Autofluorescence and Ultrastructure in the Myxomycete Diachea leucopodia (Physarales)

Autofluorescence is reported for the first time in Myxomycete fruiting bodies. Ultrastructure of stalked sporangia of Diachea leucopodia (Didymiaceae, Physarales) was studied using scanning and transmission electron microscopy, energy-dispersive X-ray microanalysis, and fluorescence microscopy. External and internal properties of the peridium that surround the spores and capillitium exhibit autofluorescence. The stalk is composed of calcareous granules and energy-dispersive X-ray microanalysis demonstrates that the elemental composition of the peridium, capillitium, and stalk has varying concentrations of calcium.     

The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery

Mitochondria typically form a reticular network radiating from the nucleus, creating an interconnected system that supplies the cell with essential energy and metabolites. These mitochondrial networks are regulated through the complex coordination of fission, fusion and distribution events. While a number of key mitochondrial morphology proteins have been identified, the precise mechanisms which govern their activity remain elusive. Moreover, post translational modifications including ubiquitination, phosphorylation and sumoylation of the core machinery are thought to regulate both fusion and division of the network. These proteins can undergo several different modifications depending on cellular signals, environment and energetic demands of the cell. Proteins involved in mitochondrial morphology may also have dual roles in both dynamics and apoptosis, with regulation of these proteins under tight control of the cell to ensure correct function. The absolute reliance of the cell on a functional mitochondrial network is highlighted in neurons, which are particularly vulnerable to any changes in organelle dynamics due to their unique biochemical requirements. Recent evidence suggests that defects in the shape or distribution of mitochondria correlate with the progression of neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's disease. This review focuses on our current understanding of the mitochondrial morphology machinery in cell homeostasis, apoptosis and neurodegeneration, and the post translational modifications that regulate these processes.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

Anthropogenic effects on interaction outcomes: examples from insect-microbial symbioses in forest and savanna ecosystems

The influence of humans on ecosystem dynamics has been, and continues to be, profound. Anthropogenic effects are expected to amplify as human populations continue to increase. Concern over these effects has given rise to a large number of studies focusing on impacts of human activities on individual species or on biotic community structure and composition. Lacking are studies on interactions, particularly mutualisms. Because of the role of mutualisms in ecosystem stability, such studies are critically needed if we are to begin to better understand and predict the responses of ecosystems to anthropogenic change. Most organisms are involved in at least one mutualism, and many in several. Mutualisms facilitate the ability of partners to exploit particular habitats and resources, and play a large role in determining ecological boundaries. When change disrupts, enhances, or introduces new organisms into a mutualism, the outcome and stability of the original partnership(s) is altered as are effects of the symbiosis on the community and ecosystem as a whole. In this paper, using examples from six microbe-insect mutualisms in forest and savanna settings, we showcase how varied and complex the responses of mutualisms can be to an equally varied set of anthropogenic influences. We also show how alterations of mutualisms may ramify throughout affected systems. We stress that researchers must be cognizant that many observed changes in the behaviors, abundances, and distributions of organisms due to human activities are likely to be mediated by mutualists which may alter predictions and actual outcomes in significant ways.     

Root hydraulic conductance,gas exchange and leaf water potential in seedlings of Pistacia lentiscus L. and Quercus suber L. grown under different fertilization and light regimes

Differences in morphology, biomass allocations and physiological responses were investigated in seedlings of Mastic tree (Pistacia lentiscus L.) and Cork oak (Quercus suber L.) submitted to contrasting fertilization and light regimes during early growth. These species are two evergreen sclerophyllous Mediterranean species frequently used in Mediterranean reforestation programmes. Fertilization was the treatment that affected most of the morphological and physiological variables evaluated in P. lentiscus and Q. suber seedlings. Leaf area and specific leaf area (SLA) were affected by shading treatment in both species, showing higher values in seedlings grown under shade. P. lentiscus seedlings showed a high capacity to modify root morphological variables and root hydraulic conductance (K_R) with the fertilization treatment. In contrast, Q. suber showed low to moderate root system changes with the treatments applied, although the fertilization level affected biomass allocation (i.e., root to shoot ratio) in both species. Under high water demand, P. lentiscus seedlings with high K_R allowed transpiration (E) to increase without increasing the water potential gradient between soil and leaves. In Q. suber, high fertilization induced significant increases in photosynthesis (A), as well as a tendency to increase E with significantly lower leaf water potential (ψ_L).     

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.     

Plant-type dependent changes in arbuscular mycorrhizal communities as soil quality indicator in semi-arid Brazil

A large remaining of dry deciduous forest (woody Caatinga) in semi-arid Brazil has been reached by successive fires and exploratory actions what leads to the invasion of low load trees and shrub mesh, called “Carrasco vegetation”. As it restrains the sprouting of woody species, land recuperation was performed using a mixed plantation of native and Eucalyptus species to both preservation and to supply the demand for wood. In order to evaluate the recuperation, a study of microbial communities was proposed. In addition to the highest soil phosphorus content found in the Carrasco area, the greatest spore density of arbuscular mycorrhizal fungi (AMF) communities occurred in the rhizosphere of the both pioneer species: Carrasco and Eucalyptus. In contrast to the DGGE bacteria profile, it was possible to group AMF species of the preserved and experimental sites which were not clustered with Carrasco species through the DGGE of Glomales DNA and also by the principal component analysis (PCA) based on diversity index. Glomus and Acaulospora were the dominant genera at both the preserved site and Carrasco. Nevertheless, Gigaspora species were preferentially found in Dry Forest, while Scutellospora were absent. In contrast, Carrasco favoured the genus Scutellospora and the species Acaulospora scrobiculata. Our results allow one to conclude that vegetation type modifies the AMF communities, which may be used as good indicator of soil quality. Based on AMF communities as soil quality indicator, the mixed forest plantation appears to be underway towards the preserved site two years after transplantation.     

Patterns of female dominance in Propithecus diadema edwardsi of Ranomafana National Park, Madagascar

Many lemur species are characterized by some form of female dominance, ranging from female feeding priority to complete female dominance, although this is a rare trait in primates and other mammals. The status of the Milne-Edwards' sifaka (Propithecus diadema edwardsi), a diurnal lemur, is ambiguous. Some short-term studies have found little or no aggression. The aim of the current, long-term study was to quantify the intersexual-dominance patterns of this sifaka. The distribution, outcome, and context of aggressive interactions were studied in four groups of wild sifakas. The majority of intersexual aggressive interactions were decided, with the loser expressing submissive behavior. Intersexual aggressive interactions occurred in all social contexts, and within all social contexts the females won the vast majority (92.7-96.0%) of aggressive interactions. While aggression rates were low (0.22/hr), this evidence suggests female dominance. We propose that female dominance exists because it provides a fitness advantage to both males and females.