Fausto Panara

The initial peopling of the Americas: a growing number of founding mitochondrial genomes from Beringia.

Pan-American mitochondrial DNA (mtDNA) haplogroup C1 has been recently subdivided into three branches, two of which (C1b and C1c) are characterized by ages and geographical distributions that are indicative of an early arrival from Beringia with Paleo-Indians. In contrast, the estimated ages of C1d--the third subset of C1--looked too young to fit the above scenario. To define the origin of this enigmatic C1 branch, we completely sequenced 63 C1d mitochondrial genomes from a wide range of geographically diverse, mixed, and indigenous American populations. The revised phylogeny not only brings the age of C1d within the range of that of its two sister clades, but reveals that there were two C1d founder genomes for Paleo-Indians. Thus, the recognized maternal founding lineages of Native Americans are at least 15, indicating that the overall number of Beringian or Asian founder mitochondrial genomes will probably increase extensively when all Native American haplogroups reach the same level of phylogenetic and genomic resolution as obtained here for C1d.

Multiple forms of barley root acid phosphatase: purification and some characteristics of the major cytoplasmic isoenzyme.

The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr approximately 38,000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was Mr 77,600 and 79,000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate (Ki = 9 x 10(-7) M). NaF, Ag(+), Hg(2+), Pb(2+) and Zn(2+) are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4-8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.

Mitochondrial Haplogroup U5b3: A Distant Echo of the Epipaleolithic in Italy and the Legacy of the Early Sardinians

There are extensive data indicating that some glacial refuge zones of southern Europe (Franco-Cantabria, Balkans, and Ukraine) were major genetic sources for the human recolonization of the continent at the beginning of the Holocene. Intriguingly, there is no genetic evidence that the refuge area located in the Italian Peninsula contributed to this process. Here we show, through phylogeographic analyses of mitochondrial DNA (mtDNA) variation performed at the highest level of molecular resolution (52 entire mitochondrial genomes), that the most likely homeland for U5b3-a haplogroup present at a very low frequency across Europe-was the Italian Peninsula. In contrast to mtDNA haplogroups that expanded from other refugia, the Holocene expansion of haplogroup U5b3 toward the North was restricted by the Alps and occurred only along the Mediterranean coasts, mainly toward nearby Provence (southern France). From there, approximately 7,000-9,000 years ago, a subclade of this haplogroup moved to Sardinia, possibly as a result of the obsidian trade that linked the two regions, leaving a distinctive signature in the modern people of the island. This scenario strikingly matches the age, distribution, and postulated geographic source of a Sardinian Y chromosome haplogroup (I2a2-M26), a paradigmatic case in the European context of a founder event marking both female and male lineages.

The effects of copper on actin and fibronectin Organization in Mytilus galloprovincialls haemocytes

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.

Geographical and seasonal evidence of cryptic diversity in the Baetis rhodani complex (Ephemeroptera, Baetidae) revealed by means of DNA taxonomy

Previous phylogenetic investigations on the mayfly Baetis rhodani Pictet from several European countries, excluding Italy, strongly suggested the presence of cryptic species. Our paper reports a DNA-taxonomy phylogenetic analysis of B. rhodani with additional populations coming from Italian and UK sites, and aims to identify potential cryptic species with a coalescent-based method (GMYC model) and to understand the mechanisms of local coexistence of cryptic species. Twenty-five haplotypes of Italian samples and five haplotypes of UK samples were identified and added to a large European dataset. A total of 11 potential cryptic species have been recognised, and three of them co-occured in one Italian area. Such cryptic species seem to be phylogenetically over-dispersed on the tree and temporally segregated, and the seasonal substitution pattern of cryptic species could explain the apparently widespread distribution of the B. rhodani complex and its ability to adapt to different temperatures and food resources, justifying some of the differences observed in the relationship between water temperature, growth rates and phenology documented from field studies.

