Crystallization of water in multilamellar vesicles

The two-step crystallization of water in multilamellar vesicles (MLVs) of phosphatidylcholines has been investigated. The main crystallization occurs near -15 degrees C and involves bulk water. Contrary to unilamellar vesicles, a sub-zero phase transition is observed for MLVs at -40 degrees C that corresponds to the crystallization of interstitial water, as proved by Fourier transform infrared absorption and differential scanning calorimetry (DSC) experiments. Furthermore, by means of the DSC method and, more specifically, using the enthalpy change values Delta H(sub) at the sub-zero transition, the number of water molecules per 1,2-dipalmitoylphosphatidylcholine (DPPC) molecule giving rise to this transition has been estimated for different H(2)O/DPPC molar ratios. The curve of the molecular fraction of water molecules involved in the sub-zero transition versus the H(2)O/DPPC molar ratio exhibits a maximum for H(2)O/DPPC equal to 27 (40% in mass of water) and tends towards zero for H(2)O/DPPC ratio values approaching that of the swelling limit of the membrane. A smaller enthalpy value of the sub-zero transition is found for 1-oleoyl-2-palmitoyl-3-phosphatidylcholine (OPPC) than for DPPC. This may be explained by the decrease of interstitial water's quantity when the lipid contains an unsaturated chain. When troxerutin, a hydrophilic drug, is added to the DPPC multilayers, the decrease of Delta H(sub) and melting enthalpy of bulk water is attributed to a decrease of the entropy of the liquid phase owing to the network of water molecules surrounding troxerutin molecules. In all cases, the experiments revealed that the sub-zero transition occurs only in the presence of excess water with respect to the swelling limit of membranes. This evidence could be, at least qualitatively, related to an increase of membrane pressure on interstitial water subsequent to bulk water crystallization.     

Cellular lipid dynamics

During the past years, the notion of microdomains at the surface of cellular membranes has been developed. These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol. To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins. These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them. They are present in surface receptors and endocytosis occurs upon binding of the specific ligands. Thus these rafts participate to major aspects of cellular dynamics. These rafts are complex structures, insoluble in non-ionic detergents. According to the detergent used, many types of rafts can be isolated. Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface. It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface. This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures. There is both a cellular and a tissue specificity of these constituents. Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer. Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function. Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism. The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease. Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.     

Cytoplasmic and nuclear phospholipase C-beta 1 relocation: role in resumption of meiosis in the mouse oocyte

The location of the phospholipase C beta 1-isoform (PLC-beta 1) in the mouse oocyte and its role in the resumption of meiosis were examined. We used specific monoclonal antibodies to monitor the in vitro dynamics of the subcellular distribution of the enzyme from the release of the oocyte from the follicle until breakdown of the germinal vesicle (GVBD) by Western blotting, electron microscope immunohistochemistry, and confocal microscope immunofluorescence. PLC-beta 1 became relocated to the oocyte cortex and the nucleoplasm during the G2/M transition, mainly in the hour preceding GVBD. The enzyme was a 150-kDa protein, corresponding to PLC-beta 1a. Its synthesis in the cytoplasm increased during this period, and it accumulated in the nucleoplasm. GVBD was dramatically inhibited by the microinjection of anti-PLC-beta1 monoclonal antibody into the germinal vesicle (GV) only when this accumulation was at its maximum. In contrast, PLC-gamma 1 was absent from the GV from the time of release from the follicle until 1 h later, and microinjection of anti-PLC-gamma 1 into the GV did not affect GVBD. Our results demonstrate a relationship between the relocation of PLC-beta 1 and its role in the first step of meiosis.     

