Spatial and temporal variations of a saxitoxin analogue (LWTX-1) in Lyngbya wollei (Cyanobacteria) mats in the St. Lawrence River (Québec, Canada).

The concentration of the saxitoxin analogue LWTX-1 was quantified in samples of the benthic filamentous cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck collected in two fluvial lakes of the St. Lawrence River (Canada) over the 2006-2013 period. The study was aimed at documenting the spatial (between fluvial lakes, between sites within each lake) and temporal (inter-annual, monthly) variations of toxin concentration in relation with hydrological (water level), physical (water temperature, conductivity, transparency), chemical (nutrients in overlying water) and biological (L. wollei biomass and mat condition) characteristics. Toxin concentration was hypothesized to vary seasonally with biomass accumulation and environmental conditions. Toxin concentrations measured in Lake Saint-Louis (51±40µg LWTX-1g-1 DM, N=29 days in 2007, 2009-2011) were double those in Lake Saint-Pierre (25±31µg LWTX-1g-1 DM, N=26 days in 2006-2008, 2012-2013); however, August 2007 measurements taken from both lakes did not differ significantly. Ten of the twelve highest values (>100µg LWTX-1g-1 DM) were obtained from Lake Saint-Louis, between April and October in 2007, 2010 or 2011. Under ice samples showed intermediate concentrations of LWTX-1 (42±9µg LWTX-1g-1 DM, N=2). Concentrations of LWTX-1 were positively correlated with Secchi depth (r=0.59, p<0.001), L. wollei biomass (Spearman r=0.31, p<0.01) and %N in filaments (r=0.48, p<0.001), suggesting toxin production was linked to mat growth and metabolism rather than water quality. Although LWTX-1 has been reported to have a low toxicity, monitoring of L. wollei abundance is required to assess the environmental and human health risks posed by this taxon in the St. Lawrence - Great Lakes system.

Potential value of fetal echocardiography in the differential diagnosis of twin pregnancy with presence of polyhydramnios-oligohydramnios syndrome.

OBJECTIVE: Our purpose was to establish whether index values of cardiac performance could discriminate between the twin-twin transfusion syndrome and placental insufficiency as the etiology of the polyhydramnios-oligohydramnios sequence in monochorionic diamniotic twins. STUDY DESIGN: Thirteen monochorionic diamniotic twin pregnancies with ultrasonographic evidence of polyhydramnios-oligohydramnios sequence had a complete echocardiography. The etiology was confirmed postnatally: placental insufficiency in eight pairs and the twin-twin transfusion syndrome in five. Intertwin comparisons were made for the following cardiac parameters: cardiothoracic index, end-diastolic thickness of the ventricular walls and septum, aortic and pulmonary artery Doppler peak velocities, ejection and acceleration times, left ventricular shortening fraction, and combined cardiac output and output indexed to fetal weight. RESULTS: All five recipient twins had thickened ventricular walls. The left ventricular shortening fractions and outputs were significantly increased in the donor twin with twin-twin transfusion syndrome and normal in placental insufficiency. CONCLUSION: In twin-twin transfusion syndrome the donor twin shows evidence of a hyperdynamic cardiac state. Intertwin comparison of cardiac parameters, especially the left ventricular shortening fraction, can be considered a useful tool in diagnosing the different etiologies of the polyhydramnios-oligohydraminos sequence.

Flow cytometric evaluation of leukocyte subpopulations in the follicular fluids of infertile patients.

