Surface-enhanced emission from single semiconductor nanocrystals.

The fluorescence behavior of single CdSe(ZnS) core-shell nanocrystal (NC) quantum dots is dramatically affected by electromagnetic interactions with a rough metal film. Observed changes include a fivefold increase in the observed fluorescence intensity of single NCs, a striking reduction in their fluorescence blinking behavior, complete conversion of the emission polarization to linear, and single NC exciton lifetimes that are >10(3) times faster. The enhanced excited state decay process for NCs coupled to rough metal substrates effectively competes with the Auger relaxation process, allowing us to observe both charged and neutral exciton emission from these NC quantum dots.

Effects of beta-estradiol and bisphenol A on heat shock protein levels and localization in the mouse uterus are antagonized by the antiestrogen ICI 182,780.

Bisphenol A (BPA) exhibits many estrogen-like effects in the rodent uterus, but not all of these can be attenuated by antiestrogens. This suggests the involvement of alternate pathways of BPA action that do not involve the estrogen receptor (ER). An examination of the in vivo effects of BPA on uterine gene expression and protein levels should contribute to an understanding of its mechanism of action. In this study we examined the dose-related effects of BPA on levels of a suite of heat shock proteins (hsps) and on the localization of hsp90alpha, a chaperone of the ER, in uteri of ovariectomized B6C3F1 mice and compared these effects with those of beta-estradiol (E2). The antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E2 in order to examine the potential role of the ER. BPA, although less potent than E2, increased hsp90alpha and grp94 to similar levels, but was much less effective than E2 in increasing levels of hsp72. Treatment with 100 mg BPA/kg/day or 2 microg E2/kg/day increased hsp90alpha to 300% of control levels and altered its tissue expression pattern. In uteri of corn oil (control)-treated mice, hsp90alpha predominantly localized in the cytoplasm and nuclei of epithelial cells. Upon treatment with BPA or E2 there was increased intensity of staining in the stroma and myometrium, and in the epithelium hsp90alpha was localized almost exclusively in the cytoplasm. The effects of BPA or E2 on hsp levels and hsp90alpha localization were attenuated by ICI. These results suggest an involvement of the ER in BPA- and E2-induced increases in uterine levels of hsp90alpha, grp94, and hsp72, and localization of hsp90alpha.

Platelet glass bead retention predicts bleeding after cardiac surgery.

OBJECTIVE: To determine if the platelet glass bead retention assay can predict bleeding after cardiac surgery. DESIGN: Prospective, observational study. SETTING: Large, tertiary care, academic medical center. PARTICIPANTS: Forty-three adult patients scheduled to undergo elective cardiac surgery employing cardiopulmonary bypass (CPB). MEASUREMENTS AND MAIN RESULTS: Whole blood samples were observed for platelet count, prothrombin time, and platelet (glass bead) retention assay. The platelet retention and prothrombin times were independent univariant and multivariant predictors of bleeding after CPB (r = 0.554, p = 0.0002 and r = 0.655, p = 0.00001). CONCLUSION: The platelet glass bead retention assay measures dynamic platelet function and is sensitive to the CPB-induced adhesion and aggregation defect and correlates with postoperative blood loss. Modification of this platelet function assay used with the prothrombin time may provide a simple and effective diagnostic approach to bleeding after CPB.

Bisphenol A-induced increase in uterine weight and alterations in uterine morphology in ovariectomized B6C3F1 mice: role of the estrogen receptor.

The ability of the environmental xenoestrogen bisphenol A (BPA) to increase uterine wet weight in the rodent remains controversial, and few studies have previously examined the effects of BPA on uterine morphology. Furthermore, it is not known whether BPA-induced uterotrophic effects are, similarly to beta-estradiol (E(2)), mediated through the estrogen receptor (ER). In this study, we compared the effects of BPA on uterine wet weight and morphology to those of E(2) in the B6C3F1 ovariectomized mouse. To examine whether these effects were mediated through the ER, the antiestrogen ICI 182, 780 (ICI) was co-administered with BPA or E(2). We report that subcutaneous administration of BPA at doses between 0.8 and 8 mg/day over 4 days significantly increased mean uterine wet weights above those of vehicle (corn oil)-treated mice. The uterine weight data suggest that BPA acts as a partial agonist with an EC(50) of 0.72 mg/day compared to 19.4 ng/day for E(2). BPA (2 mg/day) and E(2) (40 ng/day) induced a significant increase in luminal epithelial height and in the thickness of both the stromal and myometrial layers of the uterus. The effects of 40 ng E(2)/day on all endpoints studied were reversed by 20 microg ICI/day. ICI at 200, but not 20 microg/day, was able to reverse the BPA (2 mg/day)-induced increase in both uterine wet weight and luminal epithelial height. ICI alone at 200 microg/day stimulated an increase in thickness of both the stroma and myometrium and did not reverse the effects of BPA (2 mg/day) on these layers. These results suggest that the BPA-induced increase in uterine wet weight and in luminal epithelial height in the ovariectomized B6C3F1 mouse are mediated by the ER.

