Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

Elemental microanalysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease

A scanning proton microprobe has been used for the elemental microanalysis of individual fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. The cells were cultured on a thin ultra-clean nylon foil and retained on that surface for analysis. The focused high-energy proton beam was used to irradiate selected individual cells and elemental information was derived from X-ray and backscattered proton data. The sensitivity of the scanning proton microprobe to trace concentrations of heavy elements has allowed this elemental information to be used to identify individual cells as being either normal or a Menkes' mutant. The cell identification was based on the application of discriminate analysis to a data set formed from the ratios of copper to each of the macroelements present in the cell. This method of cell identification offers the promise of rapid diagnosis of Menkes' disease.     

Poly-β-Hydroxyalkanoate Exert a Protective Effect Against Carbon Starvation and Frozen Conditions in <Emphasis Type="Italic">Sphingopyxis chilensis</Emphasis>

Several bacterial species have developed physiological response to avoid the cellular damage when are exposed to carbon starvation or frozen stress. For example survival to inanition has been related to endogenous substrates consumptions. The aim of this study was to evaluate if poly-β-hydroxylkanoates (PHA) consumption enable Sphingopyxis chilensis S37 to survive under carbon starvation or frozen condition. Bacterial cells were grown in R₂A broth for 48 h, and suspended in mineral saline solutions, without carbon source. The cellular suspension was incubated for 48 or 120 h at 30°C, followed by a frozen period of 48 h at −20°C, and viable bacterial cells were evaluated by the microdrop method. The proportions of cells with PHA were also determined by flow cytometry using Nile Red dye. The results indicate that S. chilensis were able to survive under carbon starvation and frozen conditions. Simultaneously, a decrease in the number of cells containing PHA, and a decrease in the biovolume of the cells (c.a 2.5 times) were also observed under these conditions. The results suggest that consumptions of PHA contributed to the surviving of S. chilensis under frozen stress.     

A New Technique Facilitating Studies of Scant Cell Specimens

A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells. The cells are concentrated by centrifugation. The cell pellet is fixed, frozen and embedded in plastic. Thin (2-μm) sections are cut from the plastic. Thus, each cell may appear in several sections and many slides can be made from a single specimen. The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.     

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5′-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent

Summary A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing¹⁴¹Ce³⁺ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5′-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce³⁺ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.     

IMPROVED TECHNIQUES FOR THE PREPARATION OF ULTRATHIN FROZEN SECTIONS

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.     

A New Technique Facilitating Studies of Scant Cell Specimens

A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells. The cells are concentrated by centrifugation. The cell pellet is fixed, frozen and embedded in plastic. Thin (2-μm) sections are cut from the plastic. Thus, each cell may appear in several sections and many slides can be made from a single specimen. The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.     

Diaminobenzidine cytochemistry in cryo-ultramicrotomy for the detection of peroxidases.

Rat erythrocytes and lymphoid cells were fixed by glutaraldehyde and encapsulated in bovine serum albumin plus rabbit IgG globulins for cryo-ultramicrotomy. A technical procedure is described by which endogenous peroxidases of erythrocytes in ultrathin frozen sections were detected by hydrogen peroxide and diaminobenzidine as hydrogen donor. Modifications of this classical cytochemical procedure proved also useful in cryoultramicrotomy for immunoperoxidase labeling of antigenic determinants in rabbit IgG globulins which have been crosslinked within the supporting matrix of cells.     

Morphological,cytochemical, and culture study of normal human bone marrow frozen at −196 °C

A morphological, cytochemical, and agar culture study was carried out on samples of bone marrow that had been taken from 13 normal individuals and frozen at ?196 °C. The cryoprotective agent (DMSO) was removed by slow or rapid dilution. A large number of the thawed cells appeared to have been destroyed or exhibited vacuoles in the nucleus and cytoplasm, as well as numerous short cytoplasmic evaginations. The few mature cells of the neutrophilic line that had survived contained only rare, if any, specific granules; only the lymphocytes were apparently unaffected. There was a reduction in, or an irregular distribution of, the positive reactions to PAS, peroxidase, naphthol-ASD-chloro-acetate esterase, and Sudan black exhibited by cells of the neutrophilic line, and the lysozyme activity of the neutrophils and the monocytes was affected. Where the DMSO had been removed by slow dilution these changes were less severe and diffuse, the percentage of trypan blue-negative cells was higher, and a much larger number of the colony-forming cells were recovered, this number being constantly reproducible. Similar results were obtained by comparing the two dilution methods on thawed-out specimens of peripheral blood from five patients with chronic myeloid leukaemia. With both types of dilution very few cluster-forming cells were recovered and no spontaneous formation of clusters or colonies was observed. The results suggest that marrow frozen at ?196 °C and treated after thawing by slow dilution is suitable for marrow-transplant experiments, as a control for the agar culture of fresh marrow samples and for the stimulating activity of the feeder layers of peripheral blood leucocytes.     

Copper Resistance and Accumulation in the Zygomycete Mucor rouxii

Two copper-resistant (Cop^r) mutants, strains P1 and P3, were obtained from the dimorphic fungus Mucor rouxii. They were characterized as to their ability to take up copper in a growth medium supplemented with this metal ion. Detection of copper by linear sweep striping voltammetry in cell walls and in the cell wall-free fraction of disrupted cells revealed a higher content of the metal in both mutant fractions, as compared with those of the copper-sensitive (Cop^s) parental strain. Copper binding by M. rouxii growing cells was also studied through the use of a cytochemical method based on the compounds neocuproine (NCP) and sodium diethyldithiocarbamate (DTC). This method indicated that the P1 Cop^r strain accumulated more metal than the parental Cop^s strain, both on the cellular surface and in the intracellular milieu. Received: 30 January 1996 / Accepted: 7 March 1996     

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.     

Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro

Summary The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen at 1°C or 20°C/min and thawed immediately (20°C/min), a variety of ultrastructural alterations occurred. Membraneous structures were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first evident at 0°C, progressed to high amplitude swelling by −10°C (frozen). In addition, the rough endoplasmic reticulum was dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at −15°C. These included separation and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron microscopy revealed perforated plasma membranes in some cells at −10°C (frozen) and in most cells by −20°C. Cultures frozen at 20°C/min revealed mostly the same ultrastructural damage noted at 1°C/min except a higher percentage of cells exhibited alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing. Increased swelling in cytoplasmic organelles coupled with nuclear alterations at −15°C resulted in a decreased survival rate and release of significant quantities of LDH by −20°C. No unique morphological changes were temperature specific, but the total number of cells that displayed alterations increased as temperature decreased. The views, opinions or findings, or both, contained in this report are those of the authros and should not be construed as indicative of an official Department of the Army position, policy, or decision unless so designated by other official documentation.     

Cytochemical localization of pectinase activity in laticifers ofNerium oleander L.

Summary A method is described for the cytochemical localization of pectinase activity at the ultrastructural level. The procedure involves the use of Benedict's reagent to form an electron-dense copper precipitate when reacted with reducing sugars liberated from exogenously supplied pectin. Using this technique, pectinase activity was examined in the nonarticulated, branched laticifers ofNerium oleander. Electron opaque crystalline deposits indicating the presence of pectolytic enzymes were identified in laticifer central vacuoles. Smaller amounts of reaction product were distributed along the middle lamella between laticifers and adjacent cells. This report represents the first direct evidence for the involvement of pectinase in intrusive growth of nonarticulated laticifers.     

Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.     

Diaminobenzidine cytochemistry in cryo-ultramicrotomy for the detection of peroxidases

Summary Rat erythrocytes and lymphoid cells were fixed by glutaraldehyde and encapsulated in bovine serum albumin plus rabbit IgG globulins for cryo-ultramicrotomy. A technical procedure is described by which endogenous peroxidases of erythrocytes in ultrathin frozen sections were detected by hydrogen peroxide and diaminobenzidine as hydrogen donor. Modifications of this classical cytochemical procedure proved also useful in cryo-ultramicrotomy for immunoperoxidase labeling of antigenic determinants in rabbit IgG globulins which have been crosslinked within the supporting matrix of cells.Supported by grants of the Deutsche Forschungsgemeinschaft (SFB 136, publ. No. 18) Bonn, Federal Republic of Germany     

Freeze-fracture cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules

Freeze-fracture cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freeze-fracture cytochemistry with special reference to a new cytochemical labeling of replicas, the detergent-digestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freeze-fracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunocytochemistry. Freeze-fracture cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.     

Copper is taken up efficiently from albumin and alpha2-macroglobulin by cultured human cells by more than one mechanism

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and ₂-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3–6 µM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from ₂-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65–80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100–500 µM) inhibited copper uptake from albumin by 20–30% in both cell types and that from ₂-macroglobulin by 0–30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. ₂-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified. transcuprein; uptake kinetics; iron competition; silver competition; HepG2 cells; PMC42 cells     

Does the tube of a benthic chironomid larva play a role in protecting its dweller against chemical toxicants?

A laboratory culture of an Israeli benthic midge, Chironomus luridus, was exposed to two chemicals: copper and monochloramine. The objective of this study was to determine the protective nature of Chironomus' larval tube. Three experimental conditions were tested: larva with sand tubes, with silt tubes and without tubes. Larvae without tubes were significantly more sensitive to copper and chloramine than larvae that had sand or silt as tube building substrate. The tubes protected the insects against chemicals throughout 14 days of exposure time. Silt tubes had higher protective value than sand tubes, especially when exposed to copper for a short period of time (LC₅₀/ 24 h, with silt, sand, or none: 80.0, 7.0 and 3.4 mg l^–1 copper, respectively). C. luridus seemed to be better protected against copper than against chloramine (LC₅₀/ 24 h, with silt, sand, or none: 12.2, 6.4 and 3.7 mg l^–1 chloramine, respectively). The acute toxicity of copper to chironomid larvae was investigated using a cytochemical method. Larva in silt tubes had significantly higher non-specific esterase activity in midgut cells than larvae without tubes. We conclude that, in addition to its role in feeding, respiration and anti-predation shelter, the C. luridus tube protects its inhabitant from toxic substances.     

Tumor cellular proteasome inhibition and growth suppression by 8-hydroxyquinoline and clioquinol requires their capabilities to bind copper and transport copper into cells

We have previously reported that when mixed with copper, 8-hydroxyquinoline (8-OHQ) and its analog clioquinol (CQ) inhibited the proteasomal activity and proliferation in cultured human cancer cells. CQ treatment of high-copper-containing human tumor xenografts also caused cancer suppression, associated with proteasome inhibition in vivo. However, the nature of the copper dependence of these events has not been elucidated experimentally. In the current study, using chemical probe molecules that mimic the structures of 8-OHQ and CQ, but have no copper-binding capability, we dissected the complex cellular processes elicited by 8-OHQ–Cu and CQ–Cu mixtures and revealed that copper binding to 8-OHQ or CQ is required for transportation of the copper complex into human breast cancer cells and the consequent proteasome-inhibitory, growth-suppressive, and apoptosis-inducing activities. In contrast, the non-copper-binding analogs of 8-OHQ or CQ blocked the very first step—copper binding—in this chain of events mediated by 8-OHQ–Cu or CQ–Cu.