Towards a molecular understanding of polycythemia rubra vera.

Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.     

Primary (essential) thrombocythemia versus polycythemia vera rubra. A histomorphometric analysis of bone marrow features in trephine biopsies

A morphometric analysis of bone marrow biopsies was performed in 25 patients each with clinical diagnoses of primary (essential) thrombocythemia (PTH) and polycythemia vera rubra (P. vera) according to the rigid diagnostic criteria of the Polycythemia Vera Study Group to reveal significant differences in the histomorphologic features between these disorders. In comparison with control specimens of patients without any hematologic disease, megakaryocyte proliferation was most prominent in PTH, even exceeding that of P. vera with concomitant thrombocythemia (11 of 25 cases with a platelet count greater than 600 X 10(9)/L). Moreover, in P. vera there were wide ranges of megakaryocyte sizes, consisting of micro-megakaryocytes as well as giant forms with highly segmented nuclei (four nuclear lobes), which gave the cells a pleomorphic appearance. As compared with the normal bone marrow, the amount of neutrophilic granulopoiesis and erythropoiesis was not significantly increased in PTH, in contrast to P. vera. Similar results were obtainable regarding the density of reticulin (argyrophilic) fibers: a normal content was encountered in the control specimens and PTH, whereas P. vera displayed a minimal-to-slight increase. Finally, the bone marrow of P. vera was totally devoid of stainable iron while hemosiderin deposits were detected in about two-thirds of the patients without hematologic disorders and in PTH. The characteristic differences revealed by this morphometric study may lead to an improvement of the controversial histologic diagnosis in these disorders.     

Planimetric analysis of megakaryocytes in the four main groups of chronic myeloproliferative disorders.

Planimetry of megakaryocytes (MK) was performed in bone marrow biopsies (BMBs) from patients with chronic myeloproliferative disorders (CMPD) to substantiate cytomorphologic differences in this cell lineage between the four main groups of CMPD. The biopsy specimens were classified histologically prior to morphometry, according to the Hannover Classification of CMPD. Five histological groups were investigated, evaluating between 21 and 30 biopsies in each group. The five groups were as follows: (1) Chronic myelocytic leukemia (CML) of common type (CML.CT), (2) CML with megakaryocytic increase (CML.MI), (3) polycythemia vera (P. vera), (4) primary thrombocythemia (PTH), and (5) chronic megakaryocytic-granulocytic myelosis (CMGM). The results of five variables, i.e. the cellular and nuclear size, the cellular and nuclear form factor, and nuclear segmentation, were determined in at least 50 MK per BMB. The results reveal significant differences in MK nuclear and cellular size, as well as in nuclear segmentation between CML and the three other groups in that the nuclear and cellular size of the MK in CML are smaller than in P. vera, PTH, and CMGM. Moreover, the degree of nuclear segmentation or lobulation differs significantly between the three disorders characterized by large MK. Discriminant analysis permits 78-100% reliability of reclassification by morphometry compared with the histologic classification. A reduced reliability of the morphometric classification to around 80% was found between P. vera and PTH, as well as between P. vera and CMGM. In the design of this study, morphometry of MK lends added weight to the subjective classification of these disorders.     

Chromosome studies in polycythemia vera.

Polycythemia vera (PV) represents an apparent monoclonal stem cell proliferation with a frequent transition to full neoplastic behavior. Up to 26% of untreated PV patients can be expected to have some chromosome abnormalities in the marrow at the time of diagnosis, and 10--15% have an abnormal cell line or clone. Both structural and numerical aberrations occur. Aneuploidy is the most common type of chromosome abnormality, however, with hyperdiploid clones occurring more frequently than hypodiploid clones. Chromosomes 1, 8, 9 and 20 are involved in a non-random pattern, and aberrations of all the F group, or at least the No. 20 chromosome seem to be associated to some extent with diseases involving erythroid hyperplasia. Leukemia develops in a certain percentage of patients regardless of the type of treatment they have received, but the relationship, if any, between the chromosome abnormalities and the development of leukemia is still uncertain. The abnormal clones that occur in PV appear to be quite stable and there is no indication at this time that they correlate with a prognosis of leukemic transformation.     

The genetic legacy of polyploid Bolivian Daphnia: the tropical Andes as a source for the North and South American D. pulicaria complex

We investigated genetic variation in asexual polyploid members of the water flea Daphnia pulex complex from a set of 12 Bolivian high-altitude lakes. We used nuclear microsatellite markers to study genetic relationships among all encountered multilocus genotypes, and combined this with a phylogenetic approach using DNA sequence data of three mitochondrial genes. Analyses of mitochondrial gene sequence divergence showed the presence of three very distinct clades that likely represent cryptic undescribed species. Our phylogenetic results suggest that the Daphnia pulicaria group, a complex of predominantly North American species that has diversified rapidly since the Pleistocene, has its origin in South America, as specific tests of topology indicated that all three South American lineages are ancestral to the North American members of this species group. A comparison between variation of nuclear and mitochondrial markers revealed that closely related polyploid nuclear genotypes sometimes belonged to very divergent mitochondrial lineages, while distantly related nuclear genotypes often belonged to the same mitochondrial lineage. This discrepancy suggests that these South American water fleas originated through reciprocal hybridization between different endemic, sexually reproducing parental lineages. It is also likely that polyploidy of the investigated lineages resulted from this hybridization. Nevertheless, no putative diploid parental lineages were found in the studied region.     

Pruritus and severe iron deficiency in polycythaemia vera.

Six patients diagnosed as having polycythaemia vera had severe pruritus that persisted despite adequate haematological control. Iron supplementation was given when iron deficiency was noted in all six patients. The pruritus began to improve two to 10 days after the start of treatment and had completely disappeared after two to three weeks. In three patients the iron treatment was stopped because of unacceptably high haemoglobin concentrations; the pruritus recurred. Since chronic iron treatment may result in increases in red cell mass indiscriminate use of iron in patients with polycythaemia vera and pruritus is not advocated. Nevertheless, in patients with severe symptoms and evidence of iron deficiency treatment with iron, continuing for two to three weeks after the symptoms have abated, may be beneficial.     

The nucleotypic effects of cellular DNA content in cartilaginous and ray-finned fishes.

Cytological and organismal characteristics associated with cellular DNA content underpin most adaptionist interpretations of genome size variation. Since fishes are the only group of vertebrate for which relationships between genome size and key cellular parameters are uncertain, the cytological correlates of genome size were examined in this group. The cell and nuclear areas of erythrocytes showed a highly significant positive correlation with each other and with genome size across 22 cartilaginous and 201 ray-finned fishes. Regressions remained significant at all taxonomic levels, as well as among different fish lineages. However, the results revealed that cartilaginous fishes possess higher cytogenomic ratios than ray-finned fishes, as do cold-water fishes relative to their warm-water counterparts. Increases in genome size owing to ploidy shifts were found to influence cell and nucleus size in an immediate and causative manner, an effect that persists in ancient polyploid lineages. These correlations with cytological parameters known to have important influences on organismal phenotypes support an adaptive interpretation for genome size variation in fishes.     

Origin and evolution of the mitochondrial proteome.

The endosymbiotic theory for the origin of mitochondria requires substantial modification. The three identifiable ancestral sources to the proteome of mitochondria are proteins descended from the ancestral alpha-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from alpha-proteobacteria. Random mutations in the form of deletions large and small seem to have eliminated nonessential genes from the endosymbiont-mitochondrial genome lineages. This process, together with the transfer of genes from the endosymbiont-mitochondrial genome to nuclei, has led to a marked reduction in the size of mitochondrial genomes. All proteins of bacterial descent that are encoded by nuclear genes were probably transferred by the same mechanism, involving the disintegration of mitochondria or bacteria by the intracellular membranous vacuoles of cells to release nucleic acid fragments that transform the nuclear genome. This ongoing process has intermittently introduced bacterial genes to nuclear genomes. The genomes of the last common ancestor of all organisms, in particular of mitochondria, encoded cytochrome oxidase homologues. There are no phylogenetic indications either in the mitochondrial proteome or in the nuclear genomes that the initial or subsequent function of the ancestor to the mitochondria was anaerobic. In contrast, there are indications that relatively advanced eukaryotes adapted to anaerobiosis by dismantling their mitochondria and refitting them as hydrogenosomes. Accordingly, a continuous history of aerobic respiration seems to have been the fate of most mitochondrial lineages. The initial phases of this history may have involved aerobic respiration by the symbiont functioning as a scavenger of toxic oxygen. The transition to mitochondria capable of active ATP export to the host cell seems to have required recruitment of eukaryotic ATP transport proteins from the nucleus. The identity of the ancestral host of the alpha-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph. There are no indications of a specific alpha-proteobacterial origin to genes for glycolysis. In the absence of data to the contrary, it is assumed that the ancestral host cell was a heterotroph.     

Significance of mild cervical cytologic atypia in a sexually transmitted disease clinic population

While the clinical management of patients with cytologic atypia consistent with cervical intraepithelial neoplasia (CIN) is well established, the management of so-called nondyskaryotic or benign atypias is controversial. The natural history of such atypias was prospectively studied in 124 patients attending a sexually transmitted disease clinic. The benign atypias were subdivided into different categories according to the cell type involved, and the patients were followed cytologically and colposcopically without cervical biopsy until they reached one of the study end points. Benign atypias often signified the presence of CIN: 17% of the patients had a smear consistent with CIN on the first study visit, and an additional 24% had such a smear by six months of follow-up. After 30 months of follow-up, the overall cumulative rate of biopsy-confirmed CIN 2 to CIN 3 was 13.5%; it was 12.5% among those with two consecutive smears showing benign atypias. Of the ten patients who reached biopsy-confirmed CIN 2 to CIN 3, a cytologic smear consistent with CIN was obtained in less than one year of follow-up in all but one case; however, colposcopic evidence of progression was seen in only half of the ten cases, suggesting a sampling error rather than true progression in many cases. Patients with metaplastic cell atypia had a higher progression rate to biopsy-confirmed CIN 2 to CIN 3 (21%) than did those with nonmetaplastic cell atypia (3%). This study provides direct evidence of the potential significance of metaplastic atypia as a marker of CIN. We conclude that patients with mild atypia are often found to have CIN and warrant further investigation by colposcopy and biopsy.     

B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus.

We analyzed 17 B-cell lineages cloned from two patients with infectious mononucleosis and found that different B-cell lineages exhibited notable variation in the length of the fused Epstein-Barr virus (EBV) terminal region on intracellular EBV episomes. EBV termini in different B-cell clones from the same person differed by as many as 15 to 20 reiterations of the ca. 500-base-pair terminal repeat sequence. In contrast, analysis of seven B-cell lineages cloned from a patient with a fatal, oligoclonal lymphoma revealed that three of the cell clones had the same-sized EBV terminal region. These three clones had previously been shown, by immunoglobulin gene analysis, to be metastatic daughter cells descended from a common progenitor. Similarity of the EBV terminal regions in the three daughter clones suggested that EBV infected the progenitor cell before proliferation and metastasis. Individual, EBV-infected cells from a single individual showed sufficient heterogeneity in their EBV termini to allow use of terminal fragment size as a clonal marker in studies addressing the contribution of EBV to the clonal pathogenesis of tumors with which this virus has been associated.     

Multilocus and morphological analysis of south-eastern Iberian Wall lizards (Squamata,Podarcis)

The phylogenetic relationships among the wall lizards of the Podarcis hispanicus complex that inhabit the south-east (SE) of the Iberian Peninsula and other lineages of the complex remain unclear. In this study, four mitochondrial and two nuclear markers were used to study genetic relationships within this complex. The phylogenetic analyses based on mtDNA gene trees constructed with ML and BI, and a species tree using *BEAST support three divergent clades in this region: the Valencia, Galera and Albacete/Murcia lineages. These three lineages were also corroborated in species delimitation analyses based on mtDNA using bPTP, mPTP, GMYC, ABGD and BAPS. Bayesian inference species delimitation method (BPP) based on both nuclear data and a combined data set (mtDNA + nuclear) showed high posterior probabilities for these three SE lineages (≥0.94) and another Bayesian analysis (STACEY) based on combined data set recovered the same three groups in this region. Divergence time dating of the species tree provided an estimated divergence of the Galera lineage from the other SE group (Podarcis vaucheri, (Albacete/Murcia, Valencia)) at 12.48 Ma. During this period, the Betic–Rifian arc was isolated, which could have caused the isolation of the Galera form distributed to the south of the Betic Corridor. Although lizards from the Albacete/Murcia and Galera lineage are morphologically similar, they clearly represent distinct genetic lineages. The noteworthy separation of the Galera lineage enables us to conclude that this lineage must be considered as a new full species.     