OBJECTIVES: To evaluate the leukocyte subpopulations present in follicular fluid (FF) of infertile patients undergoing IVF-ET for tubal factor, idiopathic infertility, and endometriosis. PATIENTS: Sixty patients undergoing IVF-ET with a tubal factor diagnosis (n = 35), idiopathic infertility (n = 13), and endometriosis (n = 12) had their subpopulations of FF leukocytes analyzed by flow cytometry. MAIN OUTCOME MEASURE: Nonblood-contaminated samples of FF were collected under sterile conditions and centrifuged. Cells were labeled with a panel of monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD14, -CD20, -CD45, and -CD56, and analyzed by cytofluorometry. RESULTS: Follicular fluid leukocytes from patients with idiopathic infertility had a significantly higher proportion of T lymphocytes than tubal factor and endometriosis patients. Endometriosis patients had significantly higher proportions of natural killer (NK) cells, B lymphocytes, and monocytes compared with groups of idiopathic infertility and tubal factor. CONCLUSIONS: The differences observed in the leukocyte subpopulations from FF of patients with idiopathic infertility and endometriosis may affect folliculogenesis and oocyte maturation. Moreover, these modifications could be one of the factors altering their fertility.

Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome.

Several lines of evidence indicate that immunologic effectors, particularly suppressor T cells and NK cells, may play a role in the pathogenesis of idiopathic repetitive abortions. To investigate the involvement of these immune cell populations, we determined the immunophenotypic characteristics of endometrial leukocytes from nonpregnant recurrent aborters. Habitual aborters with a negative investigation underwent an endometrial biopsy during their secretory phase and were followed prospectively to assess clinical outcome. Endometrial leukocytes were evaluated by two-color flow cytometric analysis. The percentage of endometrial CD8+ T lymphocytes was significantly decreased in recurrent aborters, and their CD4:CD8 ratio was increased. In contrast, the proportion of B lymphocytes (CD20+) was strikingly increased in these patients' endometria. The proportion of NK cells was identical in recurrent aborters and normal controls, but the CD16-CD56 bright NK cell subset, which is predominant in normal decidua and endometrium, was significantly decreased in favor of an important contingent of CD16+CD56 dim NK cells in all habitual aborters. Repetitive aborters who had normal CD8+ and CD20+ cell numbers and a normal CD4:CD8 ratio subsequently underwent successful pregnancies, while patients with continuing abortions presented lymphoid populations observed in the habitual aborters group. In conclusion, endometrial lymphocytes of recurrent spontaneous aborters harbor a distinct immunophenotypic profile that antedates implantation and suggests that endometrial immunologic conditions are intrinsically altered in recurrent aborters. Also, the prognostic impact of CD8 and CD20 expression supports their predominant role in the development of fetal tolerance. Finally, a role for NK cells in the abortion process is suggested by their altered subsets in all repetitive aborters.

Flow-cytometric characterization of hematopoietic cells in non-pregnant human endometrium.

PROBLEM: Immunologic evaluation and quantitation of hematopoietic cells in human endometrium has been difficult to perform, particularly in nonpregnant subjects. In this study, we describe a method for the flow-cytometric characterization of hematopoietic cells present in the endometrium of non-pregnant women. METHOD: Endometrial biopsy samples from normal donors were first mechanically disrupted and filtered to generate a single-cell suspension of leukocyte-enriched endometrial cells. Cells were labeled with a panel of monoclonal antibodies, stained with propidium iodide (PI), and one- or two-color flow-cytometric analysis performed on cells excluding PI. RESULTS: The methodology described in this study was highly reproducible in experiments evaluating the interrun and intrarun variability. We then determined the immunophenotypic profile of endometrial leukocytes from 12 normal females. The majority of leukocytes were T cells (CD3: 47%; CD4: 24%; CD8: 28%) with an important contingent of NK cells (CD56: 32%), the majority of which harbored the unusual CD16-CD56 bright phenotype, and a minority of B cells (CD20: 6%) and monocytes (CD14: 7%). CONCLUSIONS: Flow cytometry can be used to assess antigen expression on the surface of endometrial leukocytes from nonpregnant women. In future studies, it will be possible to use this approach to investigate the role of immune cell populations in the endometrium of patients experiencing reproductive failure.

Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues.

A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold-acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 microM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 micrograms ml-1 or 0.2 microM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for cell survival in the sensitive vascular transition zone during freezing stress.

Decreased interleukin-2 receptor and cell cycle changes in murine lymphocytes exposed in vitro to low doses of cadmium chloride.