Mercury induces regional and cell-specific stress protein expression in rat kidney.

Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, i.v.). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.

In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli.

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.

Normothermic cardiopulmonary bypass increases heparin requirements necessary to maintain anticoagulation.

OBJECTIVE: With the practice of warm cardiopulmonary bypass (CPB) at our institution we have observed an apparent increase in heparin requirements. CPB temperature predictability affects pharmacokinetics and differences in drug metabolism can be expected. We hypothesized that heparin requirements would increase with increasing CPB temperature. METHODS: Following Institutional Review Board approval, we reviewed the charts of 354 patients undergoing primary coronary artery bypass graft surgery. We recorded patient demographic data, CPB duration, heparin requirements, and temperature during CPB. CPB was conducted between 24 degrees C and 37 degrees C. The Spearman's correlation coefficient, Pearson chi-square, and rank-sum tests were used for data analysis. RESULTS: Core temperature during CPB correlated with heparin requirements (r = 0.13, p < 0.02). However, CPB duration was shorter in warm patients than in cold patients (r = -0.455, p < 0.0001). Additional heparin requirements adjusted for duration of CPB (units/minute) were also significantly greater in the warm group (p = 0.018). CONCLUSIONS: Maintenance of adequate heparin anticoagulation during CPB is clinically important. Warm CPB patients required more heparin per minute than those undergoing cold CPB. More frequent assessment of anticoagulation and administration of additional heparin should be considered in patients undergoing warm CPB.

Heat-induced alterations in embryonic cytoskeletal and stress proteins precede somite malformations in rat embryos.

Previous work from this laboratory has demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development 24 hr after exposure and subsequent thoracic skeletal malformations in neonates. The purpose of the present study was to examine the effects of in vitro heat shock on de novo protein synthesis and on cytoskeletal protein levels in developing rat embryos. Explanted GD10 embryos were exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure (0-27 hr). embryos were labeled with 35S-methionine and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. The 70-kD protein was identified as a eukaryotic stress protein and the presence of this protein was detected between 2 and 27 hr posttreatment. Western blot analysis was used to detect quantitative changes in total actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments). Immediately following exposure, a reduction of total vimentin to minimal detectable levels was observed in heat-treated embryos. Levels of total vimentin remained depressed for more than 2 hr and gradually returned to control levels 4-8 hr postexposure. No change in total actin or tubulin was detected in treated embryos. The data demonstrate that heat-induced alterations in proteins comprising intermediate filaments occur concomitantly with the induction of stress proteins and precede aberrant somite morphology. These alterations in embryonic proteins may help elucidate the mechanism(s) by which skeletal malformations are produced.

Induction of hsp72 in heat-treated rat embryos: a tissue-specific response.

Previous studies have demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development in rat embryos 24 hr after exposure and in thoracic skeletal malformations in neonatal rats examined 3 days postpartum. The production of abnormal somites was correlated with the location of skeletal elements that developed from the affected somites. Heat has also been shown to induce changes in genetic expression whereby new proteins are synthesized and the expression of constituent proteins may be repressed. In the present study, heat-induced alterations in protein synthesis during rat organogenesis that may be associated with previously observed malformation was investigated. GD10 rat embryos were exposed in utero to a heat treatment previously demonstrated to produce skeletal malformations; maternal core temperature was raised and maintained at 42-42.4 degrees C for 5 min. In addition, explanted GD10 embryos were cultured in vitro and exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure, embryos were labeled with 35S-methionine and processed for SDS-PAGE. In both in vivo and in vitro heat-treated embryos, a transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. Actinomycin D studies were conducted to determine whether transcription of new mRNA was required for the enhanced synthesis of the 70- and 90-kD proteins in heat-treated embryos. Results from these studies demonstrated that the expression of these proteins was transcriptionally regulated. The 70-kD protein was identified, using Western blot analysis, as a eukaryotic inducible stress protein (hsp72), and the presence of this protein was detected between 2 and 27 hr post-treatment. Immunohistochemical results indicated that following heat shock, hsp72 accumulates in the neuroectodermal tissues of the embryos. The data demonstrate that although heat-induced expression and accumulation of the hsp72 precedes aberrant somite morphology, the lack of hsp72 accumulation in the somite mesoderm may explain the sensitivity of this tissue to heat.

