Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Environmental symbiont acquisition may not be the solution to warming seas for reef-building corals

Background

Coral reefs worldwide are in decline. Much of the mortality can be attributed to coral bleaching (loss of the coral''s intracellular photosynthetic algal symbiont) associated with global warming. How corals will respond to increasing oceanic temperatures has been an area of extensive study and debate. Recovery after a bleaching event is dependent on regaining symbionts, but the source of repopulating symbionts is poorly understood. Possibilities include recovery from the proliferation of endogenous symbionts or recovery by uptake of exogenous stress-tolerant symbionts.

Methodology/Principal Findings

To test one of these possibilities, the ability of corals to acquire exogenous symbionts, bleached colonies of Porites divaricata were exposed to symbiont types not normally found within this coral and symbiont acquisition was monitored. After three weeks exposure to exogenous symbionts, these novel symbionts were detected in some of the recovering corals, providing the first experimental evidence that scleractinian corals are capable of temporarily acquiring symbionts from the water column after bleaching. However, the acquisition was transient, indicating that the new symbioses were unstable. Only those symbiont types present before bleaching were stable upon recovery, demonstrating that recovery was from the resident in situ symbiont populations.

Conclusions/Significance

These findings suggest that some corals do not have the ability to adjust to climate warming by acquiring and maintaining exogenous, more stress-tolerant symbionts. This has serious ramifications for the success of coral reefs and surrounding ecosystems and suggests that unless actions are taken to reverse it, climate change will lead to decreases in biodiversity and a loss of coral reefs.     

Snapshot prediction of carbon productivity,carbon and protein content in a Southern Ocean diatom using FTIR spectroscopy

Diatoms, an important group of phytoplankton, bloom annually in the Southern Ocean, covering thousands of square kilometers and dominating the region''s phytoplankton communities. In their role as the major food source to marine grazers, diatoms supply carbon, nutrients and energy to the Southern Ocean food web. Prevailing environmental conditions influence diatom phenotypic traits (for example, photophysiology, macromolecular composition and morphology), which in turn affect the transfer of energy, carbon and nutrients to grazers and higher trophic levels, as well as oceanic biogeochemical cycles. The paucity of phenotypic data on Southern Ocean phytoplankton limits our understanding of the ecosystem and how it may respond to future environmental change. Here we used a novel approach to create a ‘snapshot'' of cell phenotype. Using mass spectrometry, we measured nitrogen (a proxy for protein), total carbon and carbon-13 enrichment (carbon productivity), then used this data to build spectroscopy-based predictive models. The models were used to provide phenotypic data for samples from a third sample set. Importantly, this approach enabled the first ever rate determination of carbon productivity from a single time point, circumventing the need for time-series measurements. This study showed that Chaetoceros simplex was less productive and had lower protein and carbon content during short-term periods of high salinity. Applying this new phenomics approach to natural phytoplankton samples could provide valuable insight into understanding phytoplankton productivity and function in the marine system.     

Sex differences in reproductive investment: maternal care reduces escape response capacity in the whelk Buccinum undatum

We evaluated the effect of reproductive state and biochemical status of male and female whelks Buccinum undatum on their escape responses (foot contortions) when touched by their major predator, the sea star Leptasterias polaris. Gonad growth in males was not associated with a decrease in their general energetic and metabolic capacity, nor in their capacity for escape responses, but did reduce the rate of foot contortions during recuperation from exhaustive exercise. Similarly, the energetic status of females was good when the ovary was mature, suggesting that oogenesis did not require somatic reserves. On the other hand, the energetic status and muscle metabolic capacities of females dropped with egg laying. This decrease was associated with a reduced capacity for escape responses. Thus, egg laying resulted in (1) major drops in the carbohydrate and protein contents of the foot, (2) decreased activities of several glycolytic enzymes (LDH, ADH and APK) in the foot muscle, (3) a sharp drop in the digestive gland index, (4) a decrease in the number of foot contortions and (5) a decreased ability to recover from exhaustive escape exercise. Reproductive costs are much greater for females than males (as females must produce protective egg capsules, search for egg-laying sites and lay the capsules). Females have greater escape capacities than males, except directly after egg laying when their energetic reserves are virtually depleted. The greater capacity of females for escape responses may attenuate the risks associated with their greater boldness in stealing food from their predator L. polaris, particularly prior to egg laying.     

Kinetic studies on reactions of iron-sulphur proteins. Oxidation of the reduced form of Spirulina platensis [2Fe-2S] ferredoxin with inorganic complexes.

