Detection of a natural point mutation in Potato virus A that overcomes resistance to vascular movement in Nicandra physaloides., and studies on seed-transmissibility of the mutant virus

Isolate M of Potato virus A (PVA‐M; genus Potyvirus) is avirulent in Nicandra physaloides L. (family Solanaceae). The inoculated leaves are infected but no systemic infection is observed. Forty plants of ‘Black Pod’ (BP) and ‘Black Pod Alba’ (BPA), two variants of N. physaloides described in this study, were inoculated with PVA‐M. Two plants of BP and one plant of BPA were systemically infected. Mosaic, blistering and dark green islands developed on the systemically infected leaves, and flowers showed colour‐break symptoms. PVAprogeny were sequence‐characterised for the 6K2 protein and viral genome‐linked protein (VPg) encoding regions known to control the long distance movement of PVA in N. physaloides. All virus progeny (designated as PVA‐Mm) in the systemically infected leaves of the plants inoculated with PVA‐M contained only a single amino acid substitution (Vail 16Met) in the central part of VPg due to a nucleotide substitution G6033A, as compared to PVA‐M. Other PVA isolates that infected N. physaloides systemically also contained Metll6 in VPg. In a previous study using chimeric viruses, Metl16 in VPg was shown to be a major determinant for vascular movement of PVA in N. physaloides, and this study reveals that the mutation for Metl16 can occur in vivo during replication of the avirulent PVA‐M in infected plants. Immunolocalisation studies on BP and BPA plants showed that the pods (berries) and seed coat contained PVA‐Mm in the developing seeds, but no virus was detected in embryons. Up to 27% of the mature seeds contained PVA‐Mm but no transmission to seedlings was observed in a total of 450 seeds tested, and no test plants were infected following mechanical inoculation with extracts prepared from the seeds.     

Timing of breeding of Tengmalm's Owl Aegolius funereus in relation to vole dynamics in western Finland

Timing of breeding in Tengmalm's Owl was studied in western Finland for 13 years. During 1973-85, half of the females started laying before 4 April, near the seasonal low of main food abundance (voles), and earlier than in southern Finland or as early as in southeastern Norway. The reason for this latitudinal trend is the shallow snow cover of the study area. The annual variation in the median laying dates was one month and was negatively correlated with the spring abundance of Microtus voles. The mean clutch size was related to the start of laying with early clutches being larger than late ones. These findings accord with the 'food limitation hypothesis', which states that laying begins as soon as the female can accumulate enough energy stores for forming eggs. Early breeding is adaptive, since juveniles of early clutches probably survive better during their first winter. In adults, early nesting improves the chances of rearing two broods per year, allows them to moult after breeding and gives more time to accumulate fat reserves to survive the winter. Tengmalm's Owl is one of the earliest breeders among North European birds. This is possible because of its hole-nesting and resident habits, small body-size in relation to the main prey and the greatest sexual size dimorphism among European owls.     

Interpopulation differences in pupal size and fecundity are not associated with occurrence of outbreaks in Epirrita autumnata (Lepidoptera, Geometridae)

Abstract.

• 1 Among-population differences in pupal mass were studied in a geometrid, Epirrita autumnata. Some Epirrita autumnata populations regularly reach outbreak densities while others are never known to do so. Because adults do not feed, pupal mass of females correlates strongly with fecundity.
• 2 Larvae were collected from twelve field sites. Ten of our sample populations originated within the outbreak range of the species and represented different phases of outbreaks. Two populations originated outside the outbreak range.
• 3 Pupal mass of field-collected E. autumnata varied significantly among populations. The peak phase populations had the smallest pupae and the biggest were found in low density populations outside the outbreak range.
• 4 Offspring of moths from each population were reared under identical conditions in two larval densities. Significant differences were not found in pupal mass among populations. That is, the inherent size, correlated with fecundity of moths, was not different between populations originating within and outside the outbreak range, nor among collections from different densities or phases of the outbreaks.
• 5 Rearing density did not interact in a consistent way with population.
• 6 As far as size and fecundity are concerned, the results do not support Chitty's hypothesis that differences in genetic composition of the population at low and high density phases generate cyclic fluctuations of population density.
• 7 Because no hereditary or maternal differences were found in size and fecundity between E.autumnata originating within and outside the outbreak range, variation in reproductive capacity cannot explain why outbreaks occur only in some populations.

