Degeneration pattern in somatic embryos of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.     

Expression of NADH-oxidases enhances ethylene productivity in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bacterial EFE

Ethylene is a major petrochemical for which biotechnological production methods are an attractive alternative. Here we use a system based on a bacterial ethylene forming enzyme (EFE) expressed in Saccharomyces cerevisiae. Metabolic modelling performed in a previous study identified re-oxidation of NADH as a factor limiting ethylene production in S. cerevisiae. In line with this, we here found that strains with multicopy plasmid expression of the heterologous oxidases nox and Aox1 led to significantly increased specific ethylene productivity, up 12 and 36%, respectively, compared to the control strain with empty plasmid. However the productivity and yield was only improved in the AOX expressing strain compared to that of the control strain. Both oxidase expressing strains also exhibited increased respiration rates compared to the reference strain, with specific oxygen consumption rates being roughly doubled in both strains. The AOX strain furthermore exhibited a significant increase in the EFE substrate 2-oxoglutarate formation compared to the reference strain, linking an improvement in ethylene production to both increased respiratory capacity and increased substrate availability, thereby corroborating our previous finding.     

Enantiomeric conservation of the male‐produced sex pheromone facilitates monitoring of threatened European hermit beetles (Osmoderma spp.)

Hermit beetles of the genus Osmoderma (Coleoptera: Scarabaeidae: Cetoniinae) are known for their fruity odour, which is released in large amounts by males. Two species of the genus occur in Europe, the eastern Osmoderma barnabita (Motschulsky) and the western Osmoderma eremita (Scopoli). Previous studies on Swedish populations of O. eremita showed that the compound responsible for the characteristic scent, γ‐decalactone, functions as a sex pheromone for the attraction of conspecific females. Male O. eremita only release the (R)‐enantiomer of the lactone, and both sexes are anosmic to the opposite enantiomer. As the distribution areas of the two hermit beetle species partly overlap, it may be expected that they use different enantiomeric compositions of γ‐decalactone as pheromones to promote species discrimination. This paper reports on the identification of the sex pheromone of O. barnabita. Surprisingly, males from a Polish population produce only the (R)‐enantiomer of γ‐decalactone, and conspecific females show equal attraction to the (R)‐enantiomer and a racemic mixture of the compound, indicating that O. barnabita is anosmic to the (S)‐enantiomer, similarly to what was observed for O. eremita. A mtDNA sequence analysis of the cytochrome oxidase subunit I gene of Polish and Swedish beetles confirmed their taxonomical status as O. barnabita and O. eremita, respectively, with an average sequence divergence of 10.5% between beetles from the two studied areas. Although genetic data suggest that these species diverged several million years ago, they still rely on the same enantiomer of γ‐decalactone for mate finding. Thus, the male‐produced pheromone in Osmoderma spp. may be regarded as a territorial signal being exploited by females, rather than a cue for determining species identity. Our data show that the same compound can be used to facilitate monitoring of both beetle species, which are considered indicator species of the species‐rich fauna of saproxylic insects in Europe.     

Physiological imaging of the lung: single-photon-emission computed tomography (SPECT).

Emission tomography provides three-dimensional, quantitative images of the distribution of radiotracers used to mark physiological, metabolic, or pathological processes. Quantitative single photon emission computed tomography (SPECT) requires correction for the image-degrading effects due to photon attenuation and scatter. Phantom experiments have shown that radioactive concentrations can be assessed within some percentage of the true value when relevant corrections are applied. SPECT is widely spread, and radiotracers are available that are easy to use and comparably inexpensive. Compared with other methods, SPECT suffers from a lower spatial resolution, and the time required for image acquisition is longer than for some alternative methods. In contrast to some other methods, SPECT allows simultaneous imaging of more than one process, e.g., both regional blood flow and ventilation, for the whole lung. SPECT has been used to explore the influence of posture and clinical interventions on the spatial distribution of lung blood flow and ventilation. Lung blood flow is typically imaged using macroaggregates of albumin. Both radioactive gases and particulate aerosols labeled with radioactivity have been used for imaging of regional ventilation. However, all radiotracers are not equally suited for quantitative measurements; all have specific advantages and limitations. With SPECT, both blood flow and ventilation can be marked with radiotracers that remain fixed in the lung tissue, which allows tracer administration during conditions different from those at image registration. All SPECT methods have specific features that result from the used radiotracer, the manner in which it is administered, and how images are registered and analyzed.     

Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

Boxes mimicking tree hollows can help conservation of saproxylic beetles

Old hollow trees have declined in Europe and many saproxylic (i.e. wood-dwelling) invertebrates living on them are threatened. The aim of this study was to investigate to what extent artificial habitats can be exploited by saproxylic beetles. To mimic the conditions in tree hollows, we constructed wooden boxes filled with different combinations of substrates like oak saw dust, oak leaves, a dead hen (Gallus domesticus), chicken dung, lucerne flour or potatoes and placed them on tree trunks. To investigate the importance of distance from dispersal sources, we placed boxes at different distances (0–1,800 m) from three species-rich sites with high densities of hollow oaks. Over 3 years, 3,423 specimens of 105 saproxylic beetle species were caught in 47 boxes. Among beetles found in hollow oaks that were either tree-hollow species, bird nest species, or wood rot species, 70% were also found in the boxes. A dead hen added to the artificial wood mould gave a higher number of beetle specimens. The number of species associated with tree hollows in oak decreased with distance from sites with hollow oaks. In conclusion, the prospects for using artificial environments for boosting substrate availability, or to fill spatial and temporal gaps therein, for saproxylic beetles are good.     

Effects of fibre type and diffusion distance on mouse skeletal muscle glycogen content in vitro

In vitro incubation of isolated rodent skeletal muscle is a widely used procedure in metabolic research. One concern with this method is the development of an anoxic state during the incubation period that can cause muscle glycogen depletion. Our aim was to investigate whether in vitro incubation conditions influence glycogen concentration in glycolytic extensor digitorum longus (EDL) and oxidative soleus mouse muscle. Quantitative immunohistochemistry was applied to assess glycogen content in incubated skeletal muscle. Glycogen concentration was depleted, independent of insulin‐stimulation in the incubated skeletal muscle. The extent of glycogen depletion was correlated with the oxidative fibre distribution and with the induction of hypoxia‐induced‐factor‐1‐alpha. Insulin exposure partially prevented glycogen depletion in soleus, but not in EDL muscle, providing evidence that glucose diffusion is not a limiting step to maintain glycogen content. Our results provide evidence to suggest that the anoxic milieu and the intrinsic characteristics of the skeletal muscle fibre type play a major role in inducing glycogen depletion in during in vitro incubations. J. Cell. Biochem. 107: 1189–1197, 2009. © 2009 Wiley‐Liss, Inc.     

Conflicting selection pressures on reproductive functions and speciation in plants

Recent developments in the field of genetic divergence and speciation focus more on diversifying processes than on geographic mode of speciation (i.e. allopatric versus sympatric). Some of these new theories concern speciation driven by conflicts between the sexes. Even though it is well known that the two reproductive functions in plants can have different selective optima, sexual selection in plants is by many assumed to be weak or non-existent. Here we outline potential sexual conflicts in plants and discuss how selection pressures generated by such conflicts may influence genetic divergence. There is opportunity for conflicting selection pressures between individuals, such as manipulative pollen traits that enhance male reproductive success at the expense of the female reproductive function. Within individual plants, fitness of the male function (pollen export) and fitness of the female function (pollen import) may be optimised by different traits, leading to conflicting selection pressures in relation to pollen transfer. This may affect selection for floral specialisation versus floral generalisation in animal-pollinated species. We believe that selection pressures generated by sexual conflict need to be appreciated in order to fully understand microevolutionary processes which may lead to genetic divergence and speciation in plants.     

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.