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.     

Ant–scale mutualism increases scale infestation,decreases folivory,and disrupts biological control in restored tropical forests

Ant–hemipteran mutualisms can have positive and negative effects on host plants depending on the level of hemipteran infestation and plant protection conferred by ants against folivory. Differential effects of such mutualisms on plant survival are well documented in undisturbed and ant-invaded systems, but few have explored how anthropogenic disturbance affects interactions between hemipterans and native ant species and what the consequences may be for recovering ecosystems. Within a fragmented landscape in Costa Rica, restored tropical forests harbor a mutualism between the native ant Wasmannia auropunctata and the scale insect Alecanochiton marquesi on the abundant, early-successional tree Conostegia xalapensis. I added A. marquesi scales to C. xalapensis seedlings and either allowed or excluded W. auropunctata to investigate if this mutualism leads to increased scale infestation, decreased scale mortality, and decreased folivory. I also examined whether these effects are mediated by the percentage of remnant forest cover in the landscape. I found that seedlings with ants excluded had fewer scale insects and higher herbivory than plants with ants present. I also found evidence that scale mortality due to fungal attack and parasitism was higher on ant-excluded versus ant-allowed seedlings but only at sites with high surrounding landscape forest cover. Together, these results suggest that mutualisms between scale insects and native ants can promote scale infestation, reduce folivory on native plant species, and potentially disrupt biological control of scale insects in recovering tropical forests. Further, my experiment underscores the importance of remnant tropical forests as sources of biological control in anthropogenically disturbed landscapes. Abstract in Spanish is available with online material.     

Ortholog clustering on a multipartite graph

We present a method for automatically extracting groups of orthologous genes from a large set of genomes by a new clustering algorithm on a weighted multipartite graph. The method assigns a score to an arbitrary subset of genes from multiple genomes to assess the orthologous relationships between genes in the subset. This score is computed using sequence similarities between the member genes and the phylogenetic relationship between the corresponding genomes. An ortholog cluster is found as the subset with the highest score, so ortholog clustering is formulated as a combinatorial optimization problem. The algorithm for finding an ortholog cluster runs in time O(|E| + |V| log |V|), where V and E are the sets of vertices and edges, respectively, in the graph. However, if we discretize the similarity scores into a constant number of bins, the runtime improves to O(|E| + |V|). The proposed method was applied to seven complete eukaryote genomes on which the manually curated database of eukaryotic ortholog clusters, KOG, is constructed. A comparison of our results with the manually curated ortholog clusters shows that our clusters are well correlated with the existing clusters     

Prognostic significance of CD34 expression in early cervical squamous cell carcinoma

The aim of the study was to evaluate angiogenesis as an independent prognostic factor and to determine the correlation of the microvessel density (MD) with lymph node metastases and survival rate in 73 women operated because of invasive squamous cell carcinoma (SCC) of the uterine cervix at clinical stages lb and IIa (FIGO). The patients were divided into two groups: I--25 (34.4%) with survival rate <5 years and II--48 (65.6%) with survival rate >5 years. Angiogenesis was quantified in light microscope using an assay for CD34. The CD34 antibody intensely immunostained single endothelial cells as well as larger microvessels. In the study. differences were revealed by comparing the MD between both groups. The 5-year overall survival rate for patients with high MD was significantly worse than for those with low MD (p<0.003). A correlation was found between angiogenesis intensity and vascular involvement as well as the incidence of lymph node metastases. Thus, tissue expression of CD34 in SCC appears to be a significant prognostic indicator.     

Tissue expression of VEGF as a prognostic factor in early cervical squamous cell carcinoma

The purpose of this retrospective study was to determine whether the intensity of tumor angiogenesis, expressed as microvessel density (MD), is indeed an important parameter predicting lymph node metastasis and survival rate in 73 women operated on for early invasive squamous cell carcinoma of the uterine cervix in stages Ib and IIa (FIGO). Angiogenesis was quantified by light microscope (LM) using an assay for vascular endothelial growth factor (VEGF). In the study, differences were revealed by comparing the MD between both groups. The patient survival with high MD was significantly worse than for those with low MD (p<0.01). A correlation was found between MD and the incidence of lymph node metastases. Hence, quantitative analysis of MD used as the expression of VEGF in the each cervical squamous cell carcinomas could be useful as a significant prognostic indicator.     

Expression of nucleolar organizer regions (NORs) in ovarian epithelial tumors

AgNOR staining technique was tested in ovarian epithelial tumors to evaluate its diagnostic potential in distinguishing between borderline tumors and well-differentiated carcinomas. In our opinion, the AgNOR count appears useful for assessing differences only between borderline and well-differentiated serous ovarian tumors at stage I of FIGO clinical advancement.     

Proliferative activity in endometrial hyperplasia and adenocarcinoma

Studies on the proliferative activity of cells in endometrial hyperplasia and adenocarcinoma were performed using techniques detecting Proliferating Cell Nuclear Antigen (PCNA) and Nucleolar Organizer Regions (NORs). PCNA expression was defined as the percentage of nuclei showing reactivity in 200 cells per sample. The mean AgNOR count per cell was calculated following the analysis of at least 100 nuclei per sample at a magnification of x 400. Student-t test was used for the statistical analysis. The results obtained indicate that the evaluation of cell proliferative activity expressed by AgNOR count and PCNA index can help in the distinction between atypical hyperplasia and well-differentiated adenocarcinoma, and thus can serve as a useful pathological criterion.     

Angiogenesis as a prognostic factor in invasive carcinoma of the uterine cervix

The aim of the study was to evaluate angiogenesis as an independent prognostic factor and to determine the correlation between the angiogenic index (AI) and histologic grade of the neoplastic process in patients operated on for invasive carcinoma of the uterine cervix. Angiogenesis was assessed with immunohistochemical technique using a monoclonal antibody against human factor VIII--(F8/86 M0616, DAKO, Denmark). A positive correlation was revealed between the intensification of angiogenesis and the incidence of lymph node involvement and survival rate.     

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.     

Cone contrast computations: physical versus perceived background and colour constancy

The light reflected from an object depends both on the object's surface and on the illuminant. Visual systems attempt to resolve this intrinsic ambiguity by comparing the light reflected from the object with respect to the background by computing the difference between the object-background light sampled by three cones. The cone-contrasts for the sample-background stimulus under the test illumination should correspond to the cone-contrasts for samples matched in appearance under the standard background (C). The validity of this cone-contrast rule (Whittle, 2003) and its possible link with stability of perceived colour was studied here using six test illuminants. A successive asymmetric colour-matching task with 40 simulated Munsell samples (value 7, chroma 4) on a neutral background (N7) was used. The subject adjusted the sample under standard illuminant C to match the colour appearance of the test sample under one of the test illuminants. Brunswik Ratio (BR) was used as an index of stability of colour appearance. When computed with respect to the reference illuminant C, the cone contrast rule was violated (particularly for S-cone-contrast). However, if a new reference point based on the perceived colour of the neutral background under the test illumination was used, the cone contrast rule applied. That is, when cone contrasts of the matching samples are computed with respect to this perceived background, they correspond to cone contrasts of the test stimuli. This represents a form of discounting the illuminant for the purpose of determining an object's cone-contrast against the background, which does not vary with background illumination. These cone contrasts, however, do not provide any information about the colour appearance of objects under particular viewing conditions, unless calibrated against a standard by allowing subjects to learn particular colours.