Evidence for chemically shifted water in the brine shrimp, (Artemia): possible interpretations

We report in this paper evidence for the existence of an unusually large chemical shift for intracellular water proton in brine shrimp (Artemia). The observed chemical shift, when corrected for bulk susceptibility, is down field 0.11 ppm from the pure water signal. The possible origin of such large chemical shift for intracellular water in this system is discussed.     

Cerebral oxygen utilization analyzed by the use of oxygen-17 and its nuclear magnetic resonance

In order to assess the usefulness of oxygen-17, a stable isotope of oxygen, oxygen-17, was administered to rats for studying cerebral oxygen utilization, and the produced metabolic water was detected by 17O-NMR spectroscopy in vitro and an 1H-NMR imaging system in vivo. In the vitro study, the increment in signal amplitude of oxygen-17 was observed in the brain extracted from rats that inhaled oxygen-17 gas. The in vivo study demonstrated that there were changes in the 1H-NMR image intensity of brain of rats that inhaled oxygen-17 gas. These facts indicate that oxygen-17 can serve as a tracer in the study of cerebral oxygen utilization.     

A note on the brine shrimp Artemia in Tuz Lake (Turkey)

Population density, fecundity andreproduction period of Artemia sp. in TuzLake, Turkey, beside environmental factors, such as oxygen,temperature, salinity, pH and electrical conductivity werestudied during 1993 and 1994.Artemia sp. occurred at two stations at the coast.Overwintering cysts hatched in early May, when the population was composed of 90% metanauplii (93%at Y4 and 99% at Y5) and few adult females. Peak abundanceoccurred in late May, at 106to 114 ind.m^–3.     

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.     

Molecular phylogenetics and asexuality in the brine shrimp Artemia

Explaining cases of long-term persistence of parthenogenesis has proven an arduous task for evolutionary biologists. Interpreting sexual-asexual interactions though has recently advanced owing to methodological design, increased taxon sampling and choice of model organisms. We inferred the phylogeny of Artemia, a halophilic branchiopod genus of sexual and parthenogenetic forms with cosmopolitan distribution, marked geographic patterns and ecological partitioning. Joint analysis of newly derived ITS1 sequences and 16S RFLP markers from global isolates indicates significant interspecific divergence as well as pronounced diversity for parthenogens, matching that of sexual ancestors. Maximum parsimony, maximum likelihood, and Bayesian methods were largely congruent in reconstructing the phylogeny of the genus. Given the current sampling, at least four independent origins of parthenogenesis are deduced. Molecular clock calibrations based on biogeographic landmarks indicate that the lineage leading to A. persimilis diverged from the common ancestor of all Artemia species between 80 and 90 MYA at the time of separation of Africa from South America, whereas parthenogenesis first appeared at least 3 MYA. Common mitochondrial DNA haplotypes delineate A. urmiana and A. tibetiana as possible maternal parents of several clonal lineages. A novel topological placement of A. franciscana as a sister clade to all Asian Artemia and parthenogenetic forms is proposed and also supported by ITS1 length and other existing data.     

Genetic variation in sympatric populations of diploid and polyploid brine shrimp (Artemia parthenogenetica)

We examined genetic variation in sympatric diploid and polyploid brine shrimp Artemia parthenogenetica from each of three populations (China, Italy and Spain). Italian and Spanish tetraploids are closely related (I=0.964). Diploids and tetraploids within each of the two European populations are also closely related (mean I=0.905). Most alleles found in diploids also exist in sympatric polyploids. In contrast, the asexual Artemia (2N, 4N and 5N) in our study share few alleles with their close sexual relative, A. tunisiana (mean I=0.002). These results, as well as the work of other authors, strongly suggest that at least the tetraploid Artemia in our study have an autopolyploid origin.Clonal diversity of polyploid Artemia can be very high at least in some population. Both diploids and polyploids had low clonal diversities in the populations dominated by polyploids and high clonal diversities in the population dominated by diploids.The most common genotypes of sympatric diploid and polyploid Artemia frequently differed. Some alleles occurred only in diploids, while others were restricted to polyploids. These results suggest that polyploidy in Artemia has led to genetic divergence from diploid progenitors, and that ploidy-level variation must also be considered in developing an understanding of spatial and temporal allozyme polymorphism in asexual populations.     

