The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

The feeding and ranging behaviour of Galapagos giant tortoises (Geochehne elephantopus) The Cambridge and London University Galapagos Expeditions, 1972 and 1973

Field observations on the behaviour of adult Galapagos giant tortoises ( Geochehne elephantopus ) on the islands of Santa Cruz (Indefatigable) and Pinzon (Duncan) were made by the 1972 Expedition, and on Santa Cruz by the 1973 Expedition. The animals (in all 14 on Santa Cruz and three on Pinzon) were observed continuously throughout the day for periods of 3–14 days so that their main non-reproductive activities (feeding and walking) could be quantitatively recorded. The period of potential activity was between about 08.00 hrs and 16.00–18.00 hrs, but the pattern of behaviour was extremely variable, both in a given individual and between one individual and another; it seemed little influenced by the relatively slight fluctuations of temperature and climate which occurred during the period of study (July-September) on Santa Cruz. The tortoises on Pinzon, a much more arid island, were less active, and one showed a strongly bimodal activity pattern with a resting period during the middle of the day. The animals studied in 1972 showed a strong disposition to return after several days to the same sleeping place, but this homing tendency was not observed in 1973. This discrepancy can perhaps be attributed to the fact that the two expeditions worked in different areas under different weather conditions. Observations on the plants eaten, on the association between tortoises and certain birds, and on some other aspects of tortoise behaviour such as walking speed and responses to sounds are also described.     

Slow response of soil organic matter to the reduction in atmospheric nitrogen deposition in a Norway spruce forest

Global nitrogen (N) deposition rates in terrestrial environments have quadrupled since preindustrial times, causing structural and functional changes of ecosystems. Different emission reduction policies were therefore devised. The aim of our study was to investigate if, and over what timescale, processes of soil organic matter (OM) transformation respond to a decline in atmospheric N deposition. A N‐saturated spruce forest (current N deposition: 34 kg ha^?1 yr^?1; critical N load: 14 kg ha^?1 yr^?1), where N deposition has been reduced to 11.5 kg ha^?1 yr^?1 since 1991, was studied. Besides organic C and organic and inorganic N, noncellulosic carbohydrates, amino sugars and amino acids were determined. A decline in organic N in litter indicated initial effects at plant level. However, there were no changes in biomarkers upon the reduction in N deposition. In addition, inorganic N was not affected by reduced N deposition. The results showed that OM cycling and transformation processes have not responded so far. It was concluded that no direct N deposition effects have occurred due to the large amount of stored organic N, which seems to compensate for the reduction in deposited N. Obviously, the time span of atmospheric N reduction (about 14.5 years) is too short compared with the mean turnover time of litter to cause indirect effects on the composition of organic C and N compounds. It is assumed that ecological processes, such as microbial decomposition or recycling of organic N and C, react slowly, but may start within the next decade with the incorporation of the new litter.     

Noninvasive methodology for the sampling and extraction of DNA from free‐ranging Atlantic spotted dolphins (Stenella frontalis)

Genetic sampling and molecular investigations are important parts of studying wild populations. However, collecting tissues from free‐ranging animals can be difficult or impractical. This study develops a sampling and extraction protocol for template DNA from faecal material collected in a marine environment from small cetaceans. DNA was extracted from faecal material of free‐ranging Atlantic spotted dolphins (Stenella frontalis) and subsequently tested for its suitability in molecular investigations by amplifying both mitochondrial and nuclear DNA. The resulting mitochondrial sequences were found to closely match known S. frontalis haplotypes. Three microsatellite loci were amplified and fall within the expected size range for cetaceans. Mother and calf families previously assigned by observation were genetically confirmed using both mitochondrial haplotype and allele sharing between the mother and offspring. The protocol effectively collects and extracts dolphin DNA from faecal samples and enables species identification as well as confirmation of genetic relatedness and should be considered as a noninvasive alternative to current protocols.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.