The Taming of Immunohistochemistry: The New Era of Quality Control

The most critical factor for interpreting the results of immunohistochemistry is verification of antibody sensitivity and specificity. While some manufacturers supply material data sheets with this information, many do not. This paper describes a well-defined quality assurance program for testing immune reagents. This program can be used to provide commercial suppliers of antisera with analyses of their products destined for government licensure applications. This paper illustrates the protocol and explains the testing philosophy developed over the last eight years.     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

Biogenesis of Chromaffin Granules: Incorporation of Sulfate into Chromogranin B and into a Proteoglycan

The incorporation of [35S]sulfate into the soluble proteins of chromaffin granules was studied. Isolated bovine chromaffin cells were pulse-labeled with [35S]sulfate. The radioactively labeled products were characterized by one- and two-dimensional electrophoresis. Three proteins of chromaffin granules were preferentially labeled. One was identified by immunoprecipitation as chromogranin B (Mr 100,000). This result explains why during cellular synthesis the chromogranin B precursor is converted into a significantly more acidic protein. During chase periods, the newly synthesized chromogranin B was progressively degraded by endogenous proteases. A second labeled protein, much less labeled than chromogranin B, was identified as chromogranin A. The largest portion of the radioactive label was found in a heterogeneous component (Mr 86,000-100,000; pI 4.3-5.0). Digestion experiments with chondroitinase ABC demonstrated that this labeled component and a comigrating Coomassie Blue-stained spot were selectively degraded by this enzyme. This establishes that this component is a proteoglycan.     

Non-Reducing Terminal Fucose within N-Linked Glycan Plays a Significant Role in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

MgATP-Stimulated Release of Luteinizing Hormone Releasing Hormone from Isolated Hypothalamic Granules: Evidence for a Selective Requirement for Monovalent Cations

Abstract: The purpose of this study was to characterize the kinetics and monovalent ion requirements of the MgATP-stimulated release of Iuteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. LHRH granules were obtained from homogenates of hypothalami of adult male rats by differential centrifugation and then incubated in buffered media containing KCl. Under these conditions, the pH optimum for the MgATP-stimulated release of LHRH was between 7.5 and 8.0; the rate of release was linear for the first 5 min of incubation; and the magnitude of the release was a saturable function of KCl concentration. When the potassium in the incubation mixture was replaced with various monovalent cations (Li⁺, Na⁺, Cs⁺, choline, or tetraethylammonium), the magnitude of the MgATP-stimulated release of LHRH was inversely related to the size of the cation; release in the presence of Li⁺ was 12%, whereas release in the presence of tetraethylammonium was 0% of the total LHRH. When the chloride in the incubation mixture was replaced with various monovalent anions (Br⁻, I⁻, or isethionate), the magnitude of the MgATP-stimulated release of LHRH was not related to the size of the anion. Moreover, inclusion of anion transport inhibitors in the incubation mixture did not affect LHRH release, suggesting that anion transport is not required for the MgATP-stimulated release of LHRH. In summary, we suggest that the MgATP-stimulated release of LHRH from isolated hypothalamic granules involves an enzymatic event and that this release process does not have a selective requirement for monovalent anions, but has a selective requirement for monovalent cations.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

Functional Study of miR-27a in Human Hepatic Stellate Cells by Proteomic Analysis: Comprehensive View and a Role in Myogenic Tans-Differentiation

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.     

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.     

Collection and Dissemination of Exposure Data Throughout the Chemical Value Chain: A Case Study from a Global Consumer Product Industry

The human and environmental safety of chemicals in formulated consumer products has for decades been important for product manufacturers and ingredient suppliers. However, prior to the mid-1990s, the underlying product safety data were largely not publicly available. The U.S. Environmental Protection Agency's Chemical Right-to-Know Program and a related program of the Organization of Economic Cooperation and Development have since been the catalysts for the broad dissemination of standardized hazard datasets for thousands of high production volume (HPV) chemicals in commerce around the globe. The formulated consumer products industry took the additional step of developing and summarizing the science around exposure to their products, largely cleaning products and personal care products, in order to be able to characterize and communicate the safety of their products. Perhaps even more significant was the collection and circulation of information surrounding the habits and practices of product use, and ingredient concentrations in products. The objective of this article is to detail the methods used for collection of this information by the American Cleaning Institute (ACI), and the process by which hazard and exposure information may be used in screening-level risk assessments. These data and methods appear in a number of ACI's publications (http://www.aciscience.org).     

Fluorescent labelling of Na+,K+-ATPase in intact cells by use of a fluorescent derivative of ouabain: Salinity and teleost chloride cells

Summary Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na⁺,K⁺-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 M anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 M anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na⁺,K⁺-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na⁺,K⁺-ATPase in other tissues and species.     