Relationships between in vitro cadmium-related cell cytotoxicity, ultrastructural changes and altered cell cycle were determined at 21-72 h after mitogenic stimulation of C57BL/6 mouse spleen lymphocytes with concanavalin A (Con A). Relatively low doses, 0.6-10 microM cadmium (Cd), added at 4 h after the mitogen activation, induced a significant cell cytotoxicity and reduced the lymphoblastic activity of the cells. Cytometric analysis of the lymphoid cell cycle at 72 h revealed that at concentrations > or = 0.6 microM Cd, the number of cells arrested in G0 + G1 phase increased, whereas the proportions of cells of the S and G2 + M phases were substantially reduced. Staining of cells with fluorescent anti-CD25 monoclonal antibody showed a cadmium-related decreased number and relative mean fluorescence of CD25+ cells, demonstrating a decreased level of interleukin-2 receptor (IL-2R). Furthermore, immunogold ultramicroscopic assay was developed for determination of intracellular interleukin-2 (IL-2) in cadmium-treated lymphocytes. The level of cytoplasmic and nuclear IL-2, localized in situ by colloidal gold ultraimmunocytochemical technique, has been estimated as markedly decreased in cells treated with > or = 1.2 microM Cd, as compared with the untreated controls. Disorganization/fragmentation of mitochondrion cristae and dilatation of cisternae of the Golgi apparatus appeared as the major ultrastructural change in 1.2 microM Cd-treated lymphocytes. Interestingly, addition of cadmium in the incubation medium, up to 4 h after mitogen activation, also interacted with lymphoproliferative mechanisms of cells in G0 + G1, S and G2 + M phase. Overall, multiple ultrastructural changes of Cd-treated lymphoid cells were clearly related with the reduced cell viability and reduced number of activated lymphoblasts.

Immunocytochemical evidence for a nuclear and a cytoplasmic O6-methylguanine repair mechanism in cultured rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-metG and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2-24 h to 5 mM N-nitrosodimethylamine (NDMA) or 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both cases, the O6-metG immunoreactive sites were concentrated in the nucleus and in the rough endoplasmic reticulum (RER) rich cytoplasmic regions. The highest gold labeling density measured was observed at 2 h of NDMA or MNNG treatment. However, after a 24-h exposure, very little labeling was observed in both the nuclear and the cytoplasmic compartments. The rate of disappearance of immunoreactive sites was faster in the cytoplasm than in the nucleus, Untreated control preparations showed no specific immunogold labeling. Furthermore, when cells were exposed first to NDMA and MNNG for a few hours and then to culture medium containing no genotoxin, and subsequently were reexposed to NDMA or MNNG for a few hours, very little labeling of both the nuclear and cytoplasmic compartments was observed. Control preparations without a second genotoxin exposure showed a normal labeling pattern. Control preparations without genotoxin showed no gold labeling. Our results provide evidence for the existence of a cytoplasmic O6-metG repair mechanism that behaves like its nuclear counterpart.

Effect of follicular fluid supplementation on the in vitro development of human pre-embryos.

OBJECTIVE: To evaluate the value of supplementing a standard culture medium with 10% heat-inactivated mature follicular fluid (FF). DESIGN: Prospective randomized study evaluating the in vitro development of nontransferred, nonfrozen human pre-embryos in three culture conditions from day 3 to day 8 postfertilization. Preliminary evaluation by RIA and electrophoresis of factors responsible for these results. RESULTS: Ten percent mature FF supplementation of Inra Menezo (B2 medium) was associated with a significantly higher proportion of human pre-embryos reaching the morula (95% versus 72% with 10% maternal serum, P = 0.04) and the blastocyst stage (50% versus 11% with B2 alone, P = 0.02). The concentration of insulin-like growth factors I and II did not differ significantly between serum and mature FF-supplemented medium. Protein electrophoresis showed a difference of two bands corresponding to a molecular weight of 17,000 present in the serum and not in the FF-supplemented medium. CONCLUSION: Culture medium supplementation with 10% mature FF is associated with a significantly higher proportion of pre-embryos reaching the morula and blastocyst stage. The observed difference could be explained by the presence of a low molecular weight (17,000) embryotoxic factor contained in the serum-supplemented medium.

