Transformation of a psychrotrophic bacterium with a plasmid from a mesophile

The psychrotrophic bacteriumBacillus psychrophilus was transformed with the broadhost-range plasmid pC194. The ability of the transformant to express chloramphenicol (CAM) resistance and the possible effects of such expression on the physiology of the psychrotroph were examined. The transformant exhibited growth rates, filament formation at elevated temperatures, synthesis of cold shock proteins and cold acclimation proteins, similar to the parentalB. psychrophilus.     

Living inside a sponge skeleton: the association of a sponge,a macroalga and a diatom

The relationships between sponges and macroalgae have been poorly investigated, especially in temperate waters. This work describes the consortium between the dictyoceratid sponge Dysidea pallescens and the red alga Acrochaetium spongicola permeating spongin fibres in the Mediterranean Sea; moreover, this is the first report of a diatom, Amphora pediculus, living also inside the spongin skeleton. The annual trend of the total algal biomass did not vary over time in relation to the temperature, irradiance and incorporation of foreign bodies. Our analyses, conducted by light and electron microscopy, suggest that both the macroalga and the diatom invade the skeleton of the sponge from the external environment, and that the benthic diatom, epiphyte of the macroalga, is passively carried inside the fibres through the growth of Acrochaetium spongicola. All the examined samples of D. pallescens showed that the macroalga permeated at least some fibres, while the presence of the diatom was occasional. The superficial layer of the sponge, thin and reticulate, likely allows the passage of the light and ensures the algal survival inside the sponge tissue. The high occurrence of the association with A. spongicola, together with the morphological adaptations of the sponge favouring the algal life, suggest that the relationship is mutualistic. The possible benefits for the involved partners are hypothesized. The taxonomy and ecology of endozoic Acrochaetiales are controversial due to the reduced size of thalli, the absence of peculiar diagnostic characters and unknown reproductive structures. Therefore, detailed studies on the relationships between the algae and their hosts will provide helpful information for the algal identification.     

Characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10(6) PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with >/==" BORDER="0">10(5) PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.     

Wah Soon Chow,a teacher,a friend and a colleague

Photosynthesis Research - To finish this special issue, some friends, colleagues and students of Prof. Chow (Emeritus Professor, the Research School of Biology, the Australian National University)...     

Geminiviruses: a tale of a plasmid becoming a virus

Background  

Geminiviruses (family Geminiviridae) are small single-stranded (ss) DNA viruses infecting plants. Their virion morphology is unique in the known viral world – two incomplete T = 1 icosahedra are joined together to form twinned particles. Geminiviruses utilize a rolling-circle mode to replicate their genomes. A limited sequence similarity between the three conserved motifs of the rolling-circle replication initiation proteins (RCR Reps) of geminiviruses and plasmids of Gram-positive bacteria allowed Koonin and Ilyina to propose that geminiviruses descend from bacterial replicons.     

Vegetation as a component of a non-nested hierarchy: a conceptual model

Abstract. A general conceptual model of vegetation based on hierarchy theory is presented. The model emphasizes that prediction of vegetation requires consideration of both mechanisms of vegetation change and the constraints within which it occurs. The mechanisms of vegetation change are the responses to and effects upon their surroundings of individual plants. The most general constraints upon vegetation are aspects of the environment not affected by vegetation over successional time, and the pool of species within dispersal range. Examples of such environmental factors include macroclimate and soil parent material. In some cases, vegetation may alter important labile environmental factors such as soil nutrient and water availability. Some vegetation compositions appear to be resistant to changes in the general constraints. Due to both sources, there are multiple possible vegetation compositions given the same general constraints. Disturbance is defined as an abrupt change in the constraints on the vegetation resulting in a change in the vegetation's state or dynamics. Both the recognition of disturbance and the distinction between independent and labile environmental factors depend on the spatial and temporal scale of observation. For example, a particular wildfire at a given stand may be a disturbance, whereas at a larger scale of observation the same event may contribute to the wildfire regime, part of the constraints at that scale. Similarly, levels of soil organic matter may constrain vegetation over short time scales, due to influencing availability of water and nutrients. Over long time scales, the vegetation itself is a primary determinant of soil organic matter content. This model contains elements of both the initial, holistic theory of vegetation and recent, reductionistic approaches. It reiterates the need to considerboth mechanisms and constraints, stressed by contemporary and earlier workers. Hierarchy theory provides new insights concerning sufficient conditions for prediction, possible limits on predictability, and appropriate research strategy.     

