Aqueous solutions of lead salts (1, 2) and saturated solutions of lead hydroxide (1) have been used as stains to enhance the electron-scattering properties of components of biological materials examined in the electron microscope. Saturated solutions of lead hydroxide (1), while staining more intensely than either lead acetate or monobasic lead acetate (l , 2), form insoluble lead carbonate upon exposure to air. The avoidance of such precipitates which contaminate surfaces of sections during staining has been the stimulus for the development of elaborate procedures for exclusion of air or carbon dioxide (3, 4). Several modifications of Watson's lead hydroxide stain (1) have recently appeared (5-7). All utilize relatively high pH (approximately 12) and one contains small amounts of tartrate (6), a relatively weak complexing agent (8), in addition to lead. These modified lead stains are less liable to contaminate the surface of the section with precipitated stain products. The stain reported here differs from previous alkaline lead stains in that the chelating agent, citrate, is in sufficient excess to sequester all lead present. Lead citrate, soluble in high concentrations in basic solutions, is a chelate compound with an apparent association constant (log Ka) between ligand and lead ion of 6.5 (9). Tissue binding sites, presumably organophosphates, and other anionic species present in biological components following fixation, dehydration, and plastic embedding apparently have a greater affinity for this cation than lead citrate inasmuch as cellular and extracellular structures in the section sequester lead from the staining solution. Alkaline lead citrate solutions are less likely to contaminate sections, as no precipitates form when droplets of fresh staining solution are exposed to air for periods of up to 30 minutes. The resultant staining of the sections is of high intensity in sections of Aralditeor Epon-embedded material. Cytoplasmic membranes, ribosomes, glycogen, and nuclear material are stained (Figs. 1 to 3). STAIN SOLUTION: Lead citrate is prepared by

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THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY

The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.

Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.

Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other types of cancer, independent of previous biological knowledge.

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Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction

At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

/pdf/free-radicals-in-the-physiological-control-of-cell-function-g3w3iyr0ph.pdf

Free Radicals in the Physiological Control of Cell Function

A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy

The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique. In animals killed as early as 90 sec after injection, reaction product was found on the brushborder membranes and in the apical tubular invaginations. From the latter structures it was transported to the apical vacuoles, in which it was progressively concentrated to form protein absorption droplets. The method, which employs 3,3'-diaminobenzidine as oxidizable substrate, gives sharp localization and is sensitive. This system is advantageous in studying the early stages of renal tubular protein absorption, since small amounts of protein on membranes and in tubules and vesicles can be detected easily. The method also appears promising for studying protein transport in a variety of other cells and tissues.

The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique.

219 Ins thue exheussivelv ussed Koelle ;onsd Fniederuwald histociueuusbcal nuetisod Ion cisolinsestersose, amid its several ns(odificat ions (see Pearse, Histocheun istry, 2moded., p. 478, Little, Brown & Co., Bostons, 1960), thse tiuiociso)biose ester used as substrate is hydroiysed by cholimsestenosse, oond the liberated hhiocisohinue is captured h)\ Cu icns, pnecipitatirug sos colorless copper thiocisolbuse. The latter is convented to broot-nisis CuS by treatiuseust ot-iths yellow aissosuonununsu sulfide. Voonious steps irs thse nusethod hsovebee-mu criticized (Ion discussion see Pearse, ibid.). Lsorge useedle-like (‘rystals (occur l)articUbsonly sot sites oof isigh activity, at higher pH levels, oomsd wihis cvenlcomsg incusbatioru. Under somuse cmcuuuushsonuces, the crystals coons soppanently increase ins sizeby nsonu -euszvussat besoccnetions . (Mabnsgren sonod Syivo mu, J. Histocheun., 3: 441, 1955). Tise so(lvsonstages ccl thie presemutly described nssethsood sore: colour is prooduced directly at the site of eoozyusuatic activity, facilitating estinsation of h.hue ooptinuuius imscusi)soti000s tinse; the l)necipitate is finselv grssusuulan, so) lhooot, eveos witis prolonged incusbosoh booms, needle-like deposits are not ob)served; instemuse cnol(on sond contnsost coon conseqtiently be l)rodltlce-ib, somuch sites 0)1 low activity ussoone easily detected. TIse bsosis ol hbue issethod is that hisiochcdimseis l)elieved Its recluse-c ferricysonside to lerrocyoonide prefenenstisolly. The lootter coomiuhimues with Cu#{176} iomus to lonuss the iossolusb)le copper femnocyoonide (Hsotcheht’s l3rcowns). Tise Cti ions in the ussedium sore cocosuplexetl wit Is cit nsote too prevent fonisuation oof copper fennicysonside. Fcon best restults I huenose-I hucod is penlconsused as fooiioows: boloocks cof tissue sore fixed overmuiglut in coold 10#{176} Ionossooiims coomsh.ooirsbrsg 1% CaCI2 aoud are tbue-ms kept ins 0.88 11 sucrose-1’ guns arabic (Holt et al., .1. Biophys. Biocheno. (‘ytol., 7, 383, 1960). TIne blocks are wasised for five nusinutes in distilled ot-sote-r, blotted dry, sonuch sore quick-frozen in iso-

A "direct-coloring" thiocholine method for cholinesterases

Horseradish peroxidase was administered to mice by intravenous injection, and its distribution in cerebral cortex studied with a recently available technique for localizing peroxidase with the electron microscope. Brains were fixed by either immersion or vascular perfusion 10–60 min after administration of various doses of peroxidase. Exogenous peroxidase was localized in the lumina of blood vessels and in some micropinocytotic vesicles within endothelial cells; none was found beyond the vascular endothelium. Micropinocytotic vesicles were few in number and did not appear to transport peroxidase while tight junctions between endothelial cells were probably responsible for preventing its intercellular passage. Our findings therefore localize, at a fine structural level, a "barrier" to the passage of peroxidase at the endothelium of vessels in the cerebral cortex. The significance of these findings is discussed, particularly with reference to a recent study in which similar techniques were applied to capillaries in heart and skeletal muscle.

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Fine structural localization of a blood-brain barrier to exogenous peroxidase

The transendothelial passage of horseradish peroxidase, injected intravenously into mice, was studied at the ultrastructural level in capillaries of cardiac and skeletal muscle. Peroxidase appeared to permeate endothelial intercellular clefts and cell junctions. Abnormal peroxidase-induced vascular leakage was excluded. Neutral lanthanum tracer gave similar results. The endothelial cell junctions were considered to be maculae occludentes, with gaps of about 40 A in width between the maculae, rather than zonulae occludentes. Some observations in favor of concurrent vesicular transport of peroxidase were also made. It is concluded that the endothelial cell junctions are most likely to be the morphological equivalent of the small pore system proposed by physiologists for the passage of small, lipid-insoluble molecules across the endothelium.

/pdf/the-ultrastructural-basis-of-capillary-permeability-studied-5dsina2wex.pdf

Morris J. Karnovsky

Papers

A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron-microscopy

The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique.

A "direct-coloring" thiocholine method for cholinesterases

Fine structural localization of a blood-brain barrier to exogenous peroxidase

The ultrastructural basis of capillary permeability studied with peroxidase as a tracer