Is,indeed, the prion protein a Harlequin servant of “many” masters?

Tens of putative interacting partners of the cellular prion protein (PrP^C) have been identified, yet the physiologic role of PrP^C remains unclear. For the first time, however, a recent paper has demonstrated that the absence of PrP^C produces a lethal phenotype. Starting from this evidence, here we discuss the validity of past and more recent literature supporting that, as part of protein platforms at the cell surface, PrP^C may bridge extracellular matrix molecules and/or membrane proteins to intracellular signaling pathways.Key words: prion protein, PrP^C, extracellular matrix, cell adhesion molecules, neuritogenesis, p59fyn, Ca²⁺Initially, the discovery that the prion protein was the major, if not the unique, component of the prion agent causing transmissible spongiform encephalopathies (TSE)¹ has placed the protein in an extremely unfavorable light. Thereafter, however, a wealth of evidence has supported the notion that the protein positively influences several aspects of the cell physiology, and that its duality—in harboring both lethal and beneficial potentials—could be rationalized in terms of a structural switch. Indeed, the protein exists in at least two conformational states: the cellular, α helix-rich isoform, PrP^C, and the prion-associated β sheet-rich isoform, PrP^Sc.² If it is now unquestionable that the presence of PrP^C in the cell is mandatory for prion replication and neurotoxicity to occur,^3,4 nonetheless its physiologic function is still debatable, despite the long lasting effort, and the numerous, frequently genetically advanced, animal and cell model systems dedicated to the issue. From these studies the picture of an extremely versatile protein has emerged, whereby PrP^C acts in the cell defense against oxidative and apoptotic challenges, but also in cell adhesion, proliferation and differentiation, and in synaptic plasticity.^5,6 In an effort to converge these multiple propositions in an unifying functional model, different murine lines devoid of PrP^C have been studied. These animals, however, displayed no obvious phenotype,^7–9 suggesting that either PrP^C is dispensable during development and adult life or that compensative mechanisms mask the loss of PrP^C function in these paradigms. Thus, identifying the exact role of PrP^C in the cell would not only resolve an important biological question, but would also help elucidate the cellular steps of prion pathogenesis necessary for designing early diagnostic tools and therapeutic strategies for TSE.As is often the case, the employment of a model system unprecedented in prion research has recently disclosed a most interesting scenario with regards to PrP^C physiology, having unravelled, for the first time, a lethal phenotype linked to the absence of the protein.¹⁰ The paradigm is the zebrafish, which expresses two PrP^C isoforms (PrP1 and PrP2). Similarly to mammalian PrP^C, they are glycosylated and attached to the external side of the plasma membrane through a glycolipid anchor. PrP1 and PrP2 are, however, expressed in distinct time frames of the zebrafish embryogenesis. Accordingly, the knockdown of the PrP1, or PrP2, gene very early in embryogenesis impaired development at different stages, bypassing putative compensatory mechanisms. By focusing on PrP1, Malaga-Trillo et al. showed that the protein was essential for cell adhesion, and that this event occurred through PrP1 homophilic trans-interactions and signaling. This comprised activation of the Src-related tyrosine (Tyr) kinase p59fyn, and, possibly, Ca²⁺ metabolism, leading to the regulation of the trafficking of E-cadherin, a member of surface-expressed cell adhesion molecules (CAMs) responsible for cell growth and differentiation.¹¹ It was also reported that overlapping PrP1 functions were performed by PrP^Cs from other species, while the murine PrP^C was capable to replace PrP1 in rescuing, at least in part, the knockdown developmental phenotype. Apart from providing the long-sought proof for a vital role of PrP^C, the demonstration that a mammalian isoform corrected the lethal zebrafish phenotype strongly reinforces previous results—mainly obtained in a variety of mammalian primary neurons and cell lines—pointing to a functional interplay of PrP^C with CAMs, or extra cellular matrix (ECM) proteins, and cell signaling, to promote neuritogenesis and neuronal survival. A revisit of these data is the main topic of the present minireview.As mentioned, the capacity of PrP^C to act as a cell adhesion, or recognition, molecule, and to entertain interactions with proteins implicated in growth and survival, has already been reported for the mammalian PrP^C. A case in point is the interaction, both in cis- and trans-configurations, with the neuronal adhesion protein N-CAM12 that led to neurite outgrowth.¹³ Like cadherins, N-CAM belongs to the CAM superfamily. Following homo- or heterophylic interactions, it can not only mediate adhesion of cells, or link ECM proteins to the cytoskeleton, but also act as a receptor to transduce signals ultimately resulting in modulating neurite outgrowth, neuronal survival and synaptic plasticity.