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.Magnetotactic bacteria are a metabolically, morphologically, and phylogenetically heterogeneous group of prokaryotes that passively align and actively swim along magnetic field lines (3). This behavior, called magnetotaxis, is due to the presence of intracellular, membrane-bounded, single-magnetic-domain crystals of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) (3).Most known cultured magnetotactic bacteria are mesophilic and do not grow much above 30°C (e.g., Magnetospirillum species and Desulfovibrio magneticus strains MV-1 and MC-1 [D. A. Bazylinski, unpublished data]). Uncultured magnetotactic bacteria have been observed in numerous habitats that were mostly at 30°C and below. There is only one report describing thermophilic magnetotactic bacteria despite a number of efforts to look for them (e.g., in hydrothermal vents [D. A. Bazylinski, unpublished data]). Nash (12) reported the presence of thermophilic magnetotactic bacteria in microbial mats at about 45 to 55°C adjacent to the main flow in Little Hot Creek (but not in other springs in the same area at 40 to 80°C) and in microbial mats of other springs in central California at up to 58°C, all on the east side of the Sierra Nevada mountains. Cells biomineralized bullet-shaped crystals of magnetite and were phylogenetically affiliated with the phylum Nitrospirae (12). Few additional details were provided regarding the organisms and their habitat.In this study, water and surface sediment samples were taken from the Great Boiling Springs (GBS) geothermal field in Gerlach, NV. GBS is a series of hot springs that range from ambient temperature to ∼96°C (2, 5). The geology, chemistry, and microbial ecology of the springs have been described in some detail (2, 5). The pHs of the samples ranged from 6.4 to 7.5, while the salinities were about 4 to 5 ppt, as determined with a handheld Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH). Samples were examined for the presence of magnetotactic bacteria using the hanging drop technique on-site and in the laboratory at room temperature with and without magnetic enrichment of the sample (15). Some samples taken back to the laboratory were kept at an elevated temperature (∼62°C), while others were kept at ambient temperature. There did not appear to be a significant difference in the number of magnetotactic cells in samples taken back to the laboratory and kept at these two temperatures. Only one morphotype of magnetotactic bacteria was found in samples from nine springs whose temperatures ranged from 32 to 63°C, and we estimate their numbers to be between 10³ to 10⁵ cells ml⁻¹ in surface sediments in sample bottles. We did not observe magnetotactic cells of this type in a large number of springs or pools that were at <32°C. Only one spring positive for the presence of these magnetotactic bacteria had sediment that was partially covered with a microbial mat, while sediment at most of the springs was dark gray in color. Cells were small (1.8 ± 0.4 by 0.4 ± 0.1 μm; n = 59), Gram negative, vibrioid to helicoid in morphology, and possessed a single polar flagellum (Fig. ​(Fig.1A).1A). Magnetotactic bacteria were not observed in springs that were at 67°C and above, suggesting the maximum survival and perhaps growth temperature for the organism is about 63°C. In the lab, cells remained viable and motile in samples kept at 25 to 62°C for several months. We refer to this organism as strain HSMV-1.Open in a separate windowFIG. 1.Transmission electron microscope (TEM) images of cells and magnetosomes of strain HSMV-1. (A) TEM image of unstained cell of HSMV-1 showing a single polar flagellum and a single chain of bullet-shaped magnetosomes. The electron-dense structures at the poles were found to be phosphorus-rich based on energy-dispersive X-ray analysis (data not shown) and therefore likely represent polyphosphate granules. (B) Higher-magnification TEM image of the magnetosome chain. (C) High-magnification TEM image of magnetosomes from which a selected area electron diffraction (SAED) pattern was obtained (inset of B). The SAED pattern corresponds to the [1 0−1] zone of magnetite, Fe₃O₄: reflection o, (0 0 0); reflection a, (1 −1 1) (0.48 nm); reflection b, (1 1 1) (0.48 nm); reflection c, (2 0 2) (0.30 nm); angle a-o-b, 70.5°. (D) Iron, sulfur, and oxygen elemental maps, derived from energy-filtering transmission electron microscopy (EFTEM), showing that the positions of the magnetosome crystals correlate with increased concentrations of Fe and O, but not S, consistent with the iron oxide magnetite (Fe₃O₄).Cells of HSMV-1 biomineralized a single chain of magnetosomes that traversed the cells along their long axis (Fig. 1A to C). Selected area electron diffraction (SAED) and energy-filtering transmission electron microscopy (EFTEM) elemental maps were determined on magnetosome crystals using a Tecnai model G2 F30 Super-Twin transmission electron microscope (FEI Company, Hillsboro OR). SAED patterns of HSMV-1 magnetosome crystals (Fig. ​(Fig.1B,1B, inset) indicated that they consisted of magnetite, while EFTEM elemental maps (Fe, O and S) (Fig. ​(Fig.1D)1D) clearly showed that the crystals consisted of an iron oxide and not an iron sulfide, again consistent with the mineral magnetite. Cells contained an average of 12 ± 6 magnetosome crystals per cell (n = 15 cells) that averaged 113 ± 34 by 40 ± 5 nm in size (n = 179). A plot of the length of the crystals as a function of the shape factor (width/length ratio) is provided in Figure S1 in the supplemental material and shows that the crystals fit in the theoretical single-magnetic-domain size range (4), along with all known mature magnetosome magnetite crystals from magnetotactic bacteria (3).Whole-cell