A comparison of conservative DNA extraction methods from fins and scales of freshwater fish: A useful tool for conservation genetics

Key words: brown trout, conservative DNA extraction, fish fin clip, fish scales, northern pikeNon-destructive protocols for DNA isolationfrom fresh or preserved specimens are fundamen-tal to study endangered or elusive species, breedersor samples sibship and specimens derived frompublic or private collections (Nielsen et al. 1999;Wasko et al. 2003; Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yieldand poor quality DNA, particular laboratory careand standardizations were needed to allow repro-ducible results. We compared six DNA extractionprocedures from conservative samples: three arecommercial kits [Wizard Genomic DNA Purifi-cation Kit (WGDPK) (Promega); Wizard Mag-netic DNA Purification System for Food(WMDPF) (Promega); NucleoSpin Food (NSF)(Macheray–Nagel)] and three are largelyemployed methodologies [Trizol (Life Technolo-gies); Chelex (Sigma–Aldrich); C-TAB]. For eachmethod, the standard protocol (reported on datasheets or the first published one) and severalvariations were tested varying sample storage andpre-lysis conditions, homogenization procedures,buffer solutions and concentrations, incubationsand resuspension times and temperatures. DNAwas extracted from 200 northern pikes (Esoxlucius L.) and 100 brown trout (Salmo trutta farioL.) (Table 1) from small (£10 mg) fin pieces and5–10 scales stored dried or in absolute alcoholboth at )20 C and at room temperature. DNAextraction was carried out on fresh materials andrepeated within a period of three years (Table 1),and from 34 years old dried scales culled from amuseum collection. Furthermore, DNA wasextracted from liver and muscle of both species,from individuals that had naturally died and usedas controls. Spectrophotometric readings (260 and280 nm) and densitometric evaluations (Image J)of DNAs obtained through different methodolo-gies and protocols (conservative versus controlDNA) were analyzed by means of ANOVA andt-test. DNA suitability was assayed through PCR–RFLP and microsatellite analyses (Lucentini et al.(2006) (Table 2). Microsatellites were run onABI377 DNA sequencer whereas PCR–RFLPswere assayed on 2.5% agarose electrophoresis fortwo mtDNA NADH coding regions (ND-1 ND5/6) (Cronin et al. 1993). DNA stability andamplificability was controlled every three months,for three years, in parallel with control DNA. Toevaluate genotyping errors, null alleles presenceand allelic dropout, all the experiments were rep-licated and statistically treated as suggested(Hoffman and Amos 2005; Roon et al. 2005)through MICRO-CHECKER 2.2.3 (Vanoosterhout et al. 2004). Deviations from theHardy–Weinberg equilibrium were tested byArlequin 2000 and Genepop.Our results indicate that the quality and quan-tity of DNA extracted from fin clips and scalesvaried according to extraction methods (Table 2)and storage conditions. Alcohol preservation at)20 C is fundamental for fin specimens, althoughdried scales conservation at room temperatureallowed good DNA extraction. One percent

Isolation and partial characterization of high and low molecular weight acid phosphatases from chicken liver

Two acid phosphatase forms were isolated from chicken liver by gel filtration on Sephadex G-100. These enzymes, termed I and II, have similar Km- and Vmax-values, but differ in molecular weight, optimum pH, sensitivity to various inhibitors and substrate specificity. The results were compared with the numerous literature reports of mammalian acid phosphatases.

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the musselMytilus galloprovincialis

SummaryCell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesiles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cystoskeleton inMytilus galloprovincialis haemocytes are discussed.

Functional aspects of calcium transport in sarcoplasmic reticulum vesicles derived from frog skeletal muscle treated with saponin

SummaryTo study the physiological aspects of the excitation-contraction cycle, saponin (10–100Μg ml−1) was used as a skinning agent on muscle and sarcotubular vesicles derived from fast muscles (sartorius and tibialis anterior) ofRana esculenta.The vesicles showed similar Ca2+-ATPase activity and similar protein profiles carried out by SDS-PAGE. Calcium transport in untreated vesicles and those treated with different concentrations of saponin seemed to have the same quantitative and qualitative parameters if the saponin was used in a range between 10 and 50Μg ml−1.Our results confirm that saponin may be considered to be a valid skinning agent for the external membranes of fast skeletal muscles.

Molecular and Phenotypic Evidence of a New Species of Genus Esox (Esocidae, Esociformes, Actinopterygii): The Southern Pike, Esox flaviae

We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.