A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

Somaclonal variation as a tool to develop pest resistant plants of Torenia fournieri ‘Compacta Blue’

Callus cultures of Torenia fournieri Compacta Blue were initiated on a modified Murashige and Skoog salt medium (MS) with 2.26 M 2,4-dichlorophenoxyacetic acid. Shoots were regenerated from these cultures using MS medium amended with 2.46 M indolebutyric acid and 8.88 M benzyladenine. These shoot cultures were subjected to two-spotted spidermite (Tetranychus urticae Koch.) and the greenhouse whitefly [Trialeurodes vaporariorum (Westwood)]. Pests were allowed to feed until such time that their populations started to decrease due to lack of food. The remaining live tissue of the Torenia was placed on MS medium amended with 2.28 M zeatin to induce new adventitious shoots and plantlets. Newly regenerated plantlets were acclimated to greenhouse conditions and evaluated for resistance to the pest to which they were subjected in vitro. Highly significant differences in pest numbers were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog salt medium Storrs Agricultural Research Station Scientific Publication 1641.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Calcium dynamics during fertilization in C. elegans

Background

Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans.

Results

Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed.

Conclusion

Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.     

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus Nairovirus. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus in vivo. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.      

Somatic embryogenesis and shoot proliferation of Mussaenda cultivars

Midrib sections of Mussaenda 'Queen Sirikit', 'Do?a Luz', and 'Do?a Hilaria' were cultured on Murashige and Skoog medium (MS) supplemented with 87.7 mM sucrose, 5 g agar l⁻¹, 0, 5, 10 or 20 μM indole-3-acetic acid (IAA) and 0, 0.5, 1, 2.5, 5, 10, 25 or 50 μM 6-benzyladenine (BA). In addition, aseptic 5 mm shoot tips from 'Do?a Luz' cultures were excised and cultured on MS basal salts, 0.6 mM myo-inositol, 1.2 μM thiamine-HCl, 87.7 mM sucrose, 7 g agar l⁻¹, 0, 2.5, 5, 10, 20, or 40 μM BA, 0 or 1 μM α-naphthaleneacetic acid (NAA) and 0 or 217 μM adenine sulfate at pH 5.8. Calluses began to develop after two weeks at the cut ends of midribs when cultured on a medium containing IAA. Somatic embryos first appeared at eight weeks but only on 'Queen Sirikit' callus. After 15 weeks, the average number of somatic embryos produced per tube decreased as the IAA concentration increased from 0 to 20 μM. BA concentrations between 5.0 and 10.0 μM resulted in the largest number of somatic embryos per tube. After six weeks, the total, axillary and adventitious number of 'Do?a Luz' shoots increased as the BA concentration in the culture medium increased from 0 to 20 μM. Average shoot length and fresh weight decreased from 0 to 40 μM BA. The addition of NAA to the culture medium reduced shoot number. Adenine sulfate in the presence of BA reduced the total number of shoots. An ideal medium for proliferating the largest number of 'Do?a Luz' shoots would be a MS medium supplemented with 10–20 μM BA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.