Vivid molecular divergence over volcanic remnants: the phylogeography of Megadromus guerinii on Banks Peninsula,New Zealand

Megadromus guerinii, an endemic carabid beetle (Carabidae), is the most common carabid throughout its restricted range on Banks Peninsula, a formation of extinct volcanoes in Canterbury, New Zealand. This study characterises the small-scale phylogeographic patterns of M. guerinii across the formerly volcanically active Banks Peninsula using mitochondrial and ribosomal genes. Between the eastern and western areas of the peninsula, the mitochondrial, but not nuclear, DNA has a well-defined geographic distribution. Specifically, mitochondrial cytochrome c oxidase subunit I (CO1) identifies two distinct groups (> 6% divergence between eastern and western beetles) while ribosomal genes show no discernible pattern. Whether such a pattern represents male-biased dispersal, Wolbachia infection, a recent range expansion of a divergent lineage, or a deeper historic separation is explored. There is potential that male-biased dispersal could have occurred. Wolbachia infection was not detected. We conclude that historical processes have likely separated taxa in the eastern and western peninsula.     

Effects of Resource Availability on Carbon Allocation and Developmental Instability in Cloned Birch Seedlings

Abundant nitrogen improves seedling growth and establishment. Vigorous growth brings about changes in rates and patterns of plant development and changes in the relationship between primary and secondary metabolism, which may make seedlings more susceptible to herbivores and pathogens than are slow-growing seedlings. We studied how nitrogen fertilization and manual defoliation of source leaves affect growth, carbon allocation, and developmental instability in cloned seedlings of white birch (Betula pubescens Ehrh.). Biomass was higher, whereas concentrations of most classes of phenolic compounds were lower in the nitrogen-rich environment. Interestingly, fertilization did not change the concentrations of cell wall-bound proanthocyanidins, which represent an important fraction of the group of phenolic compounds. Nitrogen enrichment increased levels of fluctuating asymmetry, an index of developmental instability. This result confirms that not only stress but also any deviation from normal resource availability may increase leaf developmental instability in birches. In contrast to fertilization, a one-time defoliation of source leaves did not shape seedling growth, development, or carbon allocation. This could be the result of compensatory growth or of the fact that the defoliation treatment was not strong enough to induce detectable effects until the end of the growing season.     

Immunohistochemical localization of irisin in mole rats (Spalax leucodon)

Irisin was first identified in skeletal muscle cells. It is an exercise protein that is secreted into the circulation; it causes conversion of white adipose tissue to brown adipose tissue. We investigated irisin immunoreactivity in mole rat (Spalax leucodon) tissues. We examined cerebellum, pituitary, heart, liver, pancreas, spleen, uterus, kidney and striated muscle in female adult mole rats. Tissues were processed, embedded in paraffin, sectioned at 5 μm and stained immunohistochemically for irisin. Irisin immunostaining was detected in the cytoplasm of stained cells; the cytoplasm of Purkinje cells was unstained. We found that irisin may be synthesized in many tissues. The function of locally synthesized irisin currently is unknown.     

gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Multiplicity of biochemical factors determining quality of growing birch leaves

