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The effect of gold trichloride on DNA synthesis of human thymocytes and peripheral blood lymphocytes was tested. A stimulation was obtained in both cell types with a concentration of 6-50 micrograms/ml. Gold trichloride may thus not be used in a lymphocyte transformation test for the diagnosis of contact allergy.
Gold-induced pulmonary disease is difficult to diagnose, especially, in the case in which interstitial pneumonia appears in the course of gold therapy for rheumatoid arthritis. We analyzed the literature to define the clinical features and prognosis of gold-induced pulmonary disease, and to identify those features that distinguish gold-induced pulmonary disease from pulmonary disease secondary to the underlying disease process of rheumatoid arthritis. Relevant articles from the medical literature were identified using a Mediline search, and the bibliographies of the articles were retrieved. These works were critically reviewed for information on the clinical, physiologic, radiographic, pathologic, and bronchoalveolar lavage (BAL) findings. A total of 140 cases of gold-induced pulmonary disease were identified from 110 reports. In 81% of the patients, gold was being used to treat rheumatoid arthritis, bronchial asthma (6%), pemphigus (5%), or other processes (9%). Side effects other than pulmonary toxicity were common, and included skin rash (38%), peripheral eosinophilia (38%), liver dysfunction (15%), and proteinuria (22%). Only the presence of pemphigus or liver dysfunction correlated with a bad prognosis. Gold-induced pulmonary disease most often followed improvement in rheumatoid arthritis, presumably induced by gold therapy. BAL lymphocytosis and computed tomography (CT) scan findings are useful in making a diagnosis of gold-induced pulmonary disease in an appropriate clinical setting. Features that distinguish gold-induced pulmonary disease from rheumatoid lung disease include female predominance, presence of fever or skin rash, absence of subcutaneous nodules or finger clubbing, low titers of rheumatoid factor at onset of lung disease, lymphocytosis in bronchoalveolar lavage fluid (BALF), and alveolar opacities along the bronchovascular bundles on chest CT scan. Gold-induced lung disease is a distinct entity that can be distinguished from rheumatoid lung disease. It usually improves with cessation of therapy or treatment with corticosteriods.
To clarify the relationships between DNA damage and Cu-MT and between DNA damage and Cu in kidneys of rats injected with Au, we examined the histochemical localization of DNA damage, metallothionein (MT), and the accumulated Cu in the kidneys of rats injected with Au, Cu, or Cu-MT. The immunoreactivity of MT was observed predominantly in the outer stripe of the outer medulla and the inner cortex of the Au-injected rat, and the signals of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) were observed in the cortex. Cu detected by Timm's method was mainly distributed in the cortex of the Au-injected rat. These results indicated that DNA damage could be caused by free Cu in the cortex but not by the Cu bound to MT in the outer stripe of the outer medulla. This consideration was supported by the data from rats injected with Cu and Cu-MT. Furthermore, we determined the Cu contents in three fractions (cytosol, organelle, and precipitate-containing nuclei) of the kidneys. Interestingly, most of the Cu content in the kidney of the rat injected with Au or Cu-MT was detected in the cytosol, whereas most of the Cu content in the kidney of the rat injected with Cu was detected in the nuclei-containing precipitate. These findings suggest that the DNA damage in the kidneys of rats injected with Au may be associated with Cu-binding proteins but not with Cu-MT.
The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.
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A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.
In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.
Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified by one-step Strep tag affinity chromatography, chemically labeled with the marker, and employed in microscopy to localize epitopes on the membrane protein bacteriorhodopsin in purple membranes of Halobacterium halobium and the cytochrome c oxidase of Paracoccus denitrificans. In both cases, methods involving directly labeled antibody fragments show results identical to those in which antibodies or Fv fragments are detected by a secondarily labeled conjugate. The multifunctional design of the recombinant Fv fragments, however, offers more all-around applications in immunocytochemistry. The directly labeled Fv fragments, half the size of an Fab fragment, are at the molecular level the smallest antibody fragments yet described for visualization of biomolecules in microscopy.
In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin alpha/beta, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.
Angiotensin III has been reported to exist in various animals and tissues. The physiological role, however, is still unclear except that brain angiotensin III is a central regulator of vasopressin release. In this study, angiotensin III as well as angiotensin II enhanced an increase in body weight of clam worms of Perinereis sp. under a hypo-osmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo- and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms.
We studied variations in gold chloride techniques to elicit neural elements within articular samples, after "in toto" staining. These techniques attempt the differentiation of neural and vascular structures. Major changes in differential staining were observed when the gold chloride concentration was empirically modified. After the rest of the technique was standardized, we selected three gold chloride solutions to perform quantitative color experiments: 1%, 0.75%, and 0.5%. Significant sections of the same thickness were acquired with a digital camera to perform computer-assisted colorimetry. Color was measured through RGB (red-green-blue) channels in vessels, nerves, and background connective tissue as an internal control. By means of multivariate regression analysis, we compared differences in color measurements after 1%, 0.75% and 0.5% gold chloride preparation. Statistically significant coefficients confirmed that red color signals in vessels after the 0.75% and the 0.5% solution were both less intense than after the 1% preparation. Green and blue signals in vessels were also significantly less intense after the 0.5% protocol than after using the 1% solution. Red color signals in nerves between the 1% and the 0.75% preparation protocols were more intense and not significantly different, while the 0.5% preparation produced significantly less intense red signals in nerves. Non-significant differences were observed in green or blue signals from nerves after any protocol. We concluded that the 0.75% gold chloride solution protocol produced more intense red signals in nerves and less intense red signals in vessels. This was the most discriminant protocol in our series, based on color signals.
Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.
Single channel and macroscopic current recording was used to investigate block of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore by the permeant anion Au(CN)2(-). Block was 1-2 orders of magnitude stronger when Au(CN)2(-) was added to the intracellular versus the extracellular solution, depending on membrane potential. A point mutation within the pore, T-338A, strongly decreased the asymmetry of block, by weakening block by intracellular Au(CN)2(-) and at the same time strengthening block by external Au(CN)2(-). Block of T-338A, but not wild-type, was strongest at the current reversal potential and weakened by either depolarization or hyperpolarization. In contrast to these effects, the T-338A mutation had no impact on block by the impermeant Pt(NO2)4(2-) ion. We suggest that the CFTR pore has at least two anion binding sites at which Au(CN)2(-) and Pt(NO2)4(2-) block Cl- permeation. The T-338A mutation decreases a barrier for Au(CN)2(-) movement between different sites, leading to significant changes in its blocking action. Our finding that apparent blocker binding affinity can be altered by mutagenesis of a residue which does not contribute to a blocker binding site has important implications for interpreting the effects of mutagenesis on channel blocker effects.
Patients with rheumatoid arthritis were investigated for contact allergy to gold in connection with treatment with gold preparations. There were 57 patients with rheumatoid arthritis previously treated with gold, with or without cutaneous side-effects, as well as 20 patients intended for such treatment; all were exposed to patch and intradermal tests with gold sodium thiosulfate, gold sodium thiomalate and auranofin. Contact allergy to gold was demonstrated in 8 out of 77 patients (10.4%). In the retrospective material, gold allergy was found in 1.8%, in the prospective material in 35.0%. Contact allergy to gold is very frequent among patients with rheumatoid arthritis before gold therapy. In order to avoid early hypersensitivity reactions skin tests should be carried out before gold therapy is instituted.
OBJECTIVE: To determine effects of cyclophotocoagulation via administration of 100 J with a neodymium:yttrium aluminum garnet (Nd:YAG) laser on corneal touch threshold (CTT), intraocular pressure (IOP), aqueous tear production, and corneal nerve morphology in eyes of dogs. ANIMALS: 15 dogs. PROCEDURE: Noncontact Nd:YAG laser was transsclerally applied (10 applications; 25 W for 0.1 seconds for each application to each of 4 quadrants) to the ciliary body of the left eye of 15 dogs; the right eye was the control eye. Corneal integrity, CTT, tear production as measured by the Schirmer tear test (STT), and IOP were evaluated for 14 days following laser treatment. On day 14, dogs were euthanatized, eyes harvested, and corneas stained with gold chloride. Major nerve bundles were analyzed by use of a drawing tube attached to a light microscope, and maximum diameters were measured by use of image analysis software. RESULTS: All laser-treated eyes had significantly higher CTT values, compared with control eyes. Six of 15 laser-treated eyes developed ulcerative keratitis. On most days, IOP was significantly lower in laser-treated eyes in both morning and evening. Laser-treated eyes had a significant decrease of approximately 1 nerve bundle/corneal quadrant. Values for STT or nerve bundle diameters did not differ significantly. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of 100 J with a Nd:YAG laser effectively reduced IOP while increasing CTT and caused a significant decrease in number, but not diameter, of major corneal nerve bundles. Nerve damage and corneal hypoesthesia are etiologic factors in ulcerative keratitis following Nd:YAG cyclophotocoagulation.
This study was performed to explore whether alveolar macrophage (AM) phagocytosis would be impaired during endotoxemia. Therefore, we characterized in vivo AM phagocytic function in rats following either intravenous (i.v.) or intratracheal (i.t.) administration of lipopolysaccharide (LPS). The i.v. administration of LPS to rats at dosages of 0, 1, 2, and 5 mg/kg showed that increasing LPS doses were significantly associated with increased AM phagocytosis of 198Au colloid (P < .01), decreased recovery of AMs in bronchoalveolar lavage (BAL) (P = .017), no significant differences in neutrophil recovery by lavage (P = .15), or in the concentration of albumin in BAL (P = .14). Across the dosages of LPS administered i.t. (i.e., 0, 1, 5, and 10 mg/kg), there was no difference in AM phagocytosis (P = .29), a significant decrease in AM recovery (P = .002), a significant increase in neutrophil number (P = .01), and little effect on the concentration of albumin (P = .06). Thus, we found that the administration of endotoxin to rats did not impair in vivo AM phagocytic function. In fact, our findings suggest that the i.v. administration of LPS may increase AM phagocytosis of 198Au.
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