A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry     

Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes

The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids. They consist of two nonequivalent polar heads, linked by a C40 alkylic component. The molecular organization of these lipids in the plasma membrane is still a matter of study. In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness. Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range. Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes. Measurements are also extended to different solvent systems. Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains. The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Incorporation into lipid bilayer membranes of a photo-sensitive pigment from the honeybee compound eye

An increase of electrical conductance up to a factor $\text{10}{}^2\text{-5·10}{}^2$ was obtained by adding, in the dark, the honeybee photopigment to a positively charged lipid bilayer. The increase in conductance was made slower by illuminating the system during the incorporation of the protein into the membrane and it was negligible when the photopigment was bleached before the incorporation. The interaction of the photopigment with the membrane is tentatively interpreted in terms of formation of channels.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Messinian Lago-Mare deposits near the Strait of Gibraltar (Malaga Basin,S Spain)

A new stratigraphic unit (named LM unit) located at the base of the Pliocene marine succession in the eastern sector of the Malaga Basin has been distinguished. This unit is composed of two fining-upward sequences bounded by a discontinuity related to synsedimentary tectonic activity. Both sequences are made up of basal alluvial deposits overlain by subaquatic deposits. The lower sequence contains rich mollusc faunas characterized by bivalves Lymnocardiinae and Dreissenidae of Paratethyan origin, and non-marine gastropods, typical of oligo-low mesohaline shallow waters, and low diversified ostracod faunas of Paratethyan origin (Loxocorniculina djafarovi, Tyrrhenocythere pontica, Euxinocythere (Maeotocythere) praebaquana and Amnicythere propinqua) typical of low mesohaline waters. Alternances of monospecific ostracod assemblages made of Cyprideis agrigentina or oligotypic assemblages made of C. agrigentina and Loxoconcha spp. and richer assemblages made of L. djafarovi accompanied by several Amnicythere spp. and Camptocypria sp.1 characterize the upper sequence, pointing to more unstable environment affected by salinity and depth changes. Some samples collected from this upper sequence yielded rare, planktonic and benthic foraminifera, which could suggest the sporadic establishment of quasi-normal marine conditions in the westernmost Mediterranean during the deposition of this sequence. The palaeontological data and the palaeomagnetic results obtained in both the LM unit and the overlying Pliocene marine sediments restrict the age of the former to the post-evaporitic late Messinian. The age and the environmental conditions correlate these deposits with the Lago-Mare event occurring throughout the Mediterranean before the quick and widespread marine flooding of the Mediterranean at the beginning of the Pliocene.     

The late Messinian Lago-Mare episode in the Mediterranean Basin: Preliminary report on the occurrence of Paratethyan ostracod fauna from central Crete (Greece)

In the Crete Island, late Messinian Lago-Mare facies are not well known. At present, the occurrence in Crete of the uppermost Messinian post evaporitic deposits is a matter of debate. According to several authors, the well-known late Messinian Lago-Mare facies does not occur in Crete. In this paper the preliminary results obtained from the biostratigraphical analysis of some sections sampled in the Messarà Plain will be shown. Nearby Faneromeni and Ano Akria villages, the Miocene/Pliocene boundary is well exposed. There, gypsum-bearing clay, laminated microcrystalline gypsum and gypsum-rudites characterize the evaporitic deposits of the Messinian stage. In these areas, above the Messinian evaporite, post-evaporitic fine-laminated polychrome clays, with intercalations of sandstones and conglomerates, have been found. In both the Faneromeni and Ano Akria area, the Pliocene grey clays and conglomerates rest unconformably on the uppermost Messinian post-evaporitic deposits. A 20 cm-spaced sampling has been performed in both the sections, for more than 100 samples collected. The results of the micropaleontological analysis performed on the Faneromeni and Ano Akria sections point to the occurrence of ostracod assemblages containing: Loxocauda limata (Schneider in Agalarova et al.), Loxocauda sp., Cytherura pyrama Schneider, Cyprideis anlavauxensis Carbonnel, Cyprideis agrigentina Decima, Amnicythere palimpsesta (Livental), Amnicythere propinqua (Livental), Amnicythere accicularia (Olteanu), Amnicythere costata (Olteanu), Amnicythere multituberculata (Livental), Amnicythere sp. D (Miculan in Bassetti et al.), Amnicythere sp. 2 Gliozzi and Grossi, Amnicythere sp., Euxinocythere (Maeotocythere) praebaquana (Livental in Agalarova et al.), Mediocytherideini indet., Pontoniella pontica (Agalarova), Camptocypria sp. 1 Gliozzi and Grossi, Caspiocypris sp., Zalanyiella venusta (Zalanyi), Tyrrhenocythere sp., Loxoconcha rhombovalis Pokorny, Loxoconcha eichwaldi Livental, Loxoconcha sp. A (Miculan in Bassetti et al.), Loxocorniculina djafarovi (Schneider in Suzin). In the analysed samples, reworked planktonic foraminifers and well-preserved charophyte gyrogonites have been also found. The ostracod assemblages found in the Messarà Plain belong to the Loxocorniculina djafarovi Zone (sensu Carbonnel, 1978), which characterizes the uppermost Messinian deposits of the whole Mediterranean Basin. At that time, the well-known Lago-Mare biofacies was also widespread on the Crete Island. The presence of Paratethyan ostracods in the post-evaporitic Messinian deposits of both Faneromeni and Ano Akria sections suggests that in the Crete Island the latest Messinian sedimentation took place in brackish water palaeoenvironments.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

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