Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.     

Cell Specificity of Molecular Changes During Memory Storage

Abstract: The aeolid nudibranch, Hermissenda crassicornis , exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B-cell photoreceptor. The present study was designed to further examine learning-specific PKC involvement in identified cellular areas, particularly those in the visual-vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [³H]phorbol-12,13-dibutyrate ([³H]-PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [³H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [³H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.     

Removal of swine zonae pellucidae with sequential exposures to pronase and acidified medium

Exposure to acidified PBS (pH 3) for 60 sec removed swine zonae pellucidae from 70.4% of 27 swine morulae, and 73.7% of these formed blastocysts in culture. Further investigations revealed that treating embryos with 0.5% pronase and acidified PBS (pH 3) for 30 sec each was more effective. Zonae were removed in 90.6% of 85 embryos (four-cell to morulae) treated. A total of 76.9% of 65 zona-free embryos and 81.6% of 38 untreated embryos formed blastocysts (P > 0.05). An additional 57 untreated and 49 zona-free embryos (morulae to blastocysts) were transferred to seven recipient sows. Four sows returned to estrus (18 to 27 days), but three others were pregnant when slaughtered at 38 to 42 days. One pregnant sow had received a combination of five zona-free and six untreated embryos and demonstrated the potential for further development of treated embryos in vivo.     

Inhibition of phosphoinositide turnover by praziquantel in Schistosoma mansoni.

The effect of praziquantel on phosphoinositide turnover was examined in Schistosoma mansoni to determine if this anthelminthic modulates signal transduction pathways in parasites. Adult worms were radiolabeled with [3H]myoinositol for 24 hr and total inositol phosphate levels determined in the presence of praziquantel. Praziquantel inhibited inositol phosphate turnover when activated with NaF plus AlCl3 or with the nonhydrolyzable guanine nucleotide-binding protein analogue GTP gamma S. Furthermore, praziquantel decreased basal turnover of inositol phosphates. Inhibition was seen in both male and female worms as well as in schistosomula. These data indicate that inhibition of phosphoinositide turnover may contribute to the effect of praziquantel on parasite survival within the definitive host.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Key Ecological Function Peaks at the Land–Ocean Transition Zone When Vertebrate Scavengers Concentrate on Ocean Beaches

Ecosystems - Ecotones can form hot spots of biodiversity by containing species from multiple ecosystems. Because biodiversity is often linked to ecological function, we posit that rates of key...     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.