PCR amplification of the 16S rRNA gene was performed by first magnetically purifying cells of HSMV-1 using the “capillary racetrack” described by Wolfe et al. (18). Purity of the collected cells was determined by microscopic examination, and contaminating cells were never observed. The 16S rRNA gene was amplified using bacteria-specific primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ (11). PCR products were cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI). Six of eight clones sequenced had identical inserts.Alignment of 16S rRNA gene sequences was performed using the CLUSTAL W multiple alignment accessory application in the BioEdit sequence alignment editor (7). Phylogenetic trees were constructed using MEGA version 4 (17) by applying the neighbor-joining method (14). Bootstrap values were calculated with 1,000 replicates. The 16S rRNA gene sequence of strain HSMV-1 places the organism in the phylum Nitrospirae (Fig. ​(Fig.2),2), with its closest relative in culture being Thermodesulfovibrio hydrogeniphilus (87.2% identity) (8). Two other uncultured magnetotactic bacteria are phylogenetically affiliated with the phylum Nitrospirae, including the unnamed rod-shaped bacterium strain MHB-1 (86.5% identity) (6) and the very large Candidatus Magnetobacterium bavaricum (86.4% identity) (16). Interestingly, all the magnetotactic bacteria associated with the phylum Nitrospirae thus far (e.g., Candidatus Magnetobacterium bavaricum) contain bullet-shaped magnetite crystals in their magnetosomes.Open in a separate windowFIG. 2.Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HSMV-1 in the phylum Nitrospirae. Bootstrap values at nodes are percentages of 1,000 replicates. The magnetotactic bacteria Desulfovibrio magneticus and Candidatus Magnetoglobus multicellularis (outgroup; deltaproteobacteria) were used to root the tree. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.Fluorescent in situ hybridization (FISH) was used to authenticate the 16S rRNA gene sequence. A specific Alexa594-labeled probe for HSMV-1 was designed (HSMVp, 5′-CCTTCGCCACAGGCCTTCTA-3′, complementary to nucleotides 690 to 709 of the 16S rRNA molecule) based on the alignment of 10 of the most similar 16S rRNA gene sequences found in GenBank after BLAST analysis (1) and on cultivated members of the phylum Nitrospirae. FISH with the Alexa594-labeled probe was carried out after fixation of magnetically concentrated cells directly on the wells of gelatin-coated hydrophobic microscope slides with 4% paraformaldehyde. FISH was performed according to the work of Pernthaler et al. (13). The hybridization solution contained 10 ng/ml of the probe, 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na₂EDTA, and 0.01% sodium dodecyl sulfate (SDS). Cells of HSMV-1 hybridized to the HSMVp probe, while other cells in the sample did not (Fig. ​(Fig.3),3), indicating that the 16S rRNA gene sequence we obtained is from the magnetotactic bacterium under study. Strain HSMV-1 clearly represents a new genus (Fig. ​(Fig.2),2), and based on the phylogeny and what we currently know phenotypically about strain HSMV-1, we propose the name Candidatus Thermomagnetovibrio paiutensis (the GBS site was originally occupied by the Paiute Indian Tribe).Open in a separate windowFIG. 3.Fluorescent in situ hybridization (FISH) of cells of strain HSMV-1 using an HSMV-1-specific oligonucleotide rRNA probe (HSMVp). Cells used for FISH were magnetically concentrated by placing a magnet next to the side of the sample bottle for 30 min and then removed with a Pasteur pipette. This technique was used rather than the magnetic racetrack method in order to have many HSMV-1 cells as well as some other cells that could be used as a negative control. (A) Differential interference contrast (DIC) image of HSMV-1 cells (filled arrows) and other cells (negative control; empty arrows) from hot spring samples; (B) cells stained with 4′,6-diamidino-2-phenylindole (DAPI); (C) cells hybridized with the specific probe HSMVp.Nash (12) first reported thermophilic magnetotactic bacteria phylogenetically affiliated with the Nitrospirae phylum in hot springs, and it would be interesting and important to compare these organisms and their habitats. However, little can be compared at this time due to lack of information. Nash (12) reported that the one spring at Little Hot Creek was freshwater and that microbial mats were present at all springs where thermophilic magnetotactic bacteria were found. The water at our sampling sites was brackish, not freshwater, and microbial mats were not an important feature of our springs. Thus, it is difficult to determine without knowing the relationship between the organisms found by Nash (12) and strain HSMV-1 what environmental parameters are important to the growth and survival of these bacteria.It is also difficult to determine the temperature ranges for the survival and growth for strain HSMV-1 without having a pure culture. Data presented here suggest that the temperature range for both is quite wide, and this would be important for the continued presence of HSMV-1 at GBS, as temperatures in the hot springs are known to fluctuate greatly (2). Even if the maximum growth temperature of HSMV-1 is slightly lower than the maximum survival temperature (a conservative estimate) that we know of (63°C), it would still be considered a moderately thermophilic bacterium.The results presented here clearly show that some magnetotactic bacteria can be considered at least moderately thermophilic. They extend the upper temperature limit for environments where magnetotactic bacteria exist and likely grow (∼63°C) and where magnetosome magnetite is deposited, a finding that may prove significant in the study and interpretation of magnetofossils (9, 10).     

