A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain

Summary Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.     

Teratocarcinoma cell adhesion: Identification of a cell-surface protein involved in calcium-dependent cell aggregation

Teratocarcinoma cells have a Ca²⁺-dependent cell-cell adhesion site (t-CDS) that is unique in being inactivated with trypsin in the absence of Ca²⁺ but not in the presence of Ca²⁺. Fab fragments of antibodies raised against teratocarcinoma F9 cells dissociated by treatment with trypsin and calcium (anti-TC-F9) inhibit the aggregation of teratocarcinoma cells mediated by t-CDS. This inhibitory effect of Fab is removed when anti-TC-F9 is absorbed with F9 cells treated with trypsin and calcium (TC-F9), but not when it is absorbed with F9 cells treated with trypsin and EGTA (TE-F9). Comparisons of cell-surface antigens reactive to anti-TC-F9 in TC-F9 cells with those in TE-F9 cells reveal that only one component, with an approximate molecular weight of 140,000 (p140), is detected specifically on the surface of TC-F9 cells. When TC-F9 cells are retrypsinized in the absence of Ca²⁺, a substance with an approximate molecular weight of 34,000 (p34) is released that can neutralize the aggregation-inhibitory effect of the Fab. This p34 interferes with the immunoprecipitation of p140 with anti-TC-F9, suggesting that p34 is a tryptic fragment of p140. Anti-TC-F9 Fab causes the dissociation of the monolayers of teratocarcinoma cells. This effect is removed by absorption of the Fab with p34 as well as with TC-F9 cells, but not with TE-F9 cells. These results suggest that p140 is essential for the function of t-CDS, and that this is an actual cell-adhesion molecule active in the establishment of monolayers of teratocarcinoma cells.     

Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies

BackgroundAn estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries.Methodology/Principal findingsA cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01–0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8–51.6%. This low coverage was principally attributed to the owners’ lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8–76.2%.Conclusions/SignificanceThis paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.     

Molecular Basis of Disease Resistance Acquired through Cold Acclimation in Overwintering Plants

Plants require substantial resistance against freezing and pathogens for overwintering. These two traits are acquired through cold acclimation. In contrast to freezing tolerance, molecular basis of disease resistance acquired through cold acclimation is poorly understood. Recent studies have suggested that pathogenesis-related (PR) proteins that are secreted into the apoplast during cold acclimation are responsible for the disease resistance. Interestingly, some of the cold-induced PR proteins display both antifungal and antifreeze activities, suggesting a dual function in protecting plants from overwintering stresses. The signaling pathway for cold-induced disease resistance is currently unknown but can be independent of pathogen-induced defense mechanisms.