¹¹ Another example is the binding of PrPC to laminin, an ECM heterotrimeric glycoprotein, which induced neuritogenesis together with neurite adhesion and maintenance,^14,15 but also learning and memory consolidation.¹⁶ Further, it has been described that PrPC interacted with the mature 67 kDa-receptor (67LR) (and its 37 kDa-precursor) for laminin, and with glycosamminoglycans (GAGs), each of which is involved in neuronal differentiation and axon growth.^17–21 More recently, Hajj et al.²² have reported that the direct interaction of PrP^C with another ECM protein, vitronectin, could accomplish the same process, and that the absence of PrP^C could be functionally compensated by the overexpression of integrin, another laminin receptor.²³ Incidentally, the latter finding may provide a plausible explanation for the absence of clear phenotypes in mammalian PrP-null paradigms. By exposing primary cultured neurons to recombinant PrPs, others have shown that trans-interactions of PrP^C are equally important for neuronal outgrowth,^24,25 including the formation of synaptic contacts.²⁵ Finally, it has been demonstrated that the binding of PrP^C with the secreted co-chaperone stress-inducible protein 1 (STI1) stimulated neuritogenesis.²⁶ This same interaction had also a pro-survival effect, as did the interaction of PrP^C with its recombinant form.²⁴ Notably, the involvement of PrP^C in cell protection has been heightened by experiments with whole animals. By applying transient or permanent focal cerebral ischemia to the animals, it was found that their reduced brain damage correlated with spontaneous or adenoviral-mediated, upregulation of PrP^C,^27–29 (reviewed in ref. ³⁰), and that PrP^C deficiency aggravated their ischemic brain injury.^30,31 Thus, now that data are available from phylogenetically distant paradigms (zebrafish and mammalian model systems), it acquires more solid grounds the advocated engagement of PrP^C in homo/heterophilic cis/trans interactions to trigger signaling events aiming at neuronal—or, in more general terms, cell—survival and neuritogenesis. The latter notion is consistent with the delayed maturation of different types of PrP^C-less neurons, observed both in vitro and in vivo.^32,33If one assumes that the interaction of PrP^C with multiple partners (45 for PrP^C and PrP^Sc, as reviewed in Aguzzi et al.,⁵ or 46 considering the homophylic interaction) are all functionally significant, the most immediate interpretation of this “sticky” behavior entails that PrP^C acts as a scaffolding protein in different membrane protein complexes.^5,6 Each complex could then activate a specific signaling pathway depending on the type and maturation of cells, the expression and glycosylation of PrP^C, and availability of extra- and intra-cellular signaling partners. At large, all these signals have been shown to be advantageous to the cell. However, because in a cell only a subtle line divides the “good” from the “bad,” instances can be envisioned in which a pro-life signal turns into a pro-death signal. A typical example of this possibility is glutamate excitotoxicity resulting in dangerous, glutamate receptor-linked, Ca²⁺ overload. Likewise, an excessive or over-stimulated signal elicited by PrP^C, or by the putative complex housing the protein could become noxious to the cell. This possibility may explain why the massive expression of PrP^C caused degeneration of the nervous system,³⁴ and of skeletal muscles,^34,35 in transgenic animals. More intriguing is the finding that—in a mouse line expressing anchorless PrP^C—PrP^Sc was capable to replicate without threatening the integrity of neurons.³⁶ This may suggest that native membrane-bound PrP^C acts as, or takes part into, a “receptor for PrP^Sc”, and that lasting PrP^Sc-PrP^C interactions distort the otherwise beneficial signal of the protein/complex and cause neurodegeneration.³⁷ Consistent with this hypothesis is the finding that the in vivo antibody-mediated ligation of PrP^C provoked apoptosis of the antibody-injected brain area.³⁸ Speculatively, the action of N-terminally, or N-proximally truncated PrPs whose expression in PrP-less transgenic mice induced extensive neurodegeneration,^39–41 may be traced back to the same hyper-activation of PrP^C signaling. Possibly, this may hold true also for the synaptic impairment that, recorded only in PrP^C-expressing neurons, was attributed to the binding of amyloid beta (Aβ) peptide oligomers implicated in Alzheimer disease, to PrP^C.^42,43But which is (are) the cellular signaling pathway(s) conveyed by the engagement of PrP^C in different signaling complexes? In line with its multifaceted behavior, several intracellular effectors have been proposed, including p59fyn, mitogen-activated kinases (MAPK) Erk1/2, PI3K/Akt and cAMP-PKA. p59fyn is the most reported downstream effector, suggesting that, in accordance with its behavior, p59fyn could serve as the sorting point for multiple incoming and outgoing signals also in the case of PrP^C. The initial evidence of the PrP^C-p59fyn connection came from cells subjected to antibody-mediated cross-linking of PrP^C.