A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality.

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos. In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24-72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%-57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%). In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

Amending reduced fish-meal feeds with marine lecithin, but not soy lecithin, improves the growth of juvenile cobia and may attenuate heightened responses to stress challenge.

Sparing of marine resources in aquafeeds can be environmentally and economically advantageous; however, fish meal (FM) replacement can affect the production performance and physiological competence. Phospholipids are increasingly understood to be involved in maintaining growth and vigour in fish and may be deficient in reduced FM formulations. Accordingly, we evaluated the growth and stress tolerance of juvenile cobia fed typical (50% FM) or reduced FM feeds (12% FM) with or without phospholipid amendment [1% marine lecithin (12% FM + Marine PL) or soy lecithin (12% FM + Soy PL)] for 6 weeks in triplicate tanks (N = 3) in a recirculation aquaculture system. The 50% FM feed yielded significantly superior growth and growth efficiency in comparison with the 12% FM and 12% FM+ Soy PL feeds, but the 12% FM+ Marine PL feed yielded comparable results to 50% FM feed. A low-water stress challenge induced elevated plasma glucose, cortisol and lactate levels in all treatments. However, a significant interaction (diet × stress) effect suggested a lesser cortisol response among fish fed the 12% FM+ Marine PL and 50% FM diets. These findings demonstrate that growth performance and, perhaps, resilience of cobia raised on reduced FM feeds may be improved by the addition of marine-origin phospholipid to the diet.

Correlated cerebral events between physically and sensory isolated pairs of subjects exposed to yoked circumcerebral magnetic fields.

In three different experiments pairs of unrelated people sitting in two different rooms were exposed simultaneously to different rates of circumcerebral rotations of weak, complex magnetic fields in order to produce "dynamic similarity". Quantitative electroencephalographic (QEEG) measurements were taken for one member of each pair in one room while the other sat in a closed chamber in another room and intermittently observed 5Hz, 8Hz, 10Hz, or 15Hz flashing lights. Reliable increases in QEEG power within specific frequencies over the right parietal region were observed during the similar-frequency light flashes when the shared temporal-spatial complexity of the circumcerebral rotating fields was based on 100ms, the average duration of normal microstates. The development of this experimental procedure could facilitate rational understanding of this class of "coincidence" phenomena.

Prenatal exposures to LTP-patterned magnetic fields: quantitative effects on specific limbic structures and acquisition of contextually conditioned fear.

Weak (<1 microT) complex magnetic fields (CMFs) may exert their behavioral influences through the hippocampus by resonating by accident or design with intrinsic electrical patterns. Rats were exposed prenatally to one of four intensities of a CMF (either <5 nanoTesla [nT], 10-50 nT, 50-500 nT, or 500-1000 nT) designed to interact with the process of Long-Term Potentiation (LTP) in the hippocampus. Rats then underwent testing in the forced swim, open field, and fear-conditioning procedures. The cell densities of all amygdaloid nuclei, specific hypothalamic structures, and the major regions of the hippocampus were quantified. Results showed that acquisition of conditioned fear was strongly inhibited in animals exposed to LTP-CMFs. Rats exposed to intensities above 10 nT showed decreased cell density in the CA2 fields of the hippocampus; more neurons were present in the CA1 fields of rats exposed to the 10-50 nT intensities compared to all other groups. A decrease in cell density in the medial preoptic nucleus was linearly dependent on field intensity. In the forced-swim test, swimming was decreased in rats that had been exposed to low (10-50 nT) and medium intensity (50-500 nT) LTP-CMFs in a manner consistent with monoamine modulation. In the open field, exposed rats were indistinguishable from controls. These findings support the hypothesis that continuous exposure during prenatal development to CMFs designed to simulate intrinsic LTP within the hippocampus can affect adult behaviors specific to this structure and produce quantitative alterations in neuronal density.

Wigner function approach to the quantum Brownian motion of a particle in a potential.