Stress protein synthesis induced by cadmium-cysteine in rat kidney.

Biomarkers are important tools which enable toxicologists to reliably predict and detect exposures to xenobiotics and resultant cell injury, ultimately improving risk assessments. Since the de novo synthesis of stress proteins can be detected early after exposure to some agents, analysis of toxicant-induced changes in gene expression, i.e. alterations in patterns of protein synthesis, may be useful to develop as biomarkers of exposure and toxicity. We are utilizing various xenobiotics as tools to study stress protein synthesis in target organs in order to evaluate the target tissue-specificity of this response. Previous data from this laboratory have demonstrated that induction of stress proteins in rat liver, but not kidney, after acute exposure to CdCl2 precedes hepatoxicity. Since kidney is a target tissue after chronic Cd exposure, it was of interest to examine stress protein synthesis in this tissue. However, dose-limiting hepatotoxicity precluded this evaluation. Cd complexed with molecules such as cysteine (cys) or metallothionein has been used in acute dosing regimens as a tool in order to study the nephrotoxicity of Cd. Therefore, this study was undertaken in order to evaluate Cd-induced stress protein synthesis in an important tissue known to be injured after chronic exposure, i.e. kidney. Specific objectives included comparing stress protein synthesis in rat kidney and liver after acute exposure to Cd-cys and CdCl2, determining the Cd threshold concentration for renal stress protein synthesis and assessing the relationship between stress protein synthesis and nephropathy. Male rats were exposed to equivalent doses of Cd as CdCl2 or Cd-cysteine (molar ratio Cd:cys = 1:15). Kidney Cd concentrations increased 5-fold after i.v. injection of Cd-cys compared to CdCl2, mimicking Cd distribution following chronic exposure. After exposure to Cd, tissue slices were incubated with 35S-methionine. Slices were subsequently homogenized and centrifuged, and the 16,000 g supernatants were subjected to SDS-polyacrylamide gel electrophoresis. Proteins which had incorporated 35S-methionine were detected by autoradiography. De novo synthesis of 70, 90 and 110 kDa proteins was enhanced in liver, but not in kidney, 4 h after injection of 2 mg Cd/kg as CdCl2. In contrast, dose-related increases in synthesis of these proteins were observed in kidney 4 h after injection of 1 and 2mg Cd/kg as Cd-cys, but not at lower dosages. In addition, synthesis of a 68 kDa kidney protein was inhibited at 2 mg Cd/kg as Cd-cys. The threshold for Cd-induced stress protein synthesis was shown to be between 4 and 8 micrograms Cd/g tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxicity.

A diverse array of chemical and physical stressors increases the synthesis of a class of proteins referred to as heat shock proteins (hsp) or stress proteins. We are investigating the potential of using altered protein synthesis patterns, including the enhanced synthesis of stress proteins, as biomarkers of exposure and cellular injury. The purpose of the present study was to evaluate the effect of a model hepatotoxicant, cadmium (Cd(II)), on stress protein synthesis in male rat liver. To assess target tissue specificity, stress protein synthesis was also studied in kidney. Liver and kidney slices from exposed rats were incubated with [35S]methionine for 1.5 hr, subjected to one-dimensional SDS-PAGE, and 35S-labeled proteins were analyzed by autoradiography. Enhanced de novo synthesis of 70-, 90-, and 110-kilodalton (kDa) relative molecular mass (M(r)) proteins was detected 2 hr after exposure to 2 mg Cd/kg, with maximum activity occurring at 2-4 hr. By 8-16 hr postinjection, synthesis of these proteins had decreased. Synthesis of a 68-kDa protein present in control liver was inhibited 2 hr after exposure with synthesis restored at 16-24 hr. Dose-related increases in synthesis of the three stress proteins were observed 4 hr after iv injection of 1.0 and 2.0 mg Cd/kg, with concomitant inhibition of synthesis of the 68-kDa protein. Mild single cell necrosis of hepatocytes was observed 8 hr after injection of 2 mg Cd/kg which progressed to mild multifocal necrotic foci at 16 hr. No lesions were evident at lower dosages. Increases in plasma sorbitol dehydrogenase activity, a clinical indicator of hepatic injury, was not apparent until 8 hr after exposure. A functional deficit, decreased hepatic microsomal N-demethylase activity, was not observed until 16 hr after iv injection of 2 mg/kg. No changes in kidney de novo stress protein synthesis were observed. No evidence of renal injury was apparent, as evaluated by histopathology, uptake of [para-3H]aminohippurate into renal slices, and blood urea nitrogen values. The 70-kDa stress protein induced early after Cd treatment was identified with a monoclonal antibody as the 72-kDa-inducible hsp. The 90-kDa protein induced by Cd reacted negatively with three monoclonal antibodies to hsp90 and was subsequently identified as a glucose regulated protein (grp94). The data demonstrate that Cd induces alterations in the expression of hepatic gene products in vivo as evidenced by enhanced stress protein synthesis and inhibition of synthesis of constitutive proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Latex-associated allergies and anaphylactic reactions.