⁴⁴ Later, it was shown that the PrP^C-p59fyn signal converged to Erk1/2 through a pathway dependent on (but also independent of) reactive oxygen species generated by NADPH oxidase.⁴⁵ A PrP^C-dependent activation of p59fyn^13,25 and Erk1/2 (but also of PI3K and cAMP-PKA)²⁴ was evident in other neuronal cell paradigms and consistent with the almost ubiquitous expression of PrP^C, in non-neuronal cells such as Jurkat and T cells.⁴⁶ Not to forget that in zebrafish embryonic cells activated p59fyn was found in the same focal adhesion sites harboring PrP1.¹⁰ Regarding the activation of the ERK1/2 pathway promoted by the PrP^C-STI1 complex, and leading to neuritogenesis, the role of p59fyn was not investigated.²⁶ The same holds true for the transduction of a neuroprotective signal by the PrP^C-STI1 complex involving the cAMP-PKA pathway.²⁶ Interestingly, this is not the only example reporting that engagement of PrP^C activates simultaneously two independent pathways. In fact, possibly after transactivating the receptor for the epidermal growth factor, the antibody-mediated clustering of PrP^C was shown to impinge on both the Erk1/2 pathway, and on a protein (stathmin) involved in controlling microtubule dynamics.⁴⁷Yet, if p59fyn is implicated in mammalian PrP^C-activated signaling cascade, a protein linking extracellular PrP^C to p59fyn is needed, given the attachment of the enzyme to the inner leaflet of the plasma membrane through palmitoylated/myristoylated anchors. In this, the PrP^C partner N-CAM (isoform 140) seems ideal to fulfill the task, given that p59fyn is part of N-CAM-mediated signaling. Indeed, after recruitment of N-CAM to lipid rafts—which may also depend on PrP^C,¹³—together with the receptor protein Tyr phosphatase α (RPTPα), the Tyr-phosphate removing activity of RPTPα allows the subsequent activation of p59fyn through an autophosphorylation step.⁴⁸ This event recruits and activates the focal adhesion kinase (FAK),¹¹ another non-receptor Tyr kinase. Finally, formation of the FAK-p59fyn complex triggers neuritogenesis through both Erk1/2 and PI3K/Akt pathways.^49,50 Parenthetically, the FAK-p59fyn and PI3K/Akt connection would be suitable to explain why aggravation of ischemic brain injury in PrP-deficient brains was linked to a depressed Akt activation.³¹ FAK-p59fyn complex, however, may be also involved in the signal triggered by the still mysterious PrP^C partner, 67LR. This protein was reported not only to act as a laminin receptor but also to facilitate the interaction of laminin with integrins,⁵¹ thereby possibly activating (through integrins) FAK-p59fyn-regulated pathways.⁴⁹ Conversely, other data have supported the candidature of caveolin-1 for coordinating the signal that from PrP^C reaches Erk1/2 through p59fyn.^44,45,52 Further scrutiny of this route has shown that it comprised players such as laminin and integrins (upstream), FAK-p59fyn, paxillin and the Src-homology-2 domain containing adaptor protein (downstream), and that caveolin-1, a substrate of the FAK-p59fyn complex, facilitated the interaction of these signaling partners by recruiting them in caveolae-like membrane domains.⁵³For the relevance they bear, we need to acknowledge recent propositions supporting the commitment of PrP^C with proteins whose function is unrelated from the above-mentioned cell adhesion or ECM molecules; namely, the β-site amyloid precursor protein (APP) cleaving enzime (BACE1) and the N-methyl-D-aspartate (NMDA)-receptor. BACE1 is a proteolytic enzyme involved in Aβ production. It has been shown that overexpressed PrP^C restricted, while depletion of PrP^C increased the access of BACE1 to APP, possibly because PrP^C interacts with BACE1 via GAGs.⁵⁴ Thus, native PrP^C reduces the production of Aβ peptides. A beneficial effect of PrP^C was also highlighted by Khosravani et al.⁵⁵ showing that, by physically associating with the subunit 2D of the NMDA-receptor, PrP^C attenuated neuronal Ca²⁺ entry and its possible excitotoxic effect. This clear example for the control of PrP^C on Ca²⁺ metabolism is particularly intriguing in light of previous reports linking Ca²⁺ homeostasis to PrP^C pathophysiology (reviewed in ref. ⁵⁶). Also, it is important to mention that a few partners of PrP^C or downstream effectors may initiate signals that increase intracellular Ca²⁺, and that, in turn, local Ca²⁺ fluctuations regulate some of the afore-mentioned pathways.^11,49,57,58In conclusion, although still somehow speculative, the implication of Ca²⁺ in PrP^C-dependent pathways raises the possibility that the different input signals originating from the interaction of PrP^C with diverse partners may all converge to the universal, highly versatile Ca²⁺ signaling. Were indeed this the case, then clearly the acting of PrP^C as Harlequin, the famous character of the 18^th century Venetian playwright Carlo Goldoni, who struggles to fill the orders of two masters, would be merely circumstantial.     