O6-methylguanine-DNA adducts in rat lymphocytes after in vivo exposure to N-nitrosodimethylamine (NDMA).

A non-invasive approach in immunopathological risk assessment was applied for analysis of the in vivo formation of DNA adducts. DNA methylation was studied in peripheral blood lymphocytes (PBLs) collected from Sprague-Dawley rats exposed to a single dose (75 mg/kg b.w.) of N-nitrosodimethylamine (NDMA). Three different techniques were applied for characterization and quantification of DNA adducts: (i) colloidal gold ultraimmunocytochemical localization of O6-methylguanosine (O6-meG)-DNA adducts, using affinity-purified, polyclonal antibody directed against O6-meG, (ii) quantitative assay using enzyme-linked immunosorbent assay (ELISA), amplified by the avidin-biotin (AB) system, and (iii) high-performance liquid chromatography (HPLC). The O6-meG-immunoreactive sites in PBLs seem to be concentrated in the nucleus. However, significant immunolabelling was also noted in the cytoplasm of the in vivo NDMA-exposed PBLs. Control preparations showed no specific gold immunolabelling. The O6-meG-DNA adduct formation in PBLs and hepatocytes, at 2-24 h following the exposure to NDMA, was analogous for both types of cells. The data showed high correlation for the ELISA and HPLC analytical methods. The data suggest an efficient O6-metG-DNA repair mechanism in lymphocytes, possibly analogous to the enzymatic repair of DNA adducts in liver cells.

Embryonic resistance to tumour necrosis factor-alpha mediated cytotoxicity: novel mechanism underlying maternal immunological tolerance to the fetal allograft.

The cytokine tumour necrosis factor-alpha (TNF alpha) has been postulated to play an essential role in the cytotoxic activity of cell-mediated immunity against allogenic or tumour cells invading the host. Several tumour cell lines, however, are resistant to TNF mediated cytotoxicity and respond paradoxically by cellular proliferation and by autocrine secretion of TNF alpha. In view of the metastatic character of the mammalian embryo, the aim of this study was to assess the potential of murine embryos to secrete TNF alpha in vitro, to express TNF receptors and to resist TNF alpha mediated cytotoxicity during their in-vitro development to the blastocyst stage. The potential of human embryos to secrete TNF alpha in vitro until the blastocyst stage was also investigated. From a total of 11 human embryos, which were allowed to proceed to blastocyst formation, seven secreted TNF alpha in the range of 2-117 pg/ml/24 h. A total of 123 C57BL/6J mouse embryos were studied of which 55% secreted TNF alpha in the range of 1.25-3.95 mg/ml/24 h. The presence of high levels of exogenous TNF alpha (10-300 IU) was not detrimental to the in-vitro development of murine embryos. Using immunohistochemical techniques, we were not able to detect the presence of type I or II TNF receptors on the surface of murine embryos. Our findings suggest that human and C57BL/6J murine embryos have the potential to secrete TNF alpha in vitro during the developmental stages leading to blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lysoplatelet-activating factor on human sperm fertilizing ability.