Kinetic results are presented for the reaction of reduced [2Fe-2S] ferredoxin from the blue-green alga Spirulina platensis with Co(NH3)6(3+), Co(edta)- and Co(acac)3 as oxidants at pH 8.0 at I0.10 (NaCl). The aim is to compare results obtained with those previously reported for the [2Fe-2S] ferredoxin from parsley, where the two ferredoxins under consideration are in evolutionary terms widely divergent (35% amino acid variations). The three oxidants chosen have different ligand sets and different charges, and are the complexes that in previous studies have given greatest diversity in behaviour. With Co(NH3)6(3+) first-order rate constants (oxidant in large excess) tend to a limiting value with increasing concentration of oxidant. With Co(edta)- and Co(acac)3 there is no similar tendency to limiting behaviour and a first-order dependence on oxidant is observed. The temperature-dependence of the Co(NH3)6(3+) reaction was investigated, and values were obtained for delta H0 [19.8kJ X mol-1 (4.7kcal X mol-1)] and delta S0 [129.3J X K-1 X mol-1 (30.9 cal X K-1 X mol-1)] for the association step that occurs before electron transfer. Whereas redox-inactive Cr(NH3)6(3+) displays competitive inhibition in the reaction of Co(NH3)6(3+), it accelerates the reaction of Co(edta)-, and only partially blocks the reaction with Co(acac)3. Results obtained are similar to those previously reported for parsley (and spinach) ferredoxin. It is concluded that electrostatics play a dominant role and that a negatively charged functional site on the protein common to all three ferredoxins is influential. Conserved negative patches at positions 67-69 and 94-96 within 1.0 nm (10A) of an Fe atom of the active site, as well as the exposed S atoms of cysteine residues 41 and 46, which are a part of the Fe2S*2(SR)4(3-) cluster, are the most likely possibilities. The various effects of Cr(NH3)6(3+) provide a means of testing for utilization of the same site in reactions of the ferredoxins with physiological partners.     

Evaluation of glucose transport and its regulation by insulin in human monocytes using flow cytometry.

BACKGROUND: We investigated the effects of insulin on glucose transport in human monocytes using flow cytometry, a method with several advantages over previously used techniques. We hypothesized that monocytes could be used as tools to study insulin action at the cellular level and facilitate the investigation of mechanisms that lead to insulin resistance. METHODS: Blood was withdrawn from 38 healthy subjects. The expression of glucose transporter (GLUT) isoforms in plasma membrane and the rates of glucose transport were determined with and without insulin (10 to 1,000 mU/L). Anti-CD14 phycoerythrin monoclonal antibody was used for monocyte gating. GLUT isoforms were determined after staining cells with specific antisera to GLUT1, GLUT3, and GLUT4. Glucose transport was monitored with 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG). RESULTS: Insulin increased the uptake of NBDG (median effective dose 20 mU/L) and the expression of GLUT3 and GLUT4 isoforms in the plasma membrane (median effective doses 20 and 35 mU/L, respectively) but had no effect on GLUT1. Maximal effects were always reached at 100 mU/L of insulin. CONCLUSIONS: Monocytes may be a valid model system to study the effects of insulin on glucose transport. Further, flow cytometry is suitable for this investigation and can be used as an alternative to radiotracer methods.     

Real-time detection of BRCA1 gene mutations using a monolithic silicon optocoupler array

The monolithic silicon optocoupler presented here offers a platform for a new generation of fully integrated devices fabricated through the mature and inexpensive silicon technology. Using the developed optocoupler, real-time detection of the most common mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was accomplished. For this purpose, oligonucleotides corresponding to wild- and mutant-type sequences were immobilized onto different optocouplers and the hybridization with fluorescently labeled complementary or non-complementary sequences was monitored in real time. Hybridization of fluorescently labeled oligonucleotides to the immobilized ones modulated the coupling efficiency between the light emitting diode and the detector in a concentration-dependent manner. Using as label the AlexaFluor 647 dye (whose absorption maximum fits the emission maximum of the light source) a detection limit of 0.9 nM (9 fmol) was achieved. Real-time signal monitoring, especially during dehybridization, improved considerably the discrimination between wild-type and mutant sequences due to the ability to calculate dissociation kinetics upon washing independently for each one mutation. The bioanalytical capabilities of the transducer along with the fact that dense transducer arrays can be fabricated on a single chip open new frontiers in the manufacturing of microsystems appropriate for point-of-care analysis.