Immunological regression of metastatic cancer in the liver as a result of “in vivo xenogenization”

We attempted to induce the regression of liver metastatic tumor cellsin vivo by the administration to rats of Friend leukemia virus (FV) (in vivo xenogenization). The virus which was used in this experiment, FV, is highly immunogenic and does not normally cause disease in an adult rat. At first, we induced a FV viremia in tumor bearing rats in order to deliver the virus to the site of the tumor cells. FV viremia was induced by injecting 60 mg/kg cyclophosphamide (CY) i.v. after the administration of FV, and by transferring syngeneic bone marrow cells so that FV would be able to infect them and then replicate.In order that the tumor cells which were infected with virus should regress, it was necessary to break down their tolerance to FV antigens. As adoptive immunotherapy we therefore, transferred syngeneic spleen cells from rats which had been immunized with FV to tumor bearing rats. The result of this experiment was that these tumor bearing rats infected with FV which had received either normal syngeneic spleen cells or no spleen cells as controls died from liver metastasis (8 out of 9 rats (89%) and 15 out of 17 (88%) respectively). On the other hand, only 4 out of the 15 (27%) tumor bearing rats which were infected with FV and which received FV-immune spleen cells died from liver metastasis.These sets of data indicate that thein vivo xenogenization of tumor cells are indeed able to induce the regression of metastic tumor cells.     

Dicarbonyl L-Xylulose Reductase (DCXR), a “Moonlighting Protein” in the Bovine Epididymis

During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process.     

Evolutionary concepts meet the neck of penguins (Aves: Sphenisciformes), towards a “survival strategy” for evo-devo

Evolutionary developmental biology (or evo-devo) is the scientific connectivity that allowed a more comprehensive and practical completeness in the contemporary conceptualisation of evolution. The links between genetics, developmental mechanics and evolution led to a better understanding of evolutionary mechanisms. An analysis of evolutionary concepts such as homology, homeoses, constraints, novelties, modularity, and selection is given through the recurring example of the variations identified in the modular repartition of the cervical vertebrae in extant and fossil penguins. The inclusion of this study about penguins in the evolutionary system also involves a reflection on the current state and the future of evo-devo. Three principles of assessment and method, applicable to many natural and conceptual scales, are introduced to define a ??survival strategy?? for evo-devo. The above-mentioned principles are intended to strengthen and continue the connectivity induced de facto. These current and future investigation challenges are discussed and connected to three main naturalist names related directly to the conceptualisation of evolution: Charles Darwin, étienne Geoffroy Saint-Hilaire, and Lamarck.     