Due to rapidly changing physical and biochemical characteristics of growing leaves, correlations between traits of foliage biochemistry and the performance indices of flush feeding herbivores may vary considerably following relatively minor changes in experimental conditions. We examined the effects of the seasonal and inter-tree variation of a comprehensive array of biochemical compounds on the success of an early season geometrid, Epirrita autumnata, feeding on maturing foliage of mountain birch, Betula pubescens ssp. czerepanovii. We monitored the concentrations of individual phenolics, sugars, total nitrogen, nitrogen of proteins, and nitrogen of soluble compounds, water and acetone-insoluble residue. Simultaneously we recorded larval consumption, physiological performance, growth, and pupal mass of E. autumnata. We found significant phenological changes in almost all leaf traits measured. In bioassays with half-grown leaves, leaf gallotannin concentrations showed a nonlinear effect: in trees with high foliar gallotannin concentrations (over 10 mg g⁻¹), physiological performance was strongly reduced by high gallotannin concentrations. In trees with lower gallotannin concentrations, on the other hand, larval growth was reduced by soluble proanthocyanidins, not gallotannins. Differences between high and low gallotannin trees largely depended on phenology, i.e., on the age of leaves. However, not all the differences in leaf traits between late (with high gallotannin concentrations at the time of the bioassay) and early flushing trees disappeared with leaf maturation, indicating that there is also phenology-independent variance in the tree population. In the full-grown leaves of all the study trees, low concentrations of water and of nitrogen of proteins (but not nitrogen of soluble compounds) were the main factors reducing pupal masses of E. autumnata, while neither gallotannin nor proanthocyanidins now played a significant role. The observed change in the factors underlying leaf quality (from gallotannins and proanthocyanidins to nitrogen and water) relate to the activity of the shikimate pathway and the formation of cell walls: gallotannins and proanthocyanidins are both produced in the pathway, and these tannins are assumed to contribute – via binding into cell walls – to tough and durable cell walls. Interestingly, low quality of leaves did not automatically translate into low foliar consumption (i.e., benefits to the tree). On the trees with young, high gallotannin leaves, larvae actually increased consumption on low quality foliage. In the group of trees with slightly more developed, low gallotannin leaves, the quality of leaves did not clearly modify amounts consumed. In full-grown leaves, low leaf quality strongly reduced leaf consumption. These results emphasize the strong influence of tree phenology on the relationships between biochemical compounds and the herbivore. Received: 30 November 1998 / Accepted: 1 March 1999     

Insights into intragenic and extragenic effectors of prion propagation using chimeric prion proteins

