Quaternary refugia of the sweet chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.): an extended palynological approach

Knowledge about the glacial refugia of the thermophilous European Castanea sativa Mill. (sweet chestnut) is still inadequate. Its original range of distribution has been masked by strong human impact. Moreover, under natural conditions the species was probably admixed with other taxa (such as Quercus, Fraxinus, Fagus, Tilia) and thus possibly represented by low percentages in pollen records. In this paper we try to overcome the difficulties related to the scarcity and irregularity of chestnut pollen records by considering 1471 sites and extending the palynological approach to develop a Castanea refugium probability index (IRP), aimed at detecting possible chestnut refugia where chestnuts survived during the last glaciation. The results are in close agreement with the current literature on the refugia of other thermophilous European trees. The few divergences are most probably due to the large amount of new data integrated in this study, rather than to fundamental disagreements about data and data interpretation. The main chestnut refugia are located in the Transcaucasian region, north-western Anatolia, the hinterland of the Tyrrhenian coast from Liguria to Lazio along the Apennine range, the region around Lago di Monticchio (Monte Vulture) in southern Italy, and the Cantabrian coast on the Iberian peninsula. Despite the high likelihood of Castanea refugia in the Balkan Peninsula and north-eastern Italy (Colli Euganei, Monti Berici, Emilia-Romagna) as suggested by the IRP, additional palaeobotanical investigations are needed to assess whether these regions effectively sheltered chestnut during the last glaciation. Other regions, such as the Isère Département in France, the region across north-west Portugal and Galicia, and the hilly region along the Mediterranean coast of Syria and Lebanon were classified as areas of medium refugium probability. Our results reveal an unexpected spatial richness of potential Castanea refugia. It is likely that other European trees had similar distribution ranges during the last glaciation. It is thus conceivable that shelter zones with favourable microclimates were probably more numerous and more widely dispersed across Europe than so far assumed. In the future, more attention should be paid to pollen traces of sporadic taxa thought to have disappeared from a given area during the last glacial and post-glacial period.Electronic Supplementary Material Supplementary material is available in the online version of this article at . A link in the frame on the left on that page takes you directly to the supplementary material.An erratum to this article can be found at     

Observation of sequence specificity in the seeding of protein amyloid fibrils

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Vimentin and leukocyte common antigen-negative molding cells in pleural effusions of patients with small cell lung carcinoma

OBJECTIVE: To determine whether the presence of vimentin and leukocyte common antigen (LCA)-negative molding cells (VLNMC) could help in identifying rare small cell lung carcinoma (SCLC) cells. STUDY DESIGN: Thirty-four cell blocks of pleural effusions (PEs) from 26 patients with confirmed SCLC were stained immunohistochemically with vimentin and LCA antibodies and compared with hematoxylin and eosin-stained preparations. RESULTS: VLNMC were present in 22/22 PEs originally diagnosed as positive or atypical/suspicious for SCLC. Focal vimentin staining was seen in SCLC in 10/22 cases, and 1 case showed many vimentin-positive SCLC cells. One of 11 PEs originally interpreted as negative showed rare groups of VLNMC. This was supported by a subsequent PE obviously positive for SCLC. CONCLUSION: Immunoperoxidase stains for vimentin and LCA highlight SCLC in PEs as VLNMC; however, morphologic criteria must prevail in making the final diagnosis.     

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.     

Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure

The structure of the precursor form of catechol oxidase from sweet potatoes (Ipomoea batatas) has been modeled on the basis of the 3D structural data of mature catechol oxidase [Nat. Struct. Biol. 5 (1998) 1084] and of hemocyanin from giant octopus (Octopus dofleini) [J. Mol. Biol. 278 (1998) 855]. A C-terminal extension peptide is found in the cDNA sequence but not in the purified, mature form of catechol oxidase. Superimposition of the 3D structures of the native hemocyanin and catechol oxidase reveals a close relationship except for an additional C-terminal domain only found in the hemocyanin structure. As sequence alignment shows good homology this domain of the hemocyanin structure was used as a template to model the 3D structure of the C-terminal extension peptide of catechol oxidase. As hemocyanins show no or only weak catecholase activity due to this domain this indicates an inhibitory function of this extension peptide. Beside this possible shielding function for the precursor form, evidence for a function in copper-uptake also increases due to the location of three histidine residues in the model.     

Normal mode analysis of macromolecular motions in a database framework: developing mode concentration as a useful classifying statistic

We investigated protein motions using normal modes within a database framework, determining on a large sample the degree to which normal modes anticipate the direction of the observed motion and were useful for motions classification. As a starting point for our analysis, we identified a large number of examples of protein flexibility from a comprehensive set of structural alignments of the proteins in the PDB. Each example consisted of a pair of proteins that were considerably different in structure given their sequence similarity. On each pair, we performed geometric comparisons and adiabatic-mapping interpolations in a high-throughput pipeline, arriving at a final list of 3,814 putative motions and standardized statistics for each. We then computed the normal modes of each motion in this list, determining the linear combination of modes that best approximated the direction of the observed motion. We integrated our new motions and normal mode calculations in the Macromolecular Motions Database, through a new ranking interface at http://molmovdb.org. Based on the normal mode calculations and the interpolations, we identified a new statistic, mode concentration, related to the mathematical concept of information content, which describes the degree to which the direction of the observed motion can be summarized by a few modes. Using this statistic, we were able to determine the fraction of the 3,814 motions where one could anticipate the direction of the actual motion from only a few modes. We also investigated mode concentration in comparison to related statistics on combinations of normal modes and correlated it with quantities characterizing protein flexibility (e.g., maximum backbone displacement or number of mobile atoms). Finally, we evaluated the ability of mode concentration to automatically classify motions into a variety of simple categories (e.g., whether or not they are "fragment-like"), in comparison to motion statistics. This involved the application of decision trees and feature selection (particular machine-learning techniques) to training and testing sets derived from merging the "list" of motions with manually classified ones.     

Free fatty acids/triglycerides increase ocular and subcutaneous blood flow

Elevated plasma free fatty acids (FFA) induce skeletal muscle insulin resistance and impair endothelial function. The aim of this study was to characterize the acute hemodynamic effects of FFA in the eye and skin. A triglyceride (Intralipid 20%, 1.5 ml/min)/heparin (bolus: 200 IU; constant infusion rate: 0.2 IU. kg(-1). min(-1)) emulsion or placebo was administered to 10 healthy subjects. Measurements of pulsatile choroidal blood flow with laser interferometry, retinal blood flow with the blue field entoptic technique, peak systolic and end diastolic blood velocity (PSV, EDV) in the ophthalmic artery with Doppler sonography, and subcutaneous blood flow with laser Doppler flowmetry were performed during an euglycemic somatostatin-insulin clamp over 405 min. Plasma FFA/triglyceride elevation induced a rise in pulsatile choroidal blood flow by 25 +/- 3% (P < 0.001) and in retinal blood flow by 60 +/- 23% (P = 0.0125). PSV increased by 27 +/- 8% (P = 0.001), whereas EDV was not affected. Skin blood flow increased by 149 +/- 38% (P = 0.001). Mean blood pressure and pulse rate remained unchanged, whereas pulse pressure amplitude increased by 17 +/- 5% (P = 0.019). Infusion of heparin alone had no hemodynamic effect in the eye or skin. In conclusion, FFA/triglyceride elevation increases subcutaneous and ocular blood flow with a more pronounced effect in the retina than in the choroid, which may play a role for early changes of ocular perfusion in the insulin resistance syndrome.     

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.