Phenotypic Effect of “Candidatus Rickettsiella viridis,” a Facultative Symbiont of the Pea Aphid (Acyrthosiphon pisum), and Its Interaction with a Coexisting Symbiont

A gammaproteobacterial facultative symbiont of the genus Rickettsiella was recently identified in the pea aphid, Acyrthosiphon pisum. Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host''s ecological characteristics, such as attractiveness to different natural enemies. In European populations of A. pisum, the majority of Rickettsiella-infected aphids also harbor another facultative symbiont, of the genus Hamiltonella. We investigated this Rickettsiella symbiont for its interactions with the coinfecting Hamiltonella symbiont, its phenotypic effects on A. pisum with and without Hamiltonella coinfection, and its infection prevalence in A. pisum populations. Histological analyses revealed that coinfecting Rickettsiella and Hamiltonella exhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, while Rickettsiella-specific localization was found in oenocytes. Rickettsiella infections consistently altered hosts'' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes. Rickettsiella-Hamiltonella coinfections also changed red aphids to green; this greenish hue tended to be enhanced by Hamiltonella coinfection. With different host genotypes, Rickettsiella infection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereas Hamiltonella infection and Rickettsiella-Hamiltonella coinfection had negative effects. Despite considerable frequencies of Rickettsiella infection in European and North American A. pisum populations, no Rickettsiella infection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction of Rickettsiella with other facultative symbionts, their effects on their hosts'' phenotypes, and their persistence in natural host populations. We propose the designation “Candidatus Rickettsiella viridis” for the symbiont.     

“As du Coeur” study: a randomized controlled trial on quality of life impact and cost effectiveness of a physical activity program in patients with cardiovascular disease

Background

Physical activity programs (PAP) in patients with cardiovascular disease require evidence of cost-utility. To assess improvement in health-related quality of life (QoL) and reduction of health care consumption of patients following PAP, a randomized trial was used.

Methods

Patients from a health insurance company who had experienced coronary artery disease or moderate heart failure were invited to participate (N?=?1891). Positive responders (N?=?50) were randomly assigned to a progressively autonomous physical activity (PAPA) program or to a standard supervised physical activity (SPA) program. The SPA group had two supervised sessions per week over 5?months. PAPA group had one session per week and support to aid habit formation (written tips, exercise program, phone call). To measure health-related quality of life EQ-5D utility score were used, before intervention, 6?months (T6) and 1 year later. Health care costs were provided from reimbursement databases.

Results

Mobility, usual activities and discomfort improved significantly in both group (T6). One year later, EQ-5D utility score was improved in the PAPA group only. Total health care consumption in the intervention group decreased, from a mean of 4097 euros per year before intervention to 2877 euros per year after (p?=?0.05), compared to a health care consumption of 4087 euros and 4180 euros per year, in the total population of patients (N?=?1891) from the health insurance company. The incremental cost effectiveness ratio was 10,928 euros per QALYs.

Conclusion

A physical activity program is cost-effective in providing a better quality of life and reducing health care consumption in cardiovascular patients.

Trial registration

ISRCTN77313697, retrospectively registered on 20 November 2015.

     

Phytoplankton communities and biomass size structure (fractionated chlorophyll “a”), along trophic gradients of the Basque coast (northern Spain)

Eutrophication is a major threat to coastal ecosystems. Within Europe, the Water Framework Directive (WFD) has established the need of developing methods of assessment. Bulk chlorophyll “a” is used world-wide as an indicator of eutrophication. However, the size structure of the phytoplankton communities has not been investigated, in detail, in relation to eutrophication pressures. This study investigates the suitability of fractionated chlorophyll “a” (<10 and >10 μm), as an indicator of eutrophication. Along the Basque coast (northern Spain) several water bodies, ranging from offshore waters to the heads of the estuaries, were surveyed during 2008. Physico-chemical conditions and chlorophyll “a” showed a distinct spatial gradient. Trophic richness increased towards the middle and inner parts of the estuaries, where nutrient inputs, from natural or anthropogenic sources, together with the residence time of the water are usually higher. In summer, phytoplankton biomass and abundance decreased, generally, in offshore and coastal waters; in estuaries, they tended to increase. The chlorophyll distribution within the two size fractions was coherent with the phytoplankton taxonomic composition. In summer, the relative abundance of small-sized cells (diatoms and non-siliceous taxa) increased. The relationships between size-fractionated chlorophyll and physico-chemical variables differed, when comparing the offshore and coastal waters, with the estuaries. In the offshore and coastal waters, a strong seasonality was observed; in estuaries, an important spatial component was found. Fractionated chlorophyll provided complementary useful information on anthropogenic pressures. However, more research is necessary to utilise this variable as a tool for ecological status assessment, within the WFD.     