Matrix metalloproteinases: a tail of a frog that became a prince

It is 40 years since the first member of what came to be known as the matrix metalloproteinase (MMP) family was described. Structural, molecular and biochemical approaches have subsequently contributed to piecing together the puzzle of how MMPs work, and how they contribute to various disease processes.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

How a G protein binds a membrane

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated Ggamma C terminus and Galpha N terminus, and another near the Galpha C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.     

Ending a decade of deception: a valiant failure, a not-so-valiant failure, and a success story

Prior studies involving two methods, Brooks Parsimony Analysis (BPA) and TreeMap, have found BPA to be the more reliable method. Recent criticisms leveled at these studies argue that the tests were unfairly created and biased in favor of BPA. The authors of a recent critique offered new exemplars to demonstrate flaws in BPA, plus a simple fix to correct the flaws found in TreeMap. A re‐evaluation of their exemplars clearly shows that the authors' calculations are incorrect, their understanding of the methods is lacking, and that their simple fix does not work. Additional analyses using TreeMap 2.02 are run to show that TreeMap 2.02, like TreeMap 1.0, cannot adequately deal with widespread parasites, contrary to the claims of its supporters. Furthermore, the exemplars corroborate previous findings that BPA, when calculated correctly, is more reliable than TreeMap1.0 and TreeMap 2.02 and therefore the method of choice in coevolutionary and biogeographic studies.     

Ergot—a blessing and a scourge

Ergot, the black mass of a particular fungus on cereal grains, especially rye, was the cause of frightful epidemics in Europe more than a thousand years ago, and the ingested contaminated grain has occasioned disease in Russia as late as 1927 and in the United States a generation earlier. Medicinally administered, however, it provides a very important drug in the science of obstetrics.     

Eo: a history of a mutation

Eighteen mouse t haplotype-carrying strains were found not to express cell-surface E molecules controlled by class II genes of the H-2 complex (= Eo strains). Northern and Southern blot analysis of these and other, non-t strains that also fail to express the E molecule, has revealed two kinds of defect. Three strains (CRO437, tw2, and presumably to) were found to transcribe the E alpha gene, but they were not able to convert the message into a functional protein. All other Eo strains fail to transcribe the E alpha gene because of a deletion encompassing the promoter region, the RNA initiation site, and the first exon. The length of the deletion is approximately 650 +/- 50 bp. These two defects closely resemble those found previously in standard inbred strains carrying the H-2f, H-2q (failure of E mRNA to be expressed functionally), H-2b, and H-2s (deletion of a part of the E alpha gene) haplotypes. In particular, the location and length of the E alpha deletion appear to be the same in the strains carrying this mutation. The E alpha deletion is in linkage disequilibrium with certain alleles at other H-2 loci in some of the strains. These observations, combined with the growing evidence that H-2 haplotypes associated with t chromosomes derive from a single ancestral haplotype, suggest that the E alpha deletion is an old mutation and that it has been disseminated in mouse populations by the t chromosomes.     

Carotenoids in floral parts of a narcissus, a daffodil and a tulip

1. The qualitative and quantitative distribution of carotenoids of the floral parts of three monocotyledons, the narcissus Scarlet Elegance, the daffodil King Alfred and the tulip Golden Harvest, were studied. β-Carotene, lutein or epoxy-β-carotenes were usually the main pigments, depending on the floral part and on the flower. When β-carotene was the major pigment there were only small amounts of, or sometimes no, epoxycarotenes. 2. Anthers, stigmas and styles of the three flowers did not possess any specific carotenoids but in some cases contained appreciable amounts of epoxycarotenoids. The possibility that these take part in reproduction is discussed. 3. The generalization that yellow flowers contained large amounts of xanthophylls and only traces of carotenes, whereas deep-orange flowers seemed to be characterized by the presence of large amounts of one carotene, was not always the correct one. It is suggested that the floral parts are yellow or orange depending on what carotenoids are present, which is the major one and the amount of total carotenoids, and also on the presence of other non-carotenoid pigments. 4. Two new probable isomers of 5,6:5′,6′-diepoxy-β-carotene were isolated and found together in various floral parts of the tulip Golden Harvest.     