The study of fungal prion proteins affords remarkable opportunities to elucidate both intragenic and extragenic effectors of prion propagation. The yeast prion protein Sup35 and the self-perpetuating [PSI+] prion state is one of the best characterized fungal prions. While there is little sequence homology among known prion proteins, one region of striking similarity exists between Sup35p and the mammalian prion protein PrP. This region is comprised of roughly five octapeptide repeats of similar composition. The expansion of the repeat region in PrP is associated with inherited prion diseases. In order to learn more about the effects of PrP repeat expansions on the structural properties of a protein that undergoes a similar transition to a self-perpetuating aggregate, we generated chimeric Sup35-PrP proteins. Using both in vivo and in vitro systems we described the effect of repeat length on protein misfolding, aggregation, amyloid formation and amyloid stability. We found that repeat expansions in the chimeric prion proteins increase the propensity to initiate prion propagation and enhance the formation of amyloid fibers without significantly altering fiber stability.Key words: prion, yeast, sup35, PrP, nonsense suppression, translation termination, amyloid, repeatWe recently described a novel chimeric prion system that was designed to elucidate the consequences of one class of inherited prion disease mutations on protein folding.^1,2 We created a fusion between the mammalian prion protein PrP and the yeast prion protein Sup35p (Fig. 1). Sup35p is an essential translation termination factor in yeast. Interestingly, the majority of the protein can be sequestered into a self-propagating aggregate, the [PSI+] prion.³ Remarkably, when yeast are grown in normal laboratory conditions, the [PSI+] prion is not detrimental. In fact, the biological consequences of the switch from the [psi−] non-prion state to the [PSI+] prion state may be beneficial in terms of adaptation and evolution.⁴ Importantly, the prion state of Sup35p can be readily detected in vivo by monitoring the reduced function of the translation termination factor when the protein is propagating as a prion aggregate.³ In addition, several methods have been developed to not only follow the propagation of the prion, but also to control the propagation and promote prion induction and loss (curing).⁵ Therefore, in addition to simply being a fascinating biological problem in of itself, the [PSI+] prion in yeast affords the ability to further elucidate both intragenic and extragenic effectors of prion biology.Open in a separate windowFigure 1Schematic representation of the yeast protein Sup35p and the mammalian prion protein PrP highlighting the position of the oligopeptide repeat domain (ORD). The amino acid sequence represents the consensus for a single repeat. Numbers shown represent the amino acid position of the beginning and the end of each ORD. The numbers above the schematic represent the original PrP amino acid positioning and the numbers below represent the original Sup35p amino acid sequence positions.Several prions have now been identified and interestingly, there is little sequence homology between the proteins to suggest that only one type of sequence can form a self-propagating aggregate.^6–8 In vitro studies suggest that many proteins can form amyloids under the appropriate conditions.⁹ The fact that only a small percentage of proteins propagate as prions in vivo may be partly a consequence of physiological conditions being adequate to promote amyloid formation with those particular sequences. It is unclear what the precise distinction between prion and amyloid is at this time, but localization alone may preclude some amyloidogenic proteins from being “prion proteins” per se.¹⁰The sequence context that permits a protein to adopt a prion conformation in vivo is unclear. Several of the identified prion proteins have a domain that is enriched in glutamine and asparagine (Q/N) residues, but this is not true of all prion proteins.⁷ Our recent study demonstrates that the Q/N character of the Sup35p prion-forming domain can be significantly reduced, yet still propagate as a prion.¹ This was also found recently in another prion protein chimera created and expressed in yeast.⁶ These studies suggest that the lack of stable secondary structure may be one of the defining features of a prion-forming domain. One of the striking sequence similarities that does exist between two prion proteins occurs in an oligopeptide repeat region found in Sup35p and PrP.¹¹ Previous data clearly demonstrated that the Sup35p repeats are important for [PSI+] prion propagation.^12–15 The deletion of a single repeat from the wild type SUP35 sequence results in the loss of normal [PSI+] prion propagation.¹² Moreover, the addition of two extra repeats of Sup35p sequence served to enhance the formation of the [PSI+] prion.¹³ The expansion of the analogous repeat domain in the mammalian prion protein PrP is associated with an inherited form of prion disease.¹⁶ Since the repeat regions of Sup35p and PrP are similar in size and character, we wanted to determine if the Sup35p oligopeptide repeat region could be substituted with that of PrP. Indeed, the PrP repeats in the context of Sup35p supported the propagation of the [PSI+] prion in yeast.^1,17 Strikingly, we found phenotypic changes that occurred in a repeat length-dependent manner that suggested that the repeat expansions associated with disease result in an increase in the aggregation propensity but do not necessarily dictate only one type of aggregate structure.¹More recently, we verified some of these results in vitro.² These data are in agreement with other studies on the effect of repeat expansions.^18,19 Taking the analysis one step further, we demonstrated that the stability of the amyloid fibers formed with the repeat-expanded proteins did not differ significantly. A very interesting observation that we made was that the formation of amyloid fibers by the longest repeat-expanded chimera (SP14NM) followed drastically different kinetics compared to the chimera containing the wild type number of repeats (SP5NM).² In unseeded reactions, SP14NM did not show a lag phase during the course of fiber formation whereas SP5NM displayed a characteristic lag phase. Furthermore, the morphology of the amyloid fibers visualized by EM was different between SP14NM and SP5NM. SP14NM fibers were curvy and clumped but SP5NM fibers were long and straight. The correlation between the kinetics and the morphology of amyloid formation of SP14NM and SP5NM is reminiscent of fibers formed by β2-microglobulin (β2m) protein in different conditions.