The PKC pathway and in particular its beta1 isoform is clearly involved in meiotic arrest maintenance but poorly in FSH-induced meiosis resumption of the mouse cumulus cell enclosed oocyte

PKC modulators were used to investigate the role of the PKC pathway either on the maintenance of meiotic arrest or on FSH-induced maturation of mouse cumulus cell enclosed oocytes (CEOs). (1) Whereas PKC activation (PMA 8 microM) overcomed clearly the HX-maintained meiotic arrest (83.7 +/- 3.6% vs. 16.1 +/- 10.6% GVBD oocytes), PKC inhibition (Calphostin C 100 nM) did not. On the contrary, it better maintained the meiotic arrest than HX alone. (2) No significant effect of PKC activation or inhibition was observed. (3) HX alone maintained PKCbeta1 in the cytoplasm, whereas FSH and PKC activation induced partly its translocation into the nucleus. The results show that whereas the PKC pathway is clearly involved in maintenance of the meiotic arrest through PKCbeta1, it is not involved in FSH-induced meiosis of CEOs.     

Virulence and resistance in malaria: who drives the outcome of the infection?

Theoretical and experimental studies have established the dynamic nature of virulence and that, like all traits, it has evolved. Understanding parasite evolution offers a conceptual framework for diverse fields and can contribute greatly to decision-making in disease control. Recently, Grech et al. investigated the effects of host genotype-by-parasite genotype interactions on the expression of virulence in an artificial rodent-malaria system. They found that both parasite and host effects explained most of the variance in the virulence, resistance and transmission potential. These findings are a major contribution to the emerging debate on the pros and cons of a coevolutionary approach of virulence evolution; they also hold great potential for more effective control strategies.     

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.     

Evidence for carry-over effects of predator exposure on pathogen transmission potential

Accumulating evidence indicates that species interactions such as competition and predation can indirectly alter interactions with other community members, including parasites. For example, presence of predators can induce behavioural defences in the prey, resulting in a change in susceptibility to parasites. Such predator-induced phenotypic changes may be especially pervasive in prey with discrete larval and adult stages, for which exposure to predators during larval development can have strong carry-over effects on adult phenotypes. To the best of our knowledge, no study to date has examined possible carry-over effects of predator exposure on pathogen transmission. We addressed this question using a natural food web consisting of the human malaria parasite Plasmodium falciparum, the mosquito vector Anopheles coluzzii and a backswimmer, an aquatic predator of mosquito larvae. Although predator exposure did not significantly alter mosquito susceptibility to P. falciparum, it incurred strong fitness costs on other key mosquito life-history traits, including larval development, adult size, fecundity and longevity. Using an epidemiological model, we show that larval predator exposure should overall significantly decrease malaria transmission. These results highlight the importance of taking into account the effect of environmental stressors on disease ecology and epidemiology.     

Forest ecosystem genomics and adaptation: EVOLTREE conference report

This article is a summary report of the international conference "Forest ecosystem genomics and adaptation" organized by the EVOLTREE Network of Excellence in San Lorenzo de El Escorial (Madrid), Spain, from 9 to 11 June 2010. Main achievements and results of the network are presented for the eight thematic sessions and a stakeholder session. The conference has shown that adaptive responses of trees to biotic or abiotic selection pressures can now be investigated at the gene level for traits of adaptive significance. Candidate genes have been catalogued for phenological and drought-related traits in important tree families (Salicaceae, Fagaceaea and Pinaceae), and their variation in natural populations is being explored. Genomics can now be integrated in ecological research to investigate evolutionary response to climate changes in a wide range of species. New avenues of research were also highlighted as the exploration of gene networks involved in adaptive responses and the combination of experimental and modelling approaches to disentangle components of evolutionary changes triggered by climate change. The main focus of the conference was the adaptation of trees to environmental changes. The conference was organized in eight thematic sessions ranging from genomic approaches aiming at identifying genes of adaptive significance to practical issues regarding mitigation options for combating climate change. A dialogue between scientists and end users took place in the form of an ad hoc stakeholder session. A panel of end users from various forest and policy-making institutions expressed their expectations, and the discussions with the scientists addressed the potential applications of research findings to the management of genetic resources in the context of climate changes. The conference was introduced by two keynote speakers Dr. Pierre Mathy from the European Commission, Directorate General of Research, and Dr. Allen Solomon, former National Program Leader for Global Change, US Forest Service. All the thematic sessions were introduced by high-level invited speakers from the respective fields.