The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylateor phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 ~). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxlde) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that--notable in the case of glutaraldehyde--was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succlnic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.In this paper seven different organic substances are introduced as fixatives for electron microscopy and cytochemistry. The work, as a means of satisfying the requirements of each, has taken into consideration the difficulties experienced when combining the two fields (1, 2). At the present time, osmium tetroxide (3) and, to a lesser extent, potassium permanganate (4) are the fixatives of choice that are currently used in morphological studies with the electron microscope. These reagents provide excellent cytological fixation, and also add density to some of the reactive components of the cells. However, their use in cytochemistry is very limited since they are heavy metal-containing, oxidizing reagents that completely destroy enzymatic activities except after very short periods of fixation (5-9).

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Periwinkle alkaloids in very low concentrations cause an intracytoplasmic sequestration of microtubule protein in the form of symmetrical, microtubular bodies. These crystals, which may measure up to 8 in length, appear within 30 min in L-strain fibroblasts in vitro, but they increase in incidence and size with time of exposure to the alkaloids. Similarly, if exposed to these compounds, human leukocytes in vitro contain identical crystalline structures. Neither colchicine nor puromycin prevents the formation of these bodies; the latter compound, however, retards crystal growth.Periwinkle (Vinca) alkaloids, helpful in the therapy of neoplastic diseases, are known to affect cell division through action similar to that of colchicine (1). Overdosage of these drugs damages primarily organs known to be rich in microtubules, such as neurons (2). Previous studies carried out by us showed a rapid (but, for vinblastine, reversible) decrease and disappearance of the birefringence of the mitotic spindle of living eggs of a marine annelid during perfusion with these compounds; electron microscopy confirmed the attrition and disappearance of microtubules by such treatment (3). The same alkaloids, when used in a study of microtubules of polymorphonuclear leukocytes, again made the microtubules disappear but produced in these cells intracellular bodies of singular regularity which fulfilled the criteria for definition of a crystal. This observation led to a systematic examination of the effects of the Vinca alkaloids vinblastine and vincristine sulfate on microtubules in mammalian cells. The results of these studies are reported here. Preliminary findings were reported briefly elsewhere (4). MATERIALS AND METHODSL-strain fibroblasts and human leukocytes were studied. The fibroblasts in tissue culture (5) were exposed for 1/-24 hr to the periwinkle alkaloids which were added directly to the medium, resulting in final alkaloid concentrations ranging from 4 X l0-4 to 1 X l0 -5 M. These cells, as well as control cultures, were subsequently fixed by addition of isotonic 2 % phosphate-buffered glutaraldehyde solution (pH 5.9, temperature 24°-30°C) at a ratio of 1:1, followed within 5-15 min by centrifugal separation of the cells from the incubation mediumfixative mixture (6). Fibroblasts, but not leukocytes, were fixed also at 4°C and pH 7.4. The pellet was then overlaid with the above described fixative. Osmification (7) was carried out 1-240 hr later, after a 12 hr rinse of the cell pellet in isotonic phosphate buffer. The tissue embedded in epoxy resin (Maraglas) (8) was examined in an Elmiskop 1. Similarly, suspensions of human leukocytes (9) were incubