Prognostic value of antigen expression in multiple myeloma: a PETHEMA/GEM study on 1265 patients enrolled in four consecutive clinical trials.

Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19pos, CD27neg, CD38lo, CD45pos, CD81pos, CD117neg and CD138lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38lowCD81posCD117neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.

Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems.

Polysaccharides have shown ideal features for their application in nanomedicine as nanoparticulated systems. Nanoparticles based on mixtures of alginate and chitosan (A/Q-50/50, formed by 50% alginate and 50% chitosan, and A/Q-70/30, formed by 70% alginate and 30% alginate) have been synthesised by an emulsification method and stabilised by amide bond formation. Tamoxifen (TMX) was loaded into these systems, and they were assayed as controlled delivery formulations. Results showed the formation of spherical nanoparticles with very small size (19-28 nm). The presence of amide bonds was determined by FT-IR and confirmed by Thermogravimetric analysis studies. TMX incorporation was achieved successfully (2-3 µg TMX per mg NP), and maximum TMX release took place between 8 and 24 h. This study shows that interaction between TMX and the system was dependent on nanoparticle composition, being the composition with higher proportion of alginate the one which showed the best release control of the drug.

Genetic analysis of the Hispano-Breton heavy horse.

Hispano-Breton (HB) is a horse breed with a recent mixed ancestry. It was developed in the 1930s by crossing local mares with Breton draught horses imported from France. Nowadays it is considered to be in a vulnerable situation due to census decline. To genetically characterize the breed and to set up the basis for a conservation programme, we have employed two types of molecular markers: a 347-bp D-loop mitochondrial DNA (mtDNA) fragment and 13 microsatellite loci. A representative sample of 53 HB individuals was analysed together with a sample of 40 Pura Raza Española horses for comparison. Both types of markers revealed a high level of genetic diversity in the HB breed, emphasizing the importance of its conservation. The construction of a phylogenetic network with mtDNA sequences including various Iberian breeds and European heavy horses provided an overall picture of the ubiquitous appearance of HB matrilines with respect to other breeds and revealed the singularity of certain HB maternal lineages. Despite the high allelic richness found in HB horses, microsatellite analysis evidenced a certain degree of inbreeding as a consequence of the type of management generally used for local breeds.

Dynamics of sperm DNA fragmentation in domestic animals II. The stallion.

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.

Correlación entre la fragmentación del ADN en el espermatozoide y otros caracteres de fertilidad en el caballo / No disponible

No disponible

Mitotic microtubule development and histone H3 phosphorylation in the holocentric chromosomes of Rhynchospora tenuis (Cyperaceae).

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.

Demografía y pesquería del erizo Loxechinus albus (Echinodermata: Echinidae) en la región sur-austral de Chile / Demography and fishery of the sea urchin Loxechinus albus (Echinodermata: Echinidae) in south-austral Chile region

In the Magellan Region of southern Chile (52 degrees 20'S - 55 degrees 30'S), the edible urchin Loxechinus albus is collected by 1200 artisanal fishermen, of whom 450 are divers. About 360 small fishing boats and 54 transport vessels carry the fresh product to 16 processing plants. Landings of about 27 000 tons were recorded between January and December 1995. Test diameters of urchins harvested monthly were measured for a total of 119 239 specimens, and 36 406 specimens were individually weighed; sex determination was carried out on 2 314 specimens. Field data indicate that the harvest was about 6.6 x 106 dozen urchins (this is a measuring method employed by fishermen in the region), with an extractive effort of 14 753 diver/days. The fisheries yield ranged from an annual minimum of 235 DUDD (dozen urchins per diver/day) to a maximum of 660 DUDD. In overall terms, the lowest average yields were between January and April (415-427 DUDD), and the highest yields between May and December (456-510 DUDD). Mean sizes increased from June to November and decreased from December to June. Size frequency of males and females were polymodal, with the most relevant modes at 72-84 mm in males, and at 79-88 mm in the females. The percentage of individuals below the minimum legal size (70 mm) did not exceed 4.9% for males and 3.6% for females. The size-weight records fit a power model which suggested that this species has a negative allometric growth (b = 2.007). Regarding weight, urchins in the size range from 80.0 to 84.9 mm were those with the maximum contribution to the regional landings. The highest values recorded for the utilized condition factor were: Average Condition Factor (ACF) = May to July, and November; Isometric (or Cubic) Condition Factor (ICF) = July; and Allometric Condition Factor (ACF) = June. Spawning occurred mainly between August and September, and ended by the end of October. Exploitation of this species represents one of the main sources of employment for the artisanal fisheries sector in the Magellan Region. The main difficulty observed in this fishery was obtaining a sufficient supply of urchins with a yellow-gold colored gonadic material, which forms the basis for demand of this urchin by the international market.

Alteration of the metaphase checkpoint by B chromosomes.

The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes.

B chromosomes: the troubles of integration.

Starting with a spontaneous B-A centric fusion found in a natural population of the grasshopper Eyprepocnemis plorans, we have obtained different strains carrying the rearrangement in various conditions and doses. Using this material, we have analyzed the meiotic behavior of the translocated chromosome in living cultured spermatocytes, simulating the successive steps of a hypothetical process of integration of a B chromosome into the standard genome via B-A centric fusion. Remarkably, the behavior of fusion heterozygotes, the initial step of the integration process, is much more regular than that of any other configuration, including homozygotes. The reasons for the failure of B chromosome integration into the normal complement by translocation are discussed.

Active role of lagging chromosomes in spindle collapse as revealed by live phase contrast and tubulin immunostaining in grasshopper spermatocytes.

Univalents, that is, chromosomes lacking an attached partner at the first meiotic division, show extremely faulty transmission. Most segregational errors stem from amphitelic (mitotic-like) orientation at metaphase I followed by anaphase I lagging. Our studies in living grasshopper spermatocytes show that amphitelic orientation may provoke spindle collapse: spindle elongation and cytokinesis are impaired and an unreduced restitution nucleus is formed. This does not prevent meiotic progression and eventually leads to the production of diploid gametes. The morphology and characteristics of spindle collapse in our material, as revealed by in vivo observation and tubulin immunostaining, indicate an active role of the chromosomes in the whole process.

The chromosomal distribution of phosphorylated histone H3 differs between plants and animals at meiosis.

Plant (Secale cereale, Triticum aestivum) and animal (Eyprepocnemis plorans) meiocytes were analyzed by indirect immunostaining with an antibody recognizing histone H3 phosphorylated at serine 10, to study the relationship between H3 phosphorylation and chromosome condensation at meiosis. To investigate whether the dynamics of histone H3 phosphorylation differs between chromosomes with a different mode of segregation, we included in this study mitotic cells and also meiotic cells of individuals forming bivalents plus three different types of univalents (A chromosomes, B chromosomes and X chromosome). During the first meiotic division, the H3 phosphorylation of the entire chromosomes initiates at the transition from leptotene to zygotene in rye and wheat, whereas in E. plorans it does so at diplotene. In all species analyzed H3 phosphorylation terminates toward interkinesis. The immunosignals at first meiotic division are identical in bivalents and univalents of A and B chromosomes, irrespective of their equational or reductional segregation at anaphase I. The grasshopper X chromosome, which always segregates reductionally, also shows the same pattern. Remarkable differences were found at second meiotic division between plant and animal material. In E. plorans H3 phosphorylation occurred all along the chromosomes, whereas in plants only the pericentromeric regions showed strong immunosignals from prophase II until telophase II. In addition, no immunolabeling was detectable on single chromatids resulting from equational segregation of plant A or B chromosome univalents during the preceding anaphase I. Simultaneous immunostaining with anti-tubulin and anti-phosphorylated H3 antibodies demonstrated that the kinetochores of all chromosomes interact with microtubules, even in the absence of detectable phosphorylated H3 immunosignals. The different pattern of H3 phosphorylation in plant and animal meiocytes suggests that this evolutionarily conserved post-translational chromatin modification might be involved in different roles in both types of organisms. The possibility that in plants H3 phosphorylation is related to sister chromatid cohesion is discussed.

Chromosomal strategies for adaptation to univalency.

The orientation and segregation behaviour of different types of univalents, namely sex chromosomes, B chromosomes and autosomal univalents, were analysed in living spermatocytes of eight evolutionarily distant grasshopper species. The meiotic behaviour of each univalent was characterized in terms of velocity of prometaphase movements, frequency of reorientations, types of final orientation at metaphase I and modes of segregation at anaphase I. All these features were found to vary between different univalents. Certain combinations of these traits, defining a 'chromosomal strategy', appear commonly together in certain chromosome types, indicating that they are the result of selection acting on the chromosomes to increase transmission effectiveness. The sex univalents show in general a strategy in which all the features favouring an eventual equational segregation at anaphase I tend to be minimized. There is much more variation in behaviour among B chromosomes than among X chromosomes, which is a reflection of their heterogeneous nature. Induced autosomal univalents are studied in Locusta migratoria. They show a very irregular behaviour, indicating their lack of adaptation to univalency.

A comparative study of orientation at behavior of univalent in living grasshopper spermatocytes.

Orientational movements and modes of segregation at anaphase I were analyzed in three different types of univalents in living spermatocytes of the grasshopper species Eyprepocnemis plorans, namely the sex univalent, three types of accessory chromosomes and spontaneous and induced autosomal univalents. When two or more univalents were present in the same spindle, their dynamics were directly compared. Chromosomes may show variable velocity and number of reorientations: the X and the most common B types (B1 and B2) are slow and rarely reorient, a more geographically restricted B (B5) is faster and reorients more often, and autosomal univalents are the fastest and show the highest frequency of reorientations. Nonetheless, the X and the accessories are rigorously reductional at anaphase I whereas autosomal univalents often fail to migrate or divide equationally. This indicates that orientational and segregational behavior are controlled mainly by chromosomal rather than cellular characteristics and that chromosomes may display a great variety of strategies to achieve regular segregation.

Evolution and the meaning of metaphase.

We used an evolutionary test to ask whether the congression of chromosomes to the spindle equator is important in itself or just a mitotic happenstance. If congression matters, then it might evolve if absent initially. Previous workers established that newly made trivalents, meiotic units of three chromosomes, generally do not congress to the spindle equator. Instead, these young trivalents lie close to the pole to which two of the three chromosomes are oriented. We studied ancient sex-chromosome trivalents that arose hundreds of thousands to several million years ago in several species of praying mantids and one grasshopper. All these old trivalents lie near the spindle equator at metaphase; some of them congress as precisely to the equator as the ordinary chromosomes in the same cells. We conclude that congression evolved independently two or three times in the materials studied. Therefore, the metaphase position of chromosomes midway between the poles appears to matter, but why? In the praying mantids, the evident answer is that metaphase is a quality-control checkpoint. Sometimes the three chromosomes are not associated in a trivalent but rather are present as a bivalent plus an unpaired chromosome, which lies near one pole. Earlier workers showed that such cells are blocked in metaphase and eventually degenerate; this prevents the formation of sperm with abnormal combinations of sex chromosomes. We suggest that the quality-control system would have trouble distinguishing an unpaired chromosome from an uncongressed, newly arisen trivalent, both of which would lie near a spindle pole. If so, the confused quality-control system would block anaphase imprudently, causing a loss of cells that would have produced normal sperm. Hence, we conclude that the congression of the trivalent to the equator probably evolved along with the metaphase quality-control checkpoint. The mechanism of congression in old trivalents is uncertain, but probably involves an interesting force-sensitive regulation of the motors associated with particular chromosomes. We also examined the congression of two newly made quadrivalents when they orient with three kinetochores to one pole and one to the other. As others have described, one of these quadrivalents does not congress, while the other quadrivalent comes closer than expected to the spindle equator. Such variation in the extent of congression may provide materials on which natural selection can act, leading to the evolution of congression. The trivalents of praying mantids are attractive materials for further studies of the mechanism of congression and of the idea that metaphase is a checkpoint for progression through the cell cycle.

Orientation and segregation of a micromanipulated multivalent: familiar principles, divergent outcomes.

We studied the orientation and segregation of a particular quadrivalent in living grasshopper spermatocytes. Quadrivalents were detached from the spindle by micromanipulation, then placed and bent as desired. The detached quadrivalents reattach and orient on the spindle. Their orientation is determined by the same principles that apply to ordinary chromosomes in mitosis and meiosis, but the outcome is different. Certain characteristics of the quadrivalent lead to a variety of orientations rather than the single one typical of ordinary chromosomes. Two kinetochores in the quadrivalent are linked to the others by unusually long, flexible chromosome arms. These kinetochores may face either the same pole or opposite poles and tend to orient initially to the pole toward which they face. Consequently, the initial orientation of the flexibly linked kinetochores is variable, and, moreover, they frequently reorient. In contrast, the other two kinetochores are as rigidly connected as those in a small bivalent and so display the typical back-to-back arrangement. Usually, this arrangement leads quickly to a stable orientation of the two kinetochores to opposite poles. Sometimes, however, the back-to-back arrangement changes to a side-by-side arrangement so that the orientation of both kinetochores to the same pole is favored. The combined effect of this diverse behavior is that the quadrivalent has four stable orientations, each leading to a different distribution of chromosomes in anaphase. The result is genetic chaos. Ironically, this chaos is produced by the same mechanisms that, in ordinary bivalents and mitotic chromosomes, produce a single stable orientation and genetically appropriate chromosome distribution.