     

Maximisation of perfusion systems and process comparison with batch-type cultures

A comparison of cell yields and monoclonal antibody productivity from the same hybridoma has been made in a wide range of cell bioreactors including both batch and continuous perfusion cultures. The most productive systems were based on porous microcarriers in fixed and fluidised beds which can be operated with a high degree of efficiency and reliability from the physico-chemical engineering point of view. Further improvements should be possible by improving the physiological environment in dense cell cultures, as indicated by the preliminary studies that are described. These include experimental data showing the relationship between monoclonal antibody production rates with glucose, glutamine, ammonia, and oxygen levels in microporous beads. The results strongly indicate that perfusion processes that are scaleable in both volume and cell density can significantly reduce production costs. Manufacturers of biologicals from animal cells now have a choice between the proven batch-type processes and reliable perfusion systems based on microporous beads.     

Relationship between chloroplast structure and O2 evolution rate of leaf discs in plants from different biotopes in South Finland

Abstract The chloroplast ultrastructure, especially the thylakoid organization, the polypeptide composition of the thylakoid membranes and photosynthetic O₂ evolution rate, chlorophyll (Chl) content and Chi a/b ratio were studied in leaves of nine plants growing in contrasting biotopes in the wild in South Finland. All the measurements were made at the beginning of the period of main growth on leaves approaching full expansion, when the CO₂-saturated O₂ evolution rate (measured at 20°C and 1500 μmol photons m^?2s^?1) was at a maximum, ranging from 19.2 to 6.9 μmol O₂ cm^?2 h^?1. Among the species, the Chi a/b ratio varied between 3.75 and 2.71. In the mesophyll chloroplasts, the ratio of the total length of appressed to non-appressed thylakoid membranes varied between 1.07 and 1.79, the number of partitions per granum varied between 2.8 and 12.0 and the grana area between 21 and 42% of the chloroplast area. There was a significant relationship between the rate of O₂ evolution of the leaf discs and the thylakoid organization in the mesophyll chloroplasts. The higher the O₂ evolution rate, the lower was the ratio of the total length of appressed to non-appressed thylakoid membranes and also the lower the grana area. Although the relationship of the photosynthetic rate with the Chi content and the Chi a/b ratio of the leaves was not as clear, a significant negative correlation existed between the Chi a/b ratio and the ratio of appressed to non-appressed thylakoid membranes, indicating lateral heterogeneity in the distribution of different Chl- protein complexes.     

A comparative spectroscopic study of tryptophan probes engineered into high- and low-affinity domains of recombinant chicken troponin C.

The spectral properties of three tryptophan-substituted mutants of recombinant chicken troponin C are compared. Site-specific mutagenesis was used to introduce a tryptophan residue into the high-affinity (Ca2+/Mg2+) domain of troponin C at residue position 105, thereby creating the mutant phenylalanine-105 to tryptophan (F105W). The spectral properties of F105W and a double mutant, F29W/F105W, were compared with the mutant phenylalanine-29 to tryptophan (F29W). Since wild-type chicken troponin C does not naturally contain either tyrosine or tryptophan residues, the tryptophan substitutions behaved as site-specific reporters of metal ion binding and conformational change. The residues that occupy positions 29 and 105 are at homologous locations in low-affinity and high-affinity domains, preceding the first liganding residues of binding loops I and III, respectively. Mutant proteins were examined by a combination of absorbance and fluorescence methods. Calcium induced significant changes in the near-UV absorbance spectra, fluorescence emission spectra, and far-UV circular dichroism of all three mutant proteins. Magnesium induced significant changes in the spectral properties of only F105W and F29W/F105W proteins. Tryptophan substitutions allowed Ca(2+)-specific and Ca(2+)/Mg(2+) sites to be titrated independently of one another. Results indicate that there is no interaction between the two binding domains under conditions where troponin C is isolated from the troponin complex. Magnesium-induced changes in the environment of the tryptophan reporter at position 105 were significantly different from those induced by calcium. This suggests that calcium and magnesium differ in their influence on the conformation of the high-affinity, Ca(2+)/Mg(2+) sites.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015     

Redescription of Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924 (Ciliophora: Choreotrichia), with Phylogenetic Analyses Based on Small Subunit rRNA Gene Sequences

ABSTRACT. The identification of Favella ehrenbergii, a marine planktonic ciliate, has largely been based on its lorica features. This approach is potentially problematic given the polymorphic lorica during this organism's life cycle. We isolated a population of F. ehrenbergii from the coastal waters of Incheon, Korea, and revealed its infraciliature using the protargol staining method. Phylogenetic analysis based on small subunit rRNA gene sequences was also performed. Results showed that this population possessed 16 collar membranelles (CM) and about 100 somatic kineties. These features are highly conserved, even in later dividers. As such, the number of CM and somatic kineties can be used as key characteristics for identification of Favella species.