Development of novel microsatellite markers to identify the different invasive lineages in the Corbicula complex and to assess androgenesis

Reliable markers are needed to identify the lineages in the invasive clam genus Corbicula. Previous studies have demonstrated that mitochondrial (mt) DNA poorly resolves Corbicula phylogeny, owing to its androgenetic reproductive mode. Moreover, hybridization and mitochondrial/nuclear mismatches occur. We developed the first eleven polymorphic markers to detect these phenomena and to investigate the nuclear identity of Corbicula populations. These microsatellite loci revealed three main lineages in Western Europe. One locus allowed rapid discrimination of these three lineages on agarose gel, saving time and money. Moreover, the eleven markers were successfully cross-amplified in the invasive Corbicula lineages found in North America.     

Microsatellite markers and cytoplasmic sequences reveal contrasting pattern of spatial genetic structure in the red algae species complex Mazzaella laminarioides

Mazzaella laminarioides is a common haploid‐diploid red alga that forms dense beds. This alga has a wide distributional range, covering 3,500 km of the Chilean coast, but is restricted to high rocky intertidal zones. Recently, the existence of three highly divergent genetic lineages was demonstrated for this taxon, and two cytoplasmic markers were used to determine that these lineages are distributed in strict parapatry. Here, using 454 next‐generation sequencing, we developed polymorphic microsatellite loci that cross amplify in all three cytoplasmic lineages. Six sites (i.e., two sites within each lineage) were analyzed using nine microsatellite loci. Our work shows that, although substantial cytoplasmic differentiation occurs within M. laminarioides, the microsatellite loci did not retrieve three nuclear genetic clusters as expected. Indeed, while the northernmost and southernmost cytoplasmic lineages form two strongly divergent nuclear groups characterized by diagnostic alleles, the third cytoplasmic lineage did not form a third nuclear independent group. It is possible that inter‐lineage gene exchange has occurred, particularly at sites along the contact zone between the different cytoplasmic lineages. This nuclear‐cytoplasmic incongruence in M. laminarioides could be explained by incomplete lineage sorting of the nuclear genes or asymmetric introgressive hybridization between the lineages. Finally, highly significant heterozygote deficiencies (suggesting occurrence of intergametophytic selfing) were observed in the three small northernmost sites while the large southernmost sites generally approached panmixia.     

Blood viscosity studies in native and reconstituted polycythemic blood

In twelve subjects with Polycythemia vera (P.V.) whole blood, plasma and relative viscosities and the main factors capable of influencing such parameters (Hct, RBCs, WBCs and platelet count, total proteins, gamma-globulins and fibrinogen) were investigated. Ten normal subjects of similar age and sex were studied as control. Whole blood viscosity was determined both in basal conditions and after reconstitution of Hct to normal values by adding some autologous plasma. After the reconstitution of Hct the subjects studied were divided into two groups on the basis of the correlation between Hct and whole blood viscosity. The first group (group A) had a good correlation between Hct and whole blood viscosity; on the other hand the second group (group B) did not show any significant correlation between these two parameters after reconstitution. In basal conditions none of the parameters capable of influencing whole blood viscosity or plasma viscosity permitted any discrimination between the two groups. The relative viscosity, which represents an indirect index of red blood cell deformability, appears to be more elevated in group B. Therefore, the different behaviour of polycythemic blood after reconstitution of Hct might be conceived to be due to a difference in red cell deformability.     

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.     