OBJECTIVE: To evaluate the penetration rates in the hamster zona-free oocyte sperm penetration assay (SPA) after exposure of spermatozoa to lysoplatelet-activating factor (LPAF) and lysophosphatidyl choline (LPC). DESIGN: Washed human spermatozoa were exposed to 100 microM of LPAF or LPC, followed by the assessment of their fertilizing ability using the SPA. The percentage of penetration, the sperm binding in the SPA, the percentage of motile spermatozoa, and the acrosome reaction rates were quantified. SETTING: Private research and university laboratories. PATIENTS, PARTICIPANTS: Fresh and frozen semen samples from fertile donors with proven fertility were used as well as fresh semen from infertile patients attending a fertility clinic. All the infertile patients had abnormal semen analysis. INTERVENTIONS: Human spermatozoa were incubated for 90 minutes in the presence or absence of LPAF or LPC at 100 microM with 0.3% albumin in Ham's F-10 (GIBCO, Dorval, Quebec, Canada), and their fertilizing ability was evaluated using the SPA. The effect of these lysophospholipids on the percentage of acrosome reaction was evaluated with a fluorescent microscopy technique. RESULTS: The penetration rates of the SPA in male factor increased significantly from 3% +/- 6% with controls to 19% +/- 9% and 34% +/- 22% after incubation with LPC and LPAF, respectively. Sperm-oocyte binding was not significantly increased in this group. Sperm penetration assay penetration rates were also increased in fertile cryopreserved spermatozoa with LPC and LPAF. In this group, the acrosome reaction was significantly increased from 2% +/- 1% in controls to 10% +/- 6% and 8% +/- 3% after incubation with LPC and LPAF, respectively. CONCLUSION: Lysoplatelet-activating factor and LPC independently increased the penetration rate of spermatozoa and the percentage of acrosome reaction. Lysophosphatidylcholine and LPAF may be beneficial in the treatment of spermatozoa with male factor infertility and may increase fertilization rates in IVF.

Mercuric chloride affects protein secretion in rat primary hepatocyte cultures: a biochemical ultrastructural, and gold immunocytochemical study.

The toxicity of mercury on hepatocytes was studied at the ultrastructural, biochemical, and immunocytochemical levels. Albumin metabolism was examined because it is a representative liver-specific function. A novel cytochemical method using the protein A-gold technique for the in situ localization of albumin in hepatocyte cultures was applied. Primary rat hepatocyte cultures were exposed to increasing HgCl2 concentrations. Cytotoxicity was assessed by measuring the release of lactic dehydrogenase from the cells. At the highest exposure concentration tested (50 microM), Hg was found to be significantly cytotoxic in contrast to what occurred at 5.0 and 0.5 microM. The level of albumin secreted, as measured by ELISA, was decreased by approximately 38% at 5.0 microM HgCl2 and was found not to be different from that of controls at lower concentrations. The ultrastructural analysis showed that hepatocytes treated with 5.0 microM HgCl2 undergo drastic morphological changes such as a decreased number of ribosomes associated with the rough endoplasmic reticulum, and the disappearance of the latter organelle, proliferation of the smooth endoplasmic reticulum, and dilatation of both the Golgi apparatus and the biliary canaliculus-like structures. Immunocytochemical detection of albumin-immunoreactive sites using protein A-gold labeling further revealed that these were less abundant in hepatocytes treated with 5.0 microM HgCl2 (-64%) as compared to control preparations. These results suggest that one of the effects of mercury on hepatocytes is to affect liver-specific functions such as albumin production, possibly through interference with ribosomal function. This study also demonstrates for the first time the applicability of the high-resolution protein A-gold technique for toxicological investigations on hepatocytes in vitro.

Recovery potential of hepatocytes from inhibition of albumin secretion by cadmium.

The aim of this study was to examine albumin production, a typical liver-specific function, in hepatocytes treated with Cd and to examine the reversibility of the perturbations induced by the toxic metal. Cultures of freshly isolated rat hepatocytes were exposed to increasing amounts of Cd in modified Leibowitz L-15 medium for 20 h; the cells were then allowed to recover by further incubation in Cd-free medium for an additional period of 20 h. The levels of albumin secreted into the extracellular medium were determined by enzyme-linked immunosorbent assay and were found to be reduced by Cd in a concentration-dependent fashion over the first 20 h. Inhibition was seen at Cd concentrations that did not cause any loss of cellular viability (up to 0.5 microM Cd), as judged from the release of lactate dehydrogenase by the cells. After replacement of the exposure medium by Cd-free medium, the same pattern of diminished albumin secretion was obtained, revealing the persistence of the cytotoxic effects when recovery conditions were applied. Moreover, hepatocytes exposed to 0.5 microM Cd for 20 h and processed for visualization of albumin immunoreactive sites using protein A-gold and electron microscopy exhibited very low albumin-specific labeling as compared to the controls (0.6 +/- 0.05 vs. 20.0 +/- 2.6 gold particles/micron2). Intracellular glutathione levels were not significantly changed by Cd either after the initial exposure or after the incubation that followed in control medium. The accumulation of Cd by the cells, as measured by graphite furnace atomic absorption spectrophotometry, was concentration dependent. It remained stable after medium change, indicating that Cd efflux was negligible upon reestablishment of normal conditions. The present data show that the perturbations in albumin metabolism caused by Cd are not readily alleviated after the cells are returned to Cd-free medium, suggesting a limited short-term recovery potential against cytotoxic damage. The data also demonstrate that hepatocyte-specific functions can be used as sensitive indicators for the detection of cellular disturbances by hepatotoxins.

