophages.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Plasma Membrane Sterols Are Essential for Sensing Osmotic Changes in the Halotolerant Alga Dunaliella

The halotolerant alga Dunaliella responds to hyperosmotic stress by synthesis of massive amounts of glycerol. The trigger for this osmotic response is the change in cell volume, but the mechanism that senses volume changes is not known. Preincubation of Dunaliella salina with tridemorph, a specific inhibitor of sterol biosynthesis, inhibits glycerol synthesis and volume recovery. The inhibition is associated with suppression of [14C]bicarbonate incorporation into sterols and is correlated with pronounced depletion of plasma membrane sterols. Incubation of sterol-depleted cells with cholesterol hemisuccinate restores the capacity for volume regulation in response to hyperosmotic stress. Tridemorph as well as lovastatin also inhibit volume changes that are induced by high light in Dunaliella bardawil, a species that responds to high light intensity by synthesis of large amounts of [beta]-carotene. These volume changes result from accumulation of glycerol and are associated with de novo synthesis of sterols. The major plasma membrane sterol in D. salina and the high-light-induced sterol in D. bardawil co-migrate with ergosterol on thin-layer chromatography and on reversed-phase high-performance liquid chromatography. These results suggest that the osmosensory mechanism in Dunaliella resides in the plasma membrane, and that sterols have an important role in sensing osmotic changes.     

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment

Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day x 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.     

The biotechnology of cultivating Dunaliella for production of β-carotene rich algae

Dunaliella accumulates massive amounts of β-carotene when cultivated under high light intensity and growth-limiting conditions. The pathway for biosynthesis of β-carotene was elucidated by analysis of the effect of selected inhibitors. The presence of the inhibitors elicited the accumulation of the following intermediates: β-zeacarotene, lycopene, ζ-carotene, phytofluene, phytoene and a few unidentified long-chain isoprenoids. Each of the accumulated intermediates was composed of about equal amounts of two stereisomers, as is the case for β-carotene in the untreated algae. It is deduced, therefore, that the isomerization reaction occurs early in the pathway of β-carotene biosynthesis, at or before phytoene.The unique caratenogenesis properties of Dunaliella led to the development of a new biotechnological process for mass-cultivation of the alga. Commercial production facilities for β-carotene rich Dunaliella exist today in Israel, USA, Australia, Spain and China. Recent developments, which indicate that the stereoisometric mixture of β-carotene present in Dunaliella is preferentially absorbed in animal tissues, coupled with new evidence for the efficacy of β-carotene in reducing the incidence of cancer, have opened new vistas of potential markets for the high β-carotene algae.     

PRODUCTION AND SELECTION OF HIGH β-CAROTENE MUTANTS OF DUNALIELLA BARDAWIL (CHLOROPHYTA)1

We describe a procedure for the selection of β-carotenerich mutants of the halotolerant alga Dunaliella bardawil Ben-Amotz & Avron. Under normal growth conditions the isolated mutants had a several-fold higher content of β-carotene than the wild type. Under carotene-induction conditions, the mutants also possessed a higher β-carotene content than the wild type. Both the production rate of phytoene and the conversion rate of phytoene to lycopene and β-carotene were accelerated in the mutants. Cycloheximide, which (in the wild type) inhibits the inductive synthesis of the proteins required for β-carotene production, had a much smaller effect on β-carotene biosynthesis in the mutants. We suggest that the mutants are affected in the regulatory path, which controls the induction of high β-carotene production in Dunaliella.