Identification and characterization of simple sequence repeat markers from Brassica napus expressed sequences

The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)‐SSR markers from Brassica napus and their cross‐amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST‐SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.     

The outcome of the invasion of Florida lakes by Daphnia lumholtzi

1. We examined 7–12 years of monthly to quarterly historical data from 15 lakes in FL, U.S.A. to determine the extent and outcome of invasion by the alien cladoceran Daphnia lumholtzi Sars. 2. The alien species was found in 10 of the 15 lakes, including Florida’s three largest lakes: Okeechobee, George and Apopka. All the surveyed lakes had resident populations of the smaller native species Daphnia ambigua Scourfield. 3. In most of the lakes, D. ambigua occurred seven to ninefold more often in plankton samples than D. lumholtzi, and at 10‐ to 100‐fold higher maximal densities. One exception was a small lake in central Florida (Lake Jesup), where D. lumholtzi attained high densities on several occasions in the 10 years of sampling. 4. In Lake Okeechobee, where data were of sufficient quality and quantity to perform statistical analyses, the results of canonical correlation analysis indicated that high densities of D. lumholtzi were correlated with lower concentrations of suspended solids, high algal biomass and higher temperature, whereas the opposite conditions were correlated with high densities of D. ambigua. 5. Based on the majority of data, D. lumholtzi has successfully invaded many lakes in Florida, yet it has not become a substantive component of the zooplankton. Additional research is needed to determine whether resources, fish predation or some other factor is responsible for this outcome of invasion.     

Mutual mate choice by mountain pine beetles: size‐dependence but not size‐assortative mating

1. Mutual mate choice may be rare, occurring when both sexes invest heavily in reproduction, mating opportunities are abundant, and individuals differ in quality. 2. Mountain pine beetles, Dendroctonus ponderosae (Curculionidae: Scolytinae) appear to meet the conditions for mutual mate choice. We introduced males to females in breeding sites and observed the occurrence and speed of a male entering a female's gallery. We tested for consequences of mutual mate choice, namely condition‐dependent choosiness and assortative mating. 3. Males were more likely to enter a female's gallery when the gallery was in a smaller tree with less resin production and when the gallery was larger. Female body size and condition did not influence the probability of entry. Larger males were less likely to enter a gallery than were smaller males, probably because of size‐dependent choosiness rather than physical limitations. 4. Small males took longer to enter galleries of large females than of small females, whereas large males entered as quickly into galleries of large females as small females. This suggests size‐dependent choosiness by females. 5. No assortative mating by body size was detected, probably because males appeared to choose on the basis of female‐associated resources rather than on female traits.     

Methane production by microbial mats under low sulphate concentrations

Cyanobacterial mats collected in hypersaline salterns were incubated in a greenhouse under low sulphate concentrations ([]) and examined for their primary productivity and emissions of methane and other major carbon species. Atmospheric greenhouse warming by gases such as carbon dioxide and methane must have been greater during the Archean than today in order to account for a record of moderate to warm palaeoclimates, despite a less luminous early sun. It has been suggested that decreased levels of oxygen and sulphate in Archean oceans could have significantly stimulated microbial methanogenesis relative to present marine rates, with a resultant increase in the relative importance of methane in maintaining the early greenhouse. We maintained modern microbial mats, models of ancient coastal marine communities, in artificial brine mixtures containing both modern [] (c. 70 mm ) and ‘Archean’[] (<0.2 mm ). At low [], primary production in the mats was essentially unaffected, while rates of sulphate reduction decreased by a factor of three, and methane fluxes increased by up to 10‐fold. However, remineralization by methanogenesis still amounted to less than 0.4% of the total carbon released by the mats. The relatively low efficiency of conversion of photosynthate to methane is suggested to reflect the particular geometry and chemical microenvironment of hypersaline cyanobacterial mats. Therefore, such mats were probably relatively weak net sources of methane throughout their 3.5 Ga history, even during periods of low environmental levels oxygen and sulphate.     