The recent reports of severe anaphylactic reactions and several fatalities caused by contact with latex-containing products raised concerns in the medical community. Although hypersensitivity to natural rubber has been widely reported in the literature, the prevalence and severity of reactions have rapidly increased in the last few years. Latex proteins, constituents of natural latex, appear to be responsible for the sensitization. Many investigators, including our laboratory, are focused on the identification of proteins in raw latex and latex products, specifically those responsible for the elicitation of allergic responses. This paper summarizes available information on the mechanism and epidemiology of latex sensitivity and reviews research efforts toward the identification of the antigen(s) responsible for the reactions. The questions of proper diagnosis and testing, heightening awareness, and prevention of reactions are also addressed.

Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity.

Adverse environmental stimuli increase the synthesis of a class of proteins referred to as stress proteins. The effect of mercuric chloride, a model nephrotoxin, on protein synthesis in male rat kidney has been evaluated. Renal slices from exposed rats were incubated with [35S]methionine for 1 hr and subjected to SDS-PAGE, after which 35S-labeled proteins were detected by autoradiography. Enhanced de novo synthesis of 70- and 90-kDa relative molecular mass (M(r)) proteins were detected 2 hr after exposure to 1 mg Hg/kg, with maximum activity occurring at 4-8 hr. By 16 hr postinjection, synthesis of these two proteins had decreased. Dose-related increases in synthesis of these proteins, and of a 110-kDa protein, were observed 4 hr after i.v. injection of 0.25, 0.5, and 1.0 mg Hg/kg, with concomitant inhibition of synthesis of proteins of M(r) 38 and 68 kDa. At a dose of 1 mg/kg, kidney proximal tubules exhibited progressive degenerative changes from 4 to 24 hr. A functional deficit, decreased uptake of [para-3H]aminohippurate into renal slices, was not observed until 16 hr after i.v. injection of 1 mg/kg. No significant histopathologic changes were observed in kidneys 4 hr after treatment with 0.25 or 0.5 mg Hg/kg, iv. No changes in liver protein synthesis were apparent until 16-24 hr, where an increase in the 70- and 90-kDa proteins was observed. A concomitant increase in plasma sorbitol dehydrogenase activity occurred at 16-24 hr; however, there was no histopathological evidence of liver injury. The 72-kDa inducible member of the 70-kDa stress protein family and the 88-kDa member of the 90-kDa protein family were detected by immunoblotting techniques using monoclonal antibodies. The data demonstrate that Hg induces alterations in the expression of renal gene products in vivo as evidenced by enhanced stress protein synthesis and inhibition of synthesis of constitutive proteins. These changes in renal protein synthesis preceded overt renal injury, occurring in the early stages of nephropathy. Altered patterns of stress protein synthesis appeared to be target organ specific. The data suggest that altered protein synthesis patterns may serve as biomarkers of renal injury.

Automated assay of alpha-amylase inhibitor proteins by continuous flow analysis.

A procedure for the automated assay of purified amylase inhibitors was developed. Samples were analyzed at a rate of 60/h using ferricyanide reagent to monitor the suppression of the release of reducing groups from a solution of starch by a calibrated alpha-amylase reagent. In addition to the sequential analysis of individual samples, the use of gradients permitted the continuous analysis of the effect of substrate and of inhibitor concentration. Also described are some of the effects of starch and inhibitor concentration and time of preincubation on the amylase-inhibitor reaction. The procedure was also suitable for the assay of samples of amylase and should be applicable to the determination of the effects of inhibitors on other enzymes which release reducing sugars.