²⁰ At pH 3.6, β2m formed curvy, worm-like fibers with no apparent lag phase. In contrast, long, straight fibers were formed at pH 2.5 and had a distinct lag phase. Analysis of the β2m fibers formed at pH 3.6 using mass spectrometric techniques identified species ranging from monomer to 13-mer. This suggested that the fibers were formed by monomer addition. On the other hand, oligomers larger than tetramers were not formed during fiber formation at pH 2.5. Based on these data the authors propose that β2m forms fibers in a nucleation-independent manner at pH 3.6, but fiber formation at pH 2.5 follows a nucleation-dependent mechanism. We suggest that the mechanism underlying SP5NM and repeat-expanded SP14NM fiber formation is similar to β2m fibers formed at pH 2.5 and pH 3.6, respectively. It will be interesting to determine if disease-associated mutations in amyloidogenic proteins alter the pathway whereby amyloid formation occurs and how that process plays a role in pathogenesis.In our in vivo study,¹ we highlighted a unique feature of the longest Sup35-PrP chimera that related to the ability of the protein to adopt multiple self-perpetuating prion conformations more readily than wild type Sup35p. We suggest that this may be an important aspect of prion biology as it relates to inherited disease. If the repeat-expanded proteins can adopt multiple conformations that aggregate, then that may contribute to the large amount of variation observed in pathology and disease progression in this class of inherited prion diseases.^21,22We also found that the spontaneous conversion of the repeat-expanded Sup35-PrP chimera into a prion state was significantly increased. However, this conversion required another aggregated protein in vivo, the [RNQ+] prion. In vitro, the prion-forming domain of the chimera showed a similar trend with the longer repeat lengths enhancing the ability of the protein to form amyloid fibers. The chimera with repeat expansions (8, 11 or 14 repeats) formed fibers very quickly as compared to that with the wild type number of repeats (5). While this correlates with the in vivo data in that both systems demonstrate an increased level of conversion with the repeat expansion, the systems are very different with respect to their requirement for a different “seed” to initiate the prion conversion. So, how does the [RNQ+] prion influence [PSI+]? At the moment, that isn''t entirely clear. Susan Liebman and colleagues discovered another epigenetic factor in yeast, [PIN+], which was important for the de novo induction of [PSI+].^23–25 Several years later, the [RNQ+] prion²⁶ was found to be that factor in the commonly used [PSI+] laboratory strains, but they also found that the overexpression of other proteins could reproduce the effect.²⁵ Hence, [RNQ+] can be [PIN+], and may be the primary epigenetic element that influences [PSI+] induction in yeast, but need not be in every case. Two models were proposed to explain the ability of [RNQ+] to influence the induction of [PSI+].^25,27 One suggested that there is a direct templating effect where the aggregated state of the Rnq1 protein in the [RNQ+] prion serves as a seed for the direct physical association and aggregation of Sup35p and initiates [PSI+]. The second postulated that there is an inhibitor of aggregation in cells that is titrated out by the presence of another aggregated protein. Recent experimental evidence suggests that the templating model may explain at least part of the mechanism of action behind the [RNQ+] prion inducing the formation of [PSI+].^28,29Why is [RNQ+] required for the in vivo conversion of the repeatexpanded chimera that forms amyloid on its own very efficiently in vitro? Interestingly, we found that the [RNQ+] prion per se is not required. We overexpressed the Rnq1 protein from a constitutive high promoter (pGPD-RNQ1) and found that Rnq1p aggregated in the cells but did not induce the [RNQ+] prion. That is, the cells were still [rnq−] and did not genetically transmit the aggregated state of the protein. However, even these non-prion aggregates of Rnq1p served to enhance the induction of the chimeric prions. Therefore, either the [RNQ+] prion or an aggregate of Rnq1 protein is sufficient, which is in line with previous studies that demonstrated that some proteins that aggregate when overexpressed can also enhance the induction of [PSI+].²⁵ Also of note, recent data suggests that the requirement of [RNQ+] for the induction of Sup35p aggregation in vivo can be overcome by very long polyglutamine or glutamine/tyrosine stretches fused to the non-prion forming domain of Sup35p.³⁰ These fusions may alter protein-protein interactions or destabilize the non-prion structure of Sup35p in such a manner that the [RNQ+] prion seed is no longer required to form [PSI+] de novo. Indeed, the non-polymerizing state of some of the fusion proteins was shown to be very unstable.So, what is the important difference between our in vitro and in vivo systems in the prion conversion? Obviously there are many candidates. First, the full length Sup35 protein may alter the conversion properties since a large part of the molecule is the structured C terminal domain. The C terminal domain may influence the initiation of prion propagation in vivo and that is not a factor in the in vitro system. Second, the influences of co-translational folding and potentially some initial unfolding of the prion-forming domain are not present since the in vitro system starts with denatured protein. Third, the environmental influences are clearly different. The molecular crowding effects and chaperones that are required for prion propagation in vivo are not required for the formation of amyloid in vitro. Finally, it is unclear if amyloid structures similar to those formed with the prion-forming domain in vitro actually exist in yeast. Certainly there is some correlation between the structures since aggregated Sup35 protein from [PSI+] cell lysates can seed amyloid formation in vitro^31,32 and the fibers formed in vitro can be transformed into [psi−] cells and cause conversion to [PSI+].³³ Nevertheless, we find it interesting that the expansion of the repeat region can have a tremendous effect on amyloid formation in vitro yet still cannot overcome the requirement for [RNQ+] for conversion in vivo. The presence of co-aggregating or cross-seeding proteins may play a role in the sporadic appearance or progression of neurodegenerative diseases and the interconnected yeast prions [RNQ+] and [PSI+] may provide a model system for elucidating the mechanism underlying such effects.     

