The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.     

Avian biodiversity across Auckland’s volcanic cone reserves

Auckland, a city with a population of approximately 1.7 million, is located directly on the Auckland Volcanic Field, a late Quaternary-era monogenetic field. There are at least 53 volcanoes across the field, many of which are of geological, cultural and ecological significance, such as for being reserves for native species; however, few assessments of the richness of avian biodiversity across the volcanoes have been made. To address this data shortfall, we conducted avian biodiversity surveys using stationary point counts within nine of Auckland's volcanic cone reserves. Thirty-eight species were detected across the sites, of which 18 were native. Our estimates of relative species abundances and detection probabilities revealed that the most common native birds within these reserves were silvereyes, tui and southern black-backed gulls, while common mynas, house sparrows, Eurasian blackbirds and eastern rosellas were the most common introduced species. In addition to tui and silvereyes, the presence of other natives critical to the functioning of native ecosystems, such as New Zealand fantails, grey warblers and New Zealand pigeon, suggest that the volcanoes possess a diverse native avifauna supported by native flora that warrant continued and intensified restoration efforts. We discuss several feasible strategies for improving faunal and floral biodiversity across the volcanic cone reserves. Continued avian biodiversity surveys are also of critical importance as they will enable us to further evaluate and prioritise restoration projects within Auckland's multitude of diverse volcanic cone reserves.     

Incorporation of 35SO 4 2− into sediments of three New York lakes

Intact sediment cores were obtained from three New York lakes in May, July, and October 1981. Radioactive S (as ³⁵SO ₄ ²⁻ ) was added to the overlying water and cores were incubated without atmospheric exchange for one week near lake bottom temperatures. Headspace flux of 0₂ as an index of sediment respiration rates varied among lakes and seasonally within lakes. Acidic South Lake had the lowest respiration rate at all seasons and also the smallest net incorporation of the ³⁵SO ₄ ²⁻ . Summer net isotope transformation into ester sulfate and non-HI reducible S (pyrite and C-bonded S) constituents was 88.6%, 89.4%, and 59.7% of total sediment isotope for Oneida, Deer, and South, respectively. Seasonal variation of net isotope incorporation was observed in each lake as were differences in ³⁵SO ₄ ²⁻ partitioning into major S pools. Of the S constituents analyzed, HCl digestible S (volatile sulfides) was the smallest pool, while ester sulfate and non-HI reducible S together accounted for greater than 50% of S isotope transformation in all lakes. In addition, ester sulfate is the major product of dissolved SO ₄ ²⁻ transformation and its formation results in less alkalinity generation than the formation of non-HI reducible S constituents. Thus ester sulfate transformation processes must be considered in calculating alkalinity generation by lake sediments. Financial support provided by Office of Water Research Technology (Project No. 13-096-NY). Financial support provided by Office of Water Research Technology (Project No. 13-096-NY).     

Exuviotrophic apostome ciliates from crustaceans of St. Andrew Bay, Florida, and a description of Gymnodinioides kozloffi n. sp

Gymnodinioides kozloffi n. sp. is described from the eelgrass broken-back shrimp Hippolyte zostericola. The species is distinct from others in the apostome genus Gymnodinioides in that the trophont ciliature has a small group of kinetosomes located to the right of Kinety 9a, and Kinety 1 and 2 are divided. Other apostome morphologies are described from many decapod crustaceans from St. Andrew Bay, Florida, including Gymnodinioides inkystans, Hyalophysa chattoni, and variants of both H. chattoni and G. kozloffi. All of these apostome ciliates are exuviotrophic, found feeding on exuvial fluid within the exoskeleton of the host after ecdysis. The hosts surveyed for this study are the following: Callinectes sapidus, Eurypanopeus depressus, Hippolyte zostericola, Farfantepenaeus spp., Palaemonetes intermedius, Palaemon floridanus, Portunus spp., Tozeuma carolinense, and Sicyonia laevigata, which revealed a number of new host-apostome records.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida)

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.     

The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W₆₀PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum,Trichophyton mentagrophytes,Trichophyton rubrum,Aspergillus fumigatus andFusarium oxysporum.None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W₆₀PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.This revised version was published online in October 2005 with corrections to the Cover Date.     

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.