Recent progress in our understanding of quantum effects on the Brownian motion in an external potential is reviewed. This problem is ubiquitous in physics and chemistry, particularly in the context of decay of metastable states, for example, the reversal of the magnetization of a single domain ferromagnetic particle, kinetics of a superconducting tunnelling junction, etc. Emphasis is laid on the establishment of master equations describing the diffusion process in phase space analogous to the classical Fokker-Planck equation. In particular, it is shown how Wigner's [E. P. Wigner, Phys. Rev., 1932, 40, 749] method of obtaining quantum corrections to the classical equilibrium Maxwell-Boltzmann distribution may be extended to the dissipative non-equilibrium dynamics governing the quantum Brownian motion in an external potential V(x), yielding a master equation for the Wigner distribution function W(x,p,t) in phase space (x,p). The explicit form of the master equation so obtained contains quantum correction terms up to o(h(4)) and in the classical limit, h --> 0, reduces to the classical Klein-Kramers equation. For a quantum oscillator, the method yields an evolution equation coinciding in all respects with that of Agarwal [G. S. Agarwal, Phys. Rev. A, 1971, 4, 739]. In the high dissipation limit, the master equation reduces to a semi-classical Smoluchowski equation describing non-inertial quantum diffusion in configuration space. The Wigner function formulation of quantum Brownian motion is further illustrated by finding quantum corrections to the Kramers escape rate, which, in appropriate limits, reduce to those yielded via quantum generalizations of reaction rate theory.

Semiclassical master equation in Wigners phase space applied to Brownian motion in a periodic potential.

The quantum Brownian motion of a particle in a cosine periodic potential V(x)= -V{0}cos(x/x{0}) is treated using the master equation for the time evolution of the Wigner distribution function W(x,p,t) in phase space (x,p) . The dynamic structure factor, escape rate, and jump-length probabilities are evaluated via matrix continued fractions in the manner customarily used for the classical Fokker-Planck equation. The escape rate so yielded is compared with that given analytically by the quantum-mechanical reaction rate solution of the Kramers turnover problem. The matrix continued fraction solution substantially agrees with the analytic solution.

Thermally activated escape rate for a Brownian particle in a tilted periodic potential for all values of the dissipation.

The translational Brownian motion of a particle in a tilted washboard potential is considered. The dynamic structure factor and longest relaxation time are evaluated from the solution of the governing Langevin equation by using the matrix continued fraction method. The longest relaxation time is compared with the Kramers theory of the escape rate of a Brownian particle from a potential well as extended to the Kramers turnover region by Mel'nikov [Physics Reports 209, 1 (1991)]. It is shown that in the low temperature limit, the universal Mel'nikov expression for the escape rate provides a good estimate of the longest relaxation time for all values of dissipation including the very low damping (VLD), very high damping (VHD), and turnover regimes. For low barriers (where the Mel'nikov method is not applicable) and zero tilt, analytic equations for the relaxation times in the VLD and VHD limits are derived.

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.

Meloidogyne incognita Surface Antigen Epitopes in Infected Arabidopsis Roots.

Surface-coat epitopes of Meloidogyne incognita were detected in root tissues of Arabidopsis thaliana during migration and feeding site formation. A whole-mount root technique was used for immunolocalization of surface coat epitopes in A. thaliana, with the aid of a monoclonal antibody raised specifically against the outer surface of infective juveniles of M. incognita. The antibody, which was Meloidogyne-specific, recognized a fucosyl-bearing glycoprotein in the surface coat. During migration in host tissues the surface coat was shed, initially accumulating in the intercellular spaces next to the juvenile and later at cell junctions farther from the nematode. Upon induction of giant cell formation, the antibody bound to proximally located companion cells and sieve elements of the phloem.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes.

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1.

Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1, a gene required for the post-meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 +/- 0.8 cM on chromosome 5. Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene. The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5-2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.

Cytological characterization of four meiotic mutants of Arabidopsis isolated from T-DNA-transformed lines.