Interactive effects of leaf maturation and phenolics on consumption and growth of a geometrid moth

Phenolic compounds are commonly regarded as the main chemical defenses of deciduous woody plants against insects. To examine how indices of leaf maturation (water content, toughness, and sugar/protein ratio) modified larval consumption and growth relative to phenolics and phenolic-related leaf traits, we measured consumption and growth of fourth-instar Epirrita autumnata (Bkh.) (Lepidoptera: Geometridae) larvae on three different days on young, normal, and mature leaves, respectively, from the same mountain birch (Betula pubescens ssp. czerepanovii (Orlova) Hämet-Ahti) trees. The larvae achieved the same growth rates on young and normal leaves, but had to consume 40% more on the latter. On more mature leaves, larval growth was poorer and was positively correlated with sugar/protein ratios (although the ratio peaked at that time). Indices of leaf maturation correlated with several phenolics in data pooled over the three study days, but poorly in any individual day. Similarly, in the pooled data, larval consumption and growth correlated with several leaf traits, but correlations between leaf and insect traits were few on any of the three days, and no trait was significant on each of the three days.We next examined whether variation in the maturation indices modified the associations of phenolics with insect consumption and growth. When interactions between phenolics and leaf maturation indices were taken into account, the number of phenolic compounds displaying significant associations with insect traits more than doubled. The relative importance of interactive versus direct associations increased with leaf maturation: on young leaves five phenolics showed direct and eleven interactive associations with insect traits, while in mature leaves we found two phenolics to display direct and thirteen phenolics interactive associations. Leaf water content, either alone or together with toughness and sugar/protein ratio, generally explained more of the variance in Epirrita growth (up to 59%) than any phenolic or phenolic-related trait alone (highest value 20%). Including interactive effects between phenolics and indices of leaf maturation in the model increased the proportion explained of variance in larval growth between 49 and 73%. Maturation indices explained 0 to 23% of variance in consumption, and the phenolic compound with the highest (positive!) correlation alone up to 28%, but taking into account interactions between phenolics and maturation indices raised the degree of explanation much (namely, 32 to 53%) over that explained by indices of leaf maturation alone. This indicates strong interactive effects on consumption between phenolics and indices of leaf maturation.     

Phenolics from the culms of five bamboo species in the Tangjiahe and Wolong Giant Panda Reserves,Sichuan, China

Phenolic compounds were studied in the culms of five bamboo species collected in China: Yushania chungii, Fargesia robusta, Fargesia denudata, Fargesia rufa and Fargesia scabrida. All the species are eaten by giant panda (Ailuropoda melanoleuca). The culms contained phenolic acids and flavonoids in small concentrations, except for F. robusta, which did not contain flavonoids in detectable amounts. The species differed from each other in their phenolic composition. For example, F. rufa with the highest number of compounds clearly differed from other species. There were also differences among sampling sites, which reflect the differences among genotypes. Furthermore, there were clear ontogenetic differences in the culms: some compounds were present in mature culms but not in young (1–2 year old) culms, while the concentrations of other compounds decreased with increasing age. Over all, the composition and concentrations of soluble phenolic compounds in the bamboo culms were affected by species, age and site.     

Ellagitannins: defences of Betula nana against Epirrita autumnata folivory?

• 1 The induced resistance of the subarctic mountain birch Betula pubescens ssp. czerepanovii is a well‐characterized phenomenon, whereas the induced responses of Betula nana L., one of the parental species of mountain birch, have not yet been characterized. Betula nana is more resistant to several classes of insectivorous herbivores than the mountain birch, although the mechanisms responsible for the better ability to resist herbivores are not known.
• 2 The present study aimed to determine the metabolic changes that are induced by early season herbivory in B. nana leaves and to study the effects of rapidly induced resistance on the growth of Epirrita autumnata larvae.
• 3 Defoliation of B. nana was accomplished by E. autumnata larvae and leaf samples for chemical analyses were collected when the defoliating larvae were at their third and fifth instar. At the same time, laboratory assays for the growth and consumption rates of E. autumnata larvae were conducted.
• 4 The wounding of leaves by E. autumna larvae induced the production of ellagitannins (ETs) in B. nana. Intriguingly, the concentrations of protein‐bound amino acids were also induced by herbivory; however, an increase in proteins was not mirrored in the growth rate of larvae, which was less on the induced foliage. The decreased growth rate of larvae was apparently linked to the increased concentrations of oxidatively‐active ETs and the high concentration of ETs may explain the better resistance of this parental species compared with the hybrid mountain birch with its lower levels of ETs.