Analysis of Time-Varying Biological Data Using Rainflow Cycle Counting

A wide range of biological investigations lead to time-history data. The characterization of such data can be difficult particularly in the presence of signal noise or superimposed signals. Several methods are described which can be brought to bear including FFT, thresholding, peak counting, and range counting. However, each of these approaches has significant disadvantages. In this paper we describe a novel method, known as rainflow cycle counting, for characterizing time varying biological time-history data in terms of spiking or oscillation amplitude and frequency. Rainflow counting is a straightforward algorithm for identifying complete cycles in the data and determining their amplitudes. The approach is simple, reliable, easily lends itself to automation, and robust even in the presence of superimposed signals or background noise. After describing the method, its use and behavior are demonstrated on three sample histories of intracellular calcium concentration in chondrocytes exposed to fluid shear stress. The method is also applied to a more challenging data set that has had an artificial random error included. The results demonstrate that the rainflow counting algorithm identifies signal oscillations and appropriately determines their amplitudes even when superimposed or distorted by background noise. These attractive properties make rainflow counting a powerful approach for quantifying and characterizing biological time histories.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

The effect of season and group size on survivorship and larval growth in Plagiodera versicolora

Abstract.

• 1 The larvae of the multivoltine imported willow leaf beetle, Plagiodera versicolora (Laicharting), are found in larval aggregations during the first half of the larval stage. An experimental study was conducted to test for seasonal (brood) and group size effects on larval fitness. For each of the three broods, egg group sizes of 1, 4, 8, 16 , 24 and 32 were established in the field and monitored for the first 6 days post-hatching.
• 2 There was no effect of group size on larval weight, but there was a significant effect of group size on survival. Individuals from group size 16 had the highest survival, which coincides with the mean natural cluster size of 15.3 eggs.
• 3 There were no significant interactions between brood and group size.
• 4 The effect of larval weight on adult fitness was determined in the laboratory and the field. Larval weight was correlated with egg production in the laboratory but not in the field.

     

Molecular Characterization of a Cytoplasmic Dynein from Dictyostelium

Cytoplasmic dynein is a high molecular weight, microtubule-based mechanochemical ATPase that is believed to provide motive force for a number of intracellular motilities, including transport of membrane-bound organelles. Cytoplasmic dynein also localizes to the mitotic spindles of some organisms and to the kinetochore regions of some condensed chromosomes, where it may play an active role in spindle assembly, spindle position, and/or chromosome movement during cell division. Despite active research efforts from a number of laboratories, little detail is yet available about dynein-based cellular activities. This paper describes our efforts to characterize cytoplasmic dynein from Dictyostelium and to use this protist as a molecular genetic factory to probe structure-function relationships of this molecule.     

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thaliana under Spaceflight Conditions

Successful development of seeds under spaceflight conditionshas been an elusive goal of numerous long-duration experimentswith plants on orbital spacecraft. Because carbohydrate metabolismundergoes changes when plants are grown in microgravity, developingseed storage reserves might be detrimentally affected duringspaceflight. Seed development in Arabidopsis thaliana plantsthat flowered during 11 d in space on shuttle mission STS-68has been investigated in this study. Plants were grown to therosette stage (13 d) on a nutrient agar medium on the groundand loaded into the Plant Growth Unit flight hardware 18 h priorto lift-off. Plants were retrieved 3 h after landing and siliqueswere immediately removed from plants. Young seeds were fixedand processed for microscopic observation. Seeds in both theground control and flight plants are similar in their morphologyand size. The oldest seeds from these plants contain completelydeveloped embryos and seed coats. These embryos developed radicle,hypocotyl, meristematic apical tissue, and differentiated cotyledons.Protoderm, procambium, and primary ground tissue had differentiated.Reserves such as starch and protein were deposited in the embryosduring tissue differentiation. The aleurone layer contains alarge quantity of storage protein and starch grains. A seedcoat developed from integuments of the ovule with gradual changein cell composition and cell material deposition. Carbohydrateswere deposited in outer integument cells especially in the outsidecell walls. Starch grains decreased in number per cell in theintegument during seed coat development. All these characteristicsduring seed development represent normal features in the groundcontrol plants and show that the spaceflight environment doesnot prevent normal development of seeds in Arabidopsis. Arabidopsis ; spaceflight; embryo; endosperm; seed coat; storage reserves     

Some Factors Influencing the in vitro Infectivity and Replication of Encephalitozoon cuniculi*

SYNOPSIS. Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.