Dynamics of a vortex with the U-shaped filament (ends “anchored”) in the heart of the ground squirrel

The dynamics of an electrical scroll wave with the U-shaped filament with both ends of the filament being “anchored” on the endocardial surface and the dependence of the structure of pseudoECG on the dynamics of the vortex during the development of polymorphic tachysystolia have been studied by applying premature stimuli to the “target phase” with subsequent registration of the spatial and temporal distribution of electrical potential throughout the surface (endocardial and epicardial) of a thin (≈1 mm) preparation. It was found that (1) the pseudoECG of the polymorphic form during the tachysystolia attack can be observed in the case that the position of the filament ends on the surfaces of the preparation does not practically change from turn to turn (filament ends are “anchored”); (2) the thread of a scroll wave during this attack can twist and untwin (twisted filament), just as it was the case for scroll waves with a straight filament; (3) in the case of pseudoECG of polymorphic form, the twisting and untwining of the filament were stronger (the angle of maximal twisting was 120 degrees and more), and the angle of twisting changed by a substantially greater value from turn to turn as compared with the pseudoECG of monomorphic form; (4) in the case of pseudoECG of polymorphic form, the time interval between the appearance of waves on the surfaces of the preparation (T _epi-endo) was substantially greater and changed to a greater extent from turn to turn of the vortex; and (5) simultaneously with the appearance of pseudoECG of polymorphic form and the onset of changes in the twisting of the scroll and the T _epi-endo interval indicated in (2–4), significant changes in the patterns of coverage of the surface by excitation occurred. Based on the results obtained, an explanation of the reasons for the appearance of excitation breakdown patterns on the surface of the myocardium was proposed, which differs from the traditional viewpoint. These patterns may be the result of reflection on myocardial surfaces of the activity of not different simultaneously occurring sources of initiation of excitation but of a single three-dimensional vortex whose filament twists when passing through the thickness of the myocardium and can closely approach one or the other surface.     

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grant-in-aid to the John Innes Institute.     

Report of an archaic human skeleton discovered at altamura (Bari), in the “Lamalunga” district

The circumstances of the discovery and the first morphological observations concerning an, archaic human skeleton found in Altamura (Bari), in the “Lamalunga” district are reported. The observations were made by anthropologists from Bari University, at the site of the discovery, in a cave (Le Solange cave), a few days after they were first spotted by speleologists from the Speleological Research Center of Altamura (C.A.R.S.) and from the “Vespertilio” group of the Italian Alpine Club (C.A.I.) of Bari. The human remains were found in a corner of a small cavity in close association with columnar stalagmitic formations. All the bones are partly covered with, or embedded in, calcareous concretion while others are visible but lined with a calcareous shell of varying thickness. It is possible to recognize the skull (lying on its back); part of the face is apparent including the orbits and all the frontal region. On the ground, very close to the skull, there are many postcranial bones, but not all immediately recognizable because of the concretions. Immediately in front of and below the frontal are the tworadii; between these and a femoral shaft is the complete, but inverted, mandible. Bothfemora, thetibiae and thefibulae are present. Furthermore, the righthumerus, the distal epiphysis of an ulna and some costal elements are recognizable. The elements furthest from thecranium appear to be the innominate bones, partly covered with calcitic crust. Inside the pelvis are heavily, concreted pieces of bone among which are a costal element and a vertebral body. From the general robusticity of the bones and the rather high and narrow iliac fossa, the skeleton can be judged that of an adult male. Considering the presence on thecranium at the same time of both progressive and ancient features the Altamura specimen can be placed in the group of fossils of the European mid-Pleistocene characterized by the presence of archaic morphological traits and by a progressive increase in Neanderthal traits.     