Introgression of mitochondrial DNA among lineages in a hybridogenetic ant

We report a remarkable pattern of incongruence between nuclear and mitochondrial variations in a social insect, the desert ant Cataglyphis hispanica. This species reproduces by social hybridogenesis. In all populations, two distinct genetic lineages coexist; non-reproductive workers develop from hybrid crosses between the lineages, whereas reproductive offspring (males and new queens) are typically produced asexually by parthenogenesis. Genetic analyses based on nuclear markers revealed that the two lineages remain highly differentiated despite constant hybridization for worker production. Here, we show that, in contrast with nuclear DNA, mitochondrial DNA (mtDNA) does not recover the two lineages as monophyletic. Rather, mitochondrial haplotypes cluster according to their geographical origin. We argue that this cytonuclear incongruence stems from introgression of mtDNA among lineages, and review the mechanisms likely to explain this pattern under social hybridogenesis.     

Two Mitochondrial Barcodes for one Biological Species: The Case of European Kuhl's Pipistrelles (Chiroptera)

The Kuhl’s pipistrelle (Pipistrellus kuhlii) is a Western Palaearctic species of bat that exhibits several deeply divergent mitochondrial lineages across its range. These lineages could represent cryptic species or merely ancient polymorphism, but no nuclear markers have been studied so far to properly assess the taxonomic status of these lineages. We examined here two lineages occurring in Western Europe, and used both mitochondrial and nuclear markers to measure degrees of genetic isolation between bats carrying them. The sampling focused on an area of strict lineage sympatry in Switzerland but also included bats from further south, in North Africa. All individuals were barcoded for the COI gene to identify their mitochondrial lineages and five highly polymorphic microsatellite loci were used to cluster them according to their nuclear genotypes. Despite this low number of nuclear markers, all North African nuclear genotypes were grouped in a highly distinct subpopulation when compared with European samples sharing the same mitochondrial barcodes. The reverse situation prevailed in Switzerland where bats carrying distinct barcodes had similar nuclear genotypes. There was a weak east/west nuclear structure of populations, but this was independent of mitochondrial lineages as bats carrying either variant were completely admixed. Thus, the divergent mitochondrial barcodes present in Western Europe do not represent cryptic species, but are part of a single biological species. We argue that these distinct barcodes evolved in allopatry and came recently into secondary contact in an area of admixture north of the Alps. Historical records from this area and molecular dating support such a recent bipolar spatial expansion. These results also highlight the need for using appropriate markers before claiming the existence of cryptic species based on highly divergent barcodes.     

Multiple cryptic species of sympatric generalists within the avian blood parasite Haemoproteus majoris

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by < 1.2% in sequence divergence. Records in a database (MalAvi) that contains global findings of haemosporidian lineages generated by universal primers suggest that these lineages vary substantially in host distribution. We confirm this pattern in a data set collected at Lake Kvismaren, Sweden, where three of the generalist lineages have local transmission. However, whether these lineages represent intraspecific mitochondrial diversity or clusters of cryptic species has previously not been examined. In this study, we developed novel molecular markers to amplify the partial segments of four nuclear genes to determine the level of genetic diversity and gene phylogenies among the five morphologically described cyt b lineages of H. majoris. All five cyt b lineages were strongly associated with unique nuclear alleles at all four nuclear loci, indicating that each mitochondrial lineage represents a distinct biological species. Within lineages, there was no apparent association between nuclear alleles and host species, indicating that they form genetically unstructured populations across multiple host species.     

Phylogeny of ground beetles subgenus Nialoe (s. lat.) Tanaka (Coleoptera: Carabidae; genus Pterostichus): A molecular phylogenetic approach

We constructed a phylogeny of the ground beetle subgenus Nialoe ( s. lat. ), genus Pterostichus (Coleoptera: Carabidae) based on two mitochondrial (cytochrome oxidase I and 16S ribosomal DNA) and one nuclear (28S ribosomal DNA) gene sequences. Thirty-three representative species of the group and three outgroup species were analyzed. The resultant trees (maximum parsimonious, maximum likelihood and Bayesian trees of the combined data of the three gene sequences) indicated that there are two large and three small lineages in the group, some of which were supported by a previous morphology-based phylogeny. In all the analyses, the small lineage composed of two Korean species is sister to the rest of the subgenus, but relationships of other four lineages differed among the analyses and remained unresolved. The implications of the present results are discussed in terms of taxonomy and biogeography of the group.