Colloidal gold ultraimmunocytochemical localization of DNA and RNA adducts in rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-methylguanine (O6-metG) and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2 h to 5 mM N-nitrosodimethylamine (NDMA). In NDMA-treated cells, the O6-metG-induced immunoreactive sites do not appear at random but seem to be concentrated in the nucleus, and in the cytoplasm, in areas rich in rough endoplasmic reticulum (RER) elements. Mitochondria were not significantly labelled. Untreated control preparations showed no specific immunogold labelling. After RNase digestion of ultrathin sections obtained from cells exposed to NDMA and subsequent immunogold labelling, most of the immunolabelling in the cytoplasm had disappeared, and that over the nucleus had only been slightly reduced, as compared to undigested specimens from NDMA-treated cultures. After similar digestion with DNase, a strong reduction of the labelling of the nucleus was observed, but labelling of the cytoplasm was practically unaffected by this enzymatic treatment, as compared to what was observed in undigested preparations of NDMA-treated hepatocytes. The results provide evidence of preferential formation of O6-metG at the DNA and RNA levels, in the nucleus and cytoplasm RER, respectively. Furthermore, this study demonstrates the applicability of the high-resolution protein A-gold technique for ultrastructural detection of nucleic acid adducts in NDMA-treated hepatocytes using affinity-purified anti-O6-metG polyclonal antibodies.

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys.

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of microfilaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cytoskeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function.

Phalloidin-gold complexes: a new tool for ultrastructural localization of F-actin.

We used a phalloidin-gold complex to study the distribution of F-actin in the myxamoebae and macroplasmodia of the slime mold Physarum polycephalum. After incubation of Lowicryl- or Quetol-embedded specimens with this complex, significantly different labeling intensities were found over the various cytoplasmic and nuclear regions of the cells. The nucleoplasm was the most heavily labeled cell compartment, followed in decreasing order of labeling intensity by the cytoplasm, the nucleolus, and the chromocenters. The labeling observed over the latter area did not appear significantly different from that of the background. Sections incubated in the phalloidin-gold complex to which an excess of F-actin was added showed no significant labeling over any of the above-mentioned cell regions. Other control experiments included incubation of the sections with a phalloidin solution followed by the phalloidin-gold complex, PEG-stabilized colloidal gold, and a bovine serum albumin-gold complex. There was no or very little labeling of the preparations.

Interaction of (dien)Pd(II) with cytidine and cytidine 5'-monophosphate. Influence of the phosphate group on the kinetics and mechanism.

The kinetics and the equilibrium of (dien)PdCl+ interaction with cytidine (C) and cytidine 5'-monophosphate (CMP) were studied by spectrophotometry and by stopped-flow methods. In both cases, the mechanism implies a (dien)Pd(H2O)2+ intermediate with a significant contribution of the solvent path at low chloride concentrations. With CMP, the rate is affected due to the addition of a mechanistic path via an intermediate formed between (dien)Pd(II) and the phosphate group of CMP. The kinetic and thermodynamic parameters have been determined and reflect the favorable electrostatic interactions due to the presence of the phosphate group of CMP. Furthermore, these parameters are in agreement with a transient (dien)Pd(II)-phosphate complex of CMP leading to the formation of the thermodynamically favored (dien)Pd(II)-N3 complex as final product.