Defining “high quality” food resources of herbivores: the case for meadow voles (Microtus pennsylvanicus)

Summary Food availability, food utilization patterns and levels of some nutritional factors in plants were studied in 1984–85 in an old-field community supporting low to moderate densities of meadow voles (Microtus pennsylvanicus). Food choice and preference indices were positively related to levels of proteins, and negatively related to levels of total phenolics and ADL fibers in plants. High quality resources for voles were resources that had the highest amount of proteins, and the lowest level of total phenolics and ADL fibers among available plant species. There were only two plants species among the available ones that possessed these characteristics, Festuca rubra and Vicia cracca and they represented 25 to 50% of the available biomass during summer. Vole densities of up to 64 animals/ha were therefore not limited by the availability of high quality resources. Fecal analyses performed on 267 animals in 1984 and 269 voles in 1985 showed that all voles ate high quality resources. Meadow voles appear to choose foods with high protein content and low levels of digestion inhibitors, and not on the basis of caloric content or availability. These observations contradict predictions arising from current antiherbivore hypotheses and lead us to reconsider these models in light of this new evidence.     

“Plants that Remind Me of Home”: Collecting,Plant Geography,and a Forgotten Expedition in the Darwinian Revolution

In 1859, Harvard botanist Asa Gray (1810–1888) published an essay of what he called “the abstract of Japan botany.” In it, he applied Charles Darwin’s evolutionary theory to explain why strong similarities could be found between the flora of Japan and that of eastern North America, which provoked his famous debate with Louis Agassiz (1807–1873) and initiated Gray’s efforts to secure a place for Darwinian biology in the American sciences. Notably, although the Gray–Agassiz debate has become one of the most thoroughly studied scientific debates, historians of science remain unable to answer one critical question: How was Gray able to acquire specimens from Japan? Making use of previously unknown archival materials, this article scrutinizes the institutional, instrumental, financial, and military settings that enabled Gray’s collector, Charles Wright (1811–1885), to travel to Japan, as well as examine Wright’s collecting practices in Japan. I argue that it is necessary to examine Gray’s diagnosis of Japan’s flora and the subsequent debate about it from the viewpoint of field sciences. The field-centered approach not only unveils an array of historical significances that have been overshadowed by the analytical framework of the Darwinian revolution and the reception of Darwinism, but also places a seemingly domestic incident in a transnational context.     

The migration of beech (Fagus sylvatica L.) up the Rhone: the Mediterranean history of a “mountain” species

New anthracological data on the beech (Fagus sylvatica L.), obtained through rescue excavations, allows the proposal of a new pattern of population by beech of the South of France since the Lateglacial. Due to its current ecology and distribution it is considered to be a mountain species in the Mediterranean area despite the presence of beech forests, usually described as glacial relicts, at low altitudes in the South of France and in the Rhone valley (Sainte-Baume, Valbonne and Grand Fays). Our results, combined with previous pollen and charcoal data, show both an early spread from glacial refuges and a late disappearance of the species. During the glacial period, the beech remained in the southernmost zones. From there, it first spread during the Preboreal towards outposts in the Rhone delta, and during the Atlantic along the rhodanian corridor. During the Subboreal, a retreat of beech towards the Pre-Alps and the Rhone delta can be traced. The Iron Age and the Classical period experienced the maximal concentration of beech along the Rhone river, while it became more rare in the North and in the South. During the Middle Ages, it first disappeared from these distal areas, and later also in the valley (around A.D. 1000), finally settling in its present mountainous distribution area (probably around the 14th century A.D.). Some crucial points of the proposed dynamics are further analysed: the paradoxical regression of beech in the valley during the Subboreal, the factors determining the expansion of beech from refuge areas during the Subatlantic and finally, the disappearance of the beech in the middle Rhone valley in rather recent times, a disappearance attributed to the strong human impact on the landscape.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Molecular Characterization of “Candidatus Parilichlamydia carangidicola,” a Novel Chlamydia-Like Epitheliocystis Agent in Yellowtail Kingfish,Seriola lalandi (Valenciennes), and the Proposal of a New Family, “Candidatus Parilichlamydiaceae” fam. nov. (Order Chlamydiales)

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “Candidatus Piscichlamydia salmonis” ({"type":"entrez-nucleotide","attrs":{"text":"AY462244","term_id":"42525281","term_text":"AY462244"}}AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “Candidatus Parilichlamydia carangidicola” and that the new family be known as “Candidatus Parilichlamydiaceae.”