Novel fluorescent oligoDNA probe bearing a multi-conjugated nucleoside with a fluorophore and a non-fluorescent intercalator as a quencher

A set of 15mer linear oligoDNA probes bearing a modified nucleoside conjugated with a polyamine/fluorescein/anthraquinone reporting moiety were synthesized. In a single-stranded form, the fluorescence generated by the excitation of fluorescein was efficiently quenched, while marked recovery of the fluorescence was observed when the probes formed duplexes with the fully complementary strand.     

From a homeostatic to a homeodynamic self

Life as an autonomous homeostatic system is discussed. A mechanism that drives a homeostatic state to an autonomous self-moving state is examined with two computational cell models. The mechanism is met with Ashby's ultrastability, where random parameter searching is activated when a system breaks a viability constraint. Such a random search process is replaced by the membrane shape in the first model and by chaotic population dynamics in the second model. Emergence of sensors, motors and the recursive coupling between them is shown to be a natural outcome of an autonomous homeostatic system.     

Parameter determination for a computer simulation model of a diver and a springboard

This study used kinematic data on springboard diving performances to estimate viscoelastic parameters of a planar model of a springboard and diver with wobbling masses in the trunk, thigh, and calf segments and spring dampers acting at the heel, ball, and toe of the foot segment. A subject-specific angle-driven eight-segment model was used with an optimization algorithm to determine viscoelastic parameter values by matching simulations to four diving performances. Using the parameters determined from the matching of a single dive in a simulation of another dive resulted in up to 31% difference between simulation and performance, indicating the danger of using too small a set of kinematic data. However, using four dives in a combined matching process to obtain a common set of parameters resulted in a mean difference of 8.6%. Because these four dives included very different rotational requirements, it is anticipated that the combined parameter set can be used with other dives from these two groups.     

Potential Facilitation Between a Commensal and a Pathogenic Microbe in a Wildlife Disease

We assessed the potential for microbial interactions influencing a well-documented host–pathogen system. Mycoplasma agassizii is the known etiological agent of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii), but disease in wild animals is extremely heterogeneous. For example, a much larger proportion of animals harbor M. agassizii than those that develop disease. With the availability of a new quantitative PCR assay for a microbe that had previously been implicated in disease, Pasteurella testudinis, we tested 389 previously collected samples of nasal microbes from tortoise populations across the Mojave desert. We showed that P. testudinis is a common commensal microbe. However, we did find that its presence was associated with higher levels of M. agassizii among the tortoises positive for this pathogen. The best predictor of P. testudinis prevalence in tortoise populations was average size of tortoises, suggesting that older populations have higher levels of P. testudinis. The prevalence of co-infection in populations was associated with the prevalence of URTD, providing additional evidence for an indirect interaction between the two microbes and inflammatory disease. We showed that URTD, like many chronic, polymicrobial diseases involving mucosal surfaces, shows patterns of a polymicrobial etiology.

     

Recognition of a flipped base in a hairpinloop DNA by a small peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Pollinator rarity as a threat to a plant with a specialized pollination system

An increasing diversity of highly specialized pollination systems are being discovered, many of which are likely to be vulnerable to anthropogenic landscape modification. Here, we investigate if a specialized pollination system limits the persistence of Caladenia huegelii (Orchidaceae), an endangered species pollinated by sexual deception of thynnine wasps. Once locally common in part of its geographical range, C. huegelii is now largely restricted to small habitat remnants in urban areas. Pollinator surveys coupled with DNA barcoding detected a single pollinator taxon, a small form of Macrothynnus insignis. Phylogenetic analysis revealed that small M. insignis from within the range of C. huegelii are strongly divergent from other wasp populations, suggesting that some reproductive isolation may exist. Although common in intact landscapes outside the range of C. huegelli, small M. insignis individuals were recorded at only 4% of sites in suitable C. huegelii habitat. Accordingly, reproductive success in C. huegelii was low compared with related Caladenia spp., with 33–60% of populations failing to set fruit in any given year. As such, populations are likely to now persist primarily through individual plant longevity rather than reproduction. Due to the low reproductive success of C. huegelii, ongoing human intervention will almost certainly be needed to sustain the species. Future research will need to focus on optimizing hand pollination to maintain reproduction and high seed fitness. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 511–525.     

What makes a type IIA topoisomerase a gyrase or a Topo IV?

Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.