Human Health Risk Assessment of Soils Contaminated with Metal(loid)s by Using DGT Uptake: A Case Study of a Former Korean Metal Refinery Site

The human health risk of soils contaminated with As, Pb, Cu, and Zn was evaluated based on pseudo-total concentrations of metal(loid)s, the physiologically based extraction test (PBET), and diffusive gradients in thin films (DGT). Non-carcinogenic (NCR) and carcinogenic (CR) risks exceeded the U.S. Environmental Protection Agency criteria under both the residential and non-residential scenarios. Human bioavailable concentrations (PBET) were much lower than pseudo-total concentrations. The Hazardous Index of NCR (HI (NCR)) for the PBET in the studied soils was 67% and 94% less than that for pseudo-total concentration, respectively, under the non-residential and residential scenarios. Similarly, CR for the PBET was also 65% and 93% less for the two soils. The concentration of metal(loid)s accumulated in the DGT resin was highly correlated with the PBET-extractable concentration (R² > 0.649). Therefore, for both the CR and HI (NCR), the DGT-calculated risk was linearly related to the PBET-calculated risk for the studied soils under both scenarios. The results suggest that DGT uptake and PBET-extracted concentrations are good surrogates for risk estimation and that both J1 and J2 soils require remediation before their use for residential or non-residential purposes.     

In Situ Detection of a PCR-Synthesized Human Pancentromeric DNA Hybridization Probe by Color Pigment Immunostaining: Application for Dicentric Assay Automation

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.     

Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.     

Optimal Design of a Series of GSTR's Performing Reversible Reactions Catalyzed by Soluble Enzymes: A Theoretical Study

The condition for the minimum overall reactor volume of a given number of CSTR's in series is theoretically determined for a reversible, single reactant-single product (Uni-Uni) enzyme catalyzed reaction. The reactor network is assumed to operate in steady-state, isothermal conditions with a single phase and a constant activity of biocatalyst. The method is based on a mathematical analysis of the discrete substrate concentration profile along the CSTR's assuming complete micromixing. The algebraic equations describing the critical loci are obtained for the general case, the mathematical proof that these equations define a minimum is presented, and an exact solution arising from an asymptotic situation is found. An approximate analytical method of optimization based on the aforementioned critical behavior is reported and its validity and usefulness discussed. The formulae introduced can be used in more general situations as tools for getting the approximate range where the optimal overall volume of the series of CSTR's lies. Hence, the reasoning developed is important for the preliminary CSTR design and relevant in the initial steps of the more involved methods of numerical optimization. Finally, the enzymatic conversion of fumarate to L-malate is examined as a model system in order to assess the usefulness and applicability of the analysis developed.     

Possible Role of Heme Oxygenase-1 and Prostaglandins in the Pathogenesis of Cerebral Malaria: Heme Oxygenase-1 Induction by Prostaglandin D2 and Metabolite by a Human Astrocyte Cell Line

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D₂ is abundantly produced in the brain and regulates the sleep response. Moreover, PGD₂ is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD₂ significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD₂ treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD₂ may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.     

A Conceptual Framework for Evaluating the Interaction of a Chemical and Nonchemical Stressor in Human Health Risk Assessments: A Case Study for Lead and Psychosocial Stress

Recent research has demonstrated that nonchemical stressors may alter the toxicity from chemical exposures. This may have public health implications for low socioeconomic status (SES) communities that may be disproportionately exposed to toxic chemicals and various types of community and personal stressors. Nonchemical stressors may introduce an important source of variability that needs to be considered by risk assessors. Herein, we propose a framework for determining if a chemical–nonchemical interaction exists and, if so, options for incorporating interaction information into risk assessments. We use the increasingly recognized interaction between lead and psychosocial stress to illustrate the framework. We found that lead exposure occurs disproportionately in low SES groups that also tend to face high levels of psychosocial stress; that stress and lead both affect neurodevelopment and that this occurs via similar pathways involving the hypothalamic-pituitary axis. Further, several epidemiological and experimental studies have provided evidence for an interaction between lead and psychosocial stress. The implications of this interaction for risk assessment are also discussed.     

Supravital Uptake of Methylene Blue by Dendritic Cells within Stratified Squamous Epithelia: a Light and Electron Microscope Study

Electron microscopic data on methylene blue staining of dendritic cells in the epithelia of the soft palate and skin of the moose after supravital dye injection are presented. The ultrastructural details were compared with corresponding light microscopic findings. Methylene blue stained tissue was fixed by immersion in a paraformaldehyde-glutaraldehyde solution containing phosphomolybdic acid. The ensuing dye precipitate was stabilized by ammonium heptamolybdate. The light microscopic investigation revealed that selective staining of dendritic cells depended on the presence of ambient oxygen. In addition, delicate morphological characteristics, like spinous structures of the dendrites, were visible. Some cells also showed terminal enlargements of the dendrites close to the surface of the epithelium. In general, visualization of morphological detail was superior to that obtained by conventional histological and immunohistochemlcal procedures. Nerve fibers were also stained within the epithelium as well as the subepithelial connective tissue. At the electron microscopic level, the dye was clearly identified as an electron dense precipitate that accumulated primarily within the cytoplasm near the plasma membrane. Furthermore, it was bound to the chromatin of the nuclei. No significant staining of mitochondria or other organelles was seen, within the cytoplasm, the oxygen-dependent binding sites may be associated with heme proteins that attract both the dye in its reduced lipophilic leuco form and oxygen, followed by generation of oxygen radicals and a reoxldation of the leuco form to the cationic blue dye. Because of its selectivity for intraepithelial dendritic cells, the method described here supplements immunocytochemical procedures at both the light and electron microscopic levels.