The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Formation of prostanoids in human umbilical vessels perfused

Four major prostanoids (6-keto-PGF_1α, PGE₂, PGF_2α and TXB₂) were measured by specific radioimmunoassays in the outputs from human umbilical vessels perfussed . As evaluated by scanning electron microscopy (SEM) only few blood platelets were attached to the vessel wall. After an initial flush with decreasing concentrations of all four prostanoids, a stable stage was reached, lasting for 4–5 hours. During this stage the production could be inhibited by indomethacin and only slightly stimulated with arachidonic acid. The TXA₂ synthetase inhibitor UK 38485 depressed the TXB₂ production, while only slightly affecting the other three prostanoids at very high concentrations. The arteries produced relatively more 6-keto-PGF_1α than did the vein.     

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 10^2.55TCID₅₀/ml, group B 10^5.25TCID₅₀/ml and group C 10^6.7TCID ₅₀/ml. A fourth group (D) was inoculated with a medium dose (10^5.25TCID₅₀/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.     

Spatial dynamics of Norwegian tetraonid populations

Different species in a given site or population of a given species in different sites may fluctuate in synchrony if they are affected similarly by factors such as spatially autocorrelated climate, predation, or by dispersal between populations of one species. We used county wise time series of hunting bag records of four Norwegian tetraonid species covering 24 years to examine patterns of interspecific and intraspecific synchrony. We estimated synchrony at three spatial scales; national, regional (consisting of counties with similar climate), and county level. Ecologically related species with overlapping distributions exhibited strong synchrony across Norway, but there was much variation between the different regions and counties. Regions with a long coastline to both the North Sea and the Norwegian Ocean exhibited an overall stronger synchrony than those consisting of more continental areas. Intraspecific synchrony was generally low across all counties, but stronger synchrony between counties within regions defined by climatic conditions. Synchrony was negatively related to distance between populations in three of four species. Only the synchrony in willow ptarmigan showed a clear negative relationship with distance, while the other species had both strong positive and negative correlations at short distances. Strong interspecific synchrony between some species pairs within regions and weak intraspecific synchrony across counties within regions suggest a stronger synchronizing effect from environmental factors such as weather or predation and less effect from dispersal. Our results suggest that the complete tetraonid community is structured by environmental factors affecting the different species similarly and causes widespread interspecific synchrony. Local factors affecting the population dynamics nevertheless frequently forces neighbouring populations out of phase.     

Trans-Atlantic genetic uniformity in the rare snowbed sedge <Emphasis Type="Italic">Carex rufina</Emphasis>

The red-listed, amphi-Atlantic sedge Carex rufina is highly specialized to certain alpine snowbeds, and threatened by current changes in snow cover duration and moisture conditions. Here we address its range-wide genetic diversity, history, and conservation using amplified fragment length polymorphisms (AFLPs). Despite extensive primer testing, we detected very low overall diversity (4.1% polymorphic markers). Only a single AFLP phenotype was found throughout Norway and across the Atlantic to Iceland and Greenland, while another was found in Canada, suggesting glacial survival in one East and one West Atlantic refugium. East Atlantic C. rufina has probably been heavily bottlenecked in a small refugium, possibly situated within the maximum limits of the ice sheets. Its lack of diversity is likely maintained through local clonal growth causing longevity of genotypes. Habitat availability appears as the main limiting factor for C. rufina, and its currently occupied habitats need to be preserved to ensure its long-time survival.     

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.     

Quantification of piscine reovirus (PRV) at different stages of Atlantic salmon Salmo salar production

The newly described piscine reovirus (PRV) appears to be associated with the development of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon Salmo salar L. PRV seems to be ubiquitous among fish in Norwegian salmon farms, but high viral loads and tissue distribution support a causal relationship between virus and disease. In order to improve understanding of the distribution of PRV in the salmon production line, we quantified PRV by using real-time PCR on heart samples collected at different points in the life cycle from pre-smolts to fish ready for slaughter. PRV positive pre-smolts were found in about 36% of the freshwater cohorts and a general increase in viral load was observed after their transfer to seawater. A reduction in viral loads was recorded when fish approached slaughter (18 mo in sea cages). Sequencing of positive samples did not support the hypothesis that outbreaks are caused by the spreading of a particular (virulent) strain of PRV.     

Kryptoperidinium foliaceum blooms in South Carolina: a multi-analytical approach to identification

Observations following the discovery of Kryptoperidinium foliaceum blooms in South Carolina (SC), USA, suggest that a multi-analytical approach, using a standard, minimal set of criteria, should be adopted for determining dinoflagellate species identity and taxonomic placement. A combination of morphological, molecular, and biochemical analyses were used to determine the identity of this “red tide” dinoflagellate, first documented in SC waters in the spring of 1998. Results from thecal plate tabulations (based on scanning electron and epifluorescence microscopy), gene sequence data, species-specific PCR probe assays, and microalgal pigment profiles were analyzed and compared to reference cultures of K. foliaceum. Comparative data showed marked inconsistencies among the K. foliaceum reference culture isolates. In addition, the SC bloom isolate was shown to be mononucleate, contrary to previous reports for K. foliaceum, suggesting a more transient endosymbiotic association than previously considered.     

中国西藏甲螨新种和新纪录种记述(蜱螨亚纲:甲螨亚目)

报道西藏甲螨4新种和6新纪录种,标本采于西藏东南色季拉山,海拔3640－4800m,新种为：粒小隅甲螨Microtegeus granulatus Wang et Shen,sp.nov.,西藏真翅背甲螨Eupterotegaeus xizangensis Wang et Solhoy,sp.nov.,头新裸甲螨Neogymnobates capitatus Wang et Solhoy,sp.nov.,山溯甲螨Birsteinius monticolus Wang et Shen,sp.nov.,新纪录种为：纽氏平懒甲螨 Platynothrus nomatai Aoki,塞氏矮汉甲螨Nanhermannia sellnicki Forsslund,杜氏小赫甲螨Hermanniella dubinini Sitnikova,多齿刀肋甲螨Cultroribula dentata Willmann,曲波甲螨Unduloribates undulatus(Berlese),毛暗色甲螨Fuscozetes setosus(C.L.koch)。本文报道的波甲螨属,溯甲螨属,新裸甲螨属和暗色甲螨属为中国首次发现。     

Discovery of the toxic dinoflagellate Pfiesteria in northern European waters.

Several dinoflagellate strains of the genus Pfiesteria were isolated by culturing techniques from sediment samples taken in the Oslofjord region of Norway. Pfiesteria piscicida, well known as a fish killer from the Atlantic coast of America, was identified by genetic methods and light microscopy. The related species Pfiesteria shumwayae was attracted from the sediment by the presence of fish, and has proved toxic. This present survey demonstrates the wide distribution of these potentially harmful species, but so far they have not been connected with fish kills in Europe.