[Molecular phylogenetic analysis of Diacyclops and Acanthocyclops (Copepoda: Cyclopoida) from Lake Baikal].

Lake Baikal is inhabited by a relatively large number of cyclopid species, many of which are endemics. Two genera, Diacyclops Kiefer, 1927 and Acanthocyclops Kiefer, 1927, are the most specious in the lake. Taxonomic discrimination of the majority of representatives of these genera is difficult owing to their high morphological similarities and poor standard description. In this study, a molecular phylogenetic analysis of Lake Baikal members of the Diacyclops/Acanthocyclops group is performed on the basis of mitochondrial cytochrome c oxidase subunit I (COI) gene. It is shown that a fragment of COI 1000 bp long is sufficient for intragenus discrimination of the cyclopids of Lake Baikal. The issues of Diacyclops/Acanthocyclops taxonomy are reflected in the obtained molecular data. Two distinct phylogenetic groups of Diacyclops genus with uncertain taxonomic status are revealed.

[A simple and effective method for assessing chromatin diminution values in copepods using qPCR].

The value of chromatin diminution (CD) in different species of freshwater cyclopoid copepods can differ significantly. The biological and evolutionary roles of these differences remain unclear. To expand the knowledge on CD distribution and magnitude in this group of copepods, a quick method for its evaluation was required. This study proposes a simple approach for CD assessment in copepods using quantitative realtime PCR (qPCR). The magnitude of changes in the genome size was assessed by comparing fluorescence curves of qPCR fragments of target genes for pre- and post-diminution materials. The method was tested on four cyclopoid copepods species. In Cyclops kolensis, CD was assessed as 95.3 ± 1.2; in Acanthocyclops vernalis it was assessed at 94.6 ± 0.8%; at C. insignis, it was 82.3 ± 5.2%; and for the first time, CD was found in Megacyclops viridis at 91.1 ± 2.6%. The advantages of our approach are its rapidity, simplicity and minimal requirements of materials studied.

Advanced modelling of the transport phenomena across horizontal clothing microclimates with natural convection.

The ability of clothing to provide protection against external environments is critical for wearer's safety and thermal comfort. It is a function of several factors, such as external environmental conditions, clothing properties and activity level. These factors determine the characteristics of the different microclimates existing inside the clothing which, ultimately, have a key role in the transport processes occurring across clothing. As an effort to understand the effect of transport phenomena in clothing microclimates on the overall heat transport across clothing structures, a numerical approach was used to study the buoyancy-driven heat transfer across horizontal air layers trapped inside air impermeable clothing. The study included both the internal flow occurring inside the microclimate and the external flow occurring outside the clothing layer, in order to analyze the interdependency of these flows in the way heat is transported to/from the body. Two-dimensional simulations were conducted considering different values of microclimate thickness (8, 25 and 52 mm), external air temperature (10, 20 and 30 °C), external air velocity (0.5, 1 and 3 m s(-1)) and emissivity of the clothing inner surface (0.05 and 0.95), which implied Rayleigh numbers in the microclimate spanning 4 orders of magnitude (9 × 10(2)-3 × 10(5)). The convective heat transfer coefficients obtained along the clothing were found to strongly depend on the transport phenomena in the microclimate, in particular when natural convection is the most important transport mechanism. In such scenario, convective coefficients were found to vary in wavy-like manner, depending on the position of the flow vortices in the microclimate. These observations clearly differ from data in the literature for the case of air flow over flat-heated surfaces with constant temperature (which shows monotonic variations of the convective heat transfer coefficients, along the length of the surface). The flow patterns and temperature fields in the microclimates were found to strongly depend on the characteristics of the external boundary layer forming along the clothing and on the distribution of temperature in the clothing. The local heat transfer rates obtained in the microclimate are in marked contrast with those found in the literature for enclosures with constant-temperature active walls. These results stress the importance of coupling the calculation of the internal and the external flows and of the heat transfer convective and radiative components, when analyzing the way heat is transported to/from the body.

The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

Protein kinases in control of the centrosome cycle.

The centrosome is the major microtubule nucleating center of the animal cell and forms the two poles of the mitotic spindle upon which chromosomes are segregated. During the cell division cycle, the centrosome undergoes a series of major structural and functional transitions that are essential for both interphase centrosome function and mitotic spindle formation. The localization of an increasing number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling many of these transitions. Here, we focus on two protein kinases, the polo-like kinase 1 and the NIMA-related kinase 2, for which recent data indicate key roles during the centrosome cycle.

C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2.

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle-regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti-C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole-centriole cohesion during the cell cycle.

Differential effects of noxious and non-noxious input on neurones according to location in ventral periaqueductal grey or dorsal raphe nucleus.

Nociceptive and non-nociceptive input to the dorsal raphe nucleus (DR) and to the surrounding periaqueductal grey (PAG) was studied in chloralose-anaesthetized rats. Single units in the midbrain responding to electrical stimulation of a coccygeal nerve were recorded with glass micropipettes. A fluorescence histochemical technique was applied to identify recording sites in the DR and PAG. 109 DR-units, 141 PAG-units and 95 units from surrounding structures were tested for responsiveness to electrical nerve stimulation. In 53% of the DR-units, but in only 20% of the PAG- and SN-units, ongoing activity was inhibited by electrical stimulation (I-units) while 42% of the PAG- and SN-units but only 24% of the DR-units were electrically excited (E-units). 40 E-units and 24 I-units were tested with repeated noxious radiant heat stimuli applied to the tail or hindpaws. 70% of the E-units were excited by heating, and in 54% of the I-units ongoing activity was inhibited by heating. The majority of the former units were located in the PAG, and most of the latter were proven to be DR-neurones. In 75% of the E-units and in 12.5% of the I-units the heat effect was in the opposite direction. The findings are discussed in terms of the now well-established role of the PAG-region in the descending control of pain. The properties of the PAG-E-units suggest that this system is involved in a negative feedback circuit by which pain transmission to the CNS limits itself. DR-I-units may be involved via an additional small loop with the PAG to disinhibit the activation of the PAG pain control system.