A secondary screen of the Feldmann collection of T-DNA transformed Arabidopsis lines identified several meiotic mutants. We used a spreading technique combined with DAPI staining in a detailed cytogenetic analysis of meiotic chromosome behaviour in four of these mutants, all of which are putatively T-DNA tagged and therefore candidates for molecular and functional analysis of the mutated genes. Two of them are defined as 'synaptic' mutants, showing greatly reduced association of homologous chromosomes at metaphase I: one is asynaptic, showing failure of synapsis during prophase I, whereas the other is desynaptic and is characterized by normal but non-maintained synapsis. Another mutant is defective in meiotic cell cycle control and undergoes a third meiotic division, resembling a second division but without an additional round of chromosome duplication. A further mutant shows meiosis-limited chromosome disruption, resulting in extensive chromosome fragmentation combined with other defects. All four mutants experience very irregular chromosome distribution during the meiotic divisions, resulting in abnormal numbers and/or sizes of microspores, with resulting reduced fertility.

Isolation of deficiencies in the Arabidopsis genome by gamma-irradiation of pollen.

Chromosomal deficiencies are a useful genetic tool in fine-scale genetic mapping and the integration of physical and visible marker genetic maps. Viable overlapping deficiencies may permit gene cloning by subtractive procedures and provide a means of analyzing the functional importance of different chromosomal regions. A method is described for isolation of deficiencies in the Arabidopsis genome which encompass specific loci and other extended chromosomal regions. The technique employs pollen mutagenized by gamma-irradiation to pollinate marker lines homozygous for recessive mutations. Deficiencies at specific loci were detected by screening for marker phenotypes in the F1. Screening for lethal mutations in the F1/F2 confirmed specific deficiencies and revealed other deficiencies that did not overlap the marker loci. Further evidence for such mutations was provided by distorted F2 segregation of the chromosomal markers linked to putative deficiencies. Maintainable (transmissible) and non-transmissible deficiencies were demonstrated by their pattern of inheritance in subsequent generations.

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.

Roots induced on cucumber cotyledons by the agropine Ri plasmid TR-DNA exhibit the transformed phenotype.

Cucumber explants were transformed by Agrabacterium strains carrying Ri plasmids with functional TL and TR-DNAs, and by strains whose pRi had an intact TR-DNA but a disarmed TL-DNA lacking open reading frames (ORFs) 3 to 9, 10 (rol A), 11 (rol B), 12 (rol C), 13, 14, 15 (rol D), 16 and 17. Roots induced by all strains exhibited extensive root hair formation under axenic conditions, synthesised opines, and contained TR-specific DNA. These results confirm that the TR-DNA of an agropine Ri plasmid is able to elicit the transformed root phenotype in this plant.

Effects of Pluronic F-68 on callus growth and protoplast plating efficiency of Solanum dulcamara.

The effects of the non-ionic surfactant, Pluronic F-68, on the growth of callus and protoplasts from Solanum dulcamara L. have been studied. Growth of callus was stimulated by addition of 0.1% (w/v) commercial grade Pluronic to culture medium, whereas lower concentrations (0.01% w/v) had no corresponding effect. In contrast, higher concentrations (1.0% w/v) of Pluronic inhibited callus growth. The mean plating efficiency of protoplasts grown at different densities (15 days after plating) was increased up to 26% following culture with 0.1% (w/v) Pluronic, while 0.01% (w/v) Pluronic was ineffective. Mean protoplast plating efficiency decreased by up to 32% following culture with 1.0% (w/v) Pluronic.

Listening, priming and emotion under a neuroleptic drug.

The authors previously found that emotional arousal increased the asymmetry of facial and other lateralised expressive behaviours. Chlorpromazine equalized the symmetry of behaviour by reducing emotional arousal rather than by blocking all behaviour. These results raise the question of whether the perception of emotion is also lateralised and similarly influenced by chlorpromazine. Sixteen student volunteers were administered placebo and 50 mg of chlorpromazine in tablet form on separate occasions two hours before carrying out two tests of lateralised perception. Dichotic recognition of positive and neutral but not negative emotional words revealed a right ear advantage. No drug effect was observed. The speed of recognition of words and nonwords was also examined following emotionally toned priming words lateralised to the visual hemifields. This task showed some parallel findings to the dichotic test but some lateralised effect of the drug was observed.