Causes of a non-random pairing by size in the brine shrimp,Artemia salina: (Crustacea: Anostraca)

Summary Brine shrimp (Artemia salina) males and females entered precopula assortatively by size in the laboratory; large males also had a pairing advantage over smaller males. We investigated the causes of such nonrandom pairing to test hypotheses on size-assortative mating.We found precopulatory biases with respect to male size in the absence of direct competition among males (which produces pairing biases in other species). Large males encountered females significantly more often than did small males. Similarly, large females encountered males more often than did small females, but showed less willingness than small females to enter precopula when housed with small males. Consequently, large females took longer than small females to enter precopula with small males. Although large males entered precopula readily with small females, such size-mismatched pairs appeared short-lived.We conclude that non-random pairing by size in A. salina is determined by several factors including: encounter rates between males and females of different sizes, female behavior, and time following initial pair formation. Our results are likely applicable to other species and can help explain variation for selection on size or other traits.     

In vivo oxygen-17 nuclear magnetic resonance for the estimation of cerebral blood flow and oxygen consumption

To explore the feasibility of in vivo 17O NMR for the estimation of cerebral blood flow and oxygen consumption, in vivo 17O NMR spectroscopy and imaging were employed in animal models. In the spectroscopy, the changes in the 17O NMR signal intensity after the injection of H2(17)O and the inhalation of 17O2 gas were obtained every 4 seconds with sufficient signal-to-noise ratios for the quantification of cerebral blood flow and oxygen consumption. In the imaging, although the time and spatial resolutions were insufficient for the quantification of H2(17)O, 17O NMR images of rabbit brain could be obtained, indicating that it is possible to map cerebral blood flow and oxygen consumption by 17O NMR imaging.     

Characterization of polymorphic microsatellite markers in the brine shrimp Artemia (Branchiopoda, Anostraca)

The brine shrimp Artemia is a complex genus containing sexual species and parthenogenetic lineages. Artemia franciscana is native to America and its cysts (diapausing eggs) are used worldwide as a food source in aquaculture. As a consequence, this anostracan has become an invasive species in many hypersaline aquatic ecosystems of other continents. Parthenogenetic Artemia lineages occur only in the Old World. Ten and five microsatellite markers were developed to characterize two populations for A. franciscana and two populations for diploid parthenogenetic Artemia, respectively. For A. franciscana the number of alleles ranged from 11 to 58 per locus, while for parthenogens the number of alleles ranged from three to 10. The levels of heterozygosity in A. franciscana and in parthenogens ranged from 0.115 to 0.976 and from 0.000 to 0.971, respectively. These microsatellite loci showed a high population assignment power, which will be useful for future studies of population genetics and invasive processes in Artemia.     

Phylogeography and local endemism of the native Mediterranean brine shrimp Artemia salina (Branchiopoda: Anostraca)

There has been a recent appreciation of the ecological impacts of zooplanktonic species invasions. The North American brine shrimp Artemia franciscana is one such alien invader in hyper-saline water ecosystems at a global scale. It has been shown to outcompete native Artemia species, leading to their local extinction. We used partial sequences of the mitochondrial Cytochrome c Oxidase Subunit 1 (COI or cox1) gene to investigate the genetic diversity and phylogeography of A. salina, an extreme halophilic sexual brine shrimp, over its known distribution range (Mediterranean Basin and South Africa) and to assess the extent of local endemism, the degree of population structure and the potential impact of traditional human saltpan management on this species. We also examined the phylogenetic relationships in the genus Artemia using COI sequences. Our results show extensive regional endemism and indicate an early Pleistocene expansion of A. salina in the Mediterranean Basin. Subsequent population isolation in a mosaic of Pleistocene refugia is suggested, with two or three refugia located in the Iberian Peninsula. Two instances of long-distance colonization were also observed. Surprisingly, given its strong phylogeographical structure, A. salina showed a signature of correlation between geographical and genetic distance. Owing to strong 'priority effects', extensive population differentiation is retained, despite dispersal via migrant birds and human management of saltpans. The foreseeable expansion of A. franciscana is likely to be followed by substantial loss of genetic diversity in Mediterranean A. salina. Large genetic divergences between Mediterranean and South African A. salina suggest that the latter deserves species status.     

Investigation of phosphatidylethanolamine bilayers by deuterium and phosphorus-31 nuclear magnetic resonance.

The motion of the ethanolamine head group in unsonicated lipid bilayers above and below the phase transition is studied by means of deuterium and phosphorus magnetic resonance. For this purpose, dipalmitoyl-3-sn-phosphatidylethanolamine is selectively deuterated at the two ethanolamine carbon atoms. The deuterium quadrupole splittings of the corresponding bilayer phases are measured at pH 5.5 as a function of temperature. In addition, the phosphorus-31 chemical shift anisotropies of planor-oriented and randomly dispersed samples of dipalmitoyl-3-sn-phosphatidylethanolamine are measured at pH 5.5 and 11 by applying a proton-decoupling field. The knowledge of the static chemical shift tensor (Kohler, S.J., and Klein, M.P. (1976), Biochemistry 15, 967) provides the basis for a quantitive analysis of the head-group motion. The nuclear magnetic resonance data are consistent with a model in which the ethanolamine group is rotating flat on the surface of the bilayer with rapid transitions occurring between two enantiomeric conformations.     

Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.     

The development of the brine shrimp Artemia salina (L.): a morphometric approach

Growth allomentry in the development of the brine shrimp Artemia salina (L.) was studied. Body width and abdomen length show a negative allometric relation to body length ( k =0.35 and 0.72 respectively) over th eight instars studied. Antennule length shows a negative allometric relation in respect to body length, with a change in the value of the growth constant at Instar Five from 0.31 to 0.75. Thoracic limb length shows a high positive relation to body length ( k =3.0) until Instar Six, when a negative allometric phase ( k =0.4) follows. A change in the sign of the growth constant from positive ( k =0.45) to negative ( k =-1.4) is noted for the length of the 2nd antenna in relation to body length.     

Calcium binding to mixed cardiolipin-phosphatidylcholine bilayers as studied by deuterium nuclear magnetic resonance

Calcium binding to bilayer membranes containing cardiolipin (CDL) mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using phosphorus-31 and deuterium nuclear magnetic resonance (NMR) spectroscopy. The destabilizing effect of Ca2+ on CDL bilayers, including the formation of hexagonal H11 and isotropic phases, was eliminated when CDL was mixed with sufficiently large proportion of POPC. Thus, for the mixture CDL-POPC (1:9 M/M), 31P NMR spectra retained a line shape typical of fluid bilayer lipids even in the presence of 1.0 M Ca2+. Specifically head-group-deuteriated CDL or POPC showed in this mixture 2H NMR spectra indicating that both lipids remained in a fluidlike bilayer at Ca2+ concentrations up to 1.0 M. Any phase separation of Ca2-CDL clusters could be excluded. The residence time of Ca2+ at an individual head group binding site was shorter than 10(-6) s. The deuterium quadrupole splitting, delta nu Q, of POPC deuteriated at the alpha-methylene segment of the choline head group was found to be linearly related to the number of bound calcium ions, X2, for the CDL-POPC (1:9 M/M) mixture. The effective surface charge density, sigma, could be determined from the measured amount of bound Ca2+. Subsequently, the surface potential, psi 0, and the concentration of free Ca2+ ions at the plane of ion binding were calculated by employing the Gouy-Chapman theory. Various possible models of the equilibrium binding of Ca2+ could then be tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amyloid fibril dynamics revealed by combined hydrogen/deuterium exchange and nuclear magnetic resonance

A general method to explore the dynamic nature of amyloid fibrils is described, combining hydrogen/deuterium exchange and nuclear magnetic resonance spectroscopy to determine the exchange rates of individual amide protons within an amyloid fibril. Our method was applied to fibrils formed by the amyloid-β(1-40) peptide, the major protein component of amyloid plaques in Alzheimer’s disease. The fastest exchange rates were detected among the first 14 residues of the peptide, a stretch known to be poorly structured within the fibril. Considerably slower exchange rates were observed in the remainder of the peptide within the β-strand-turn-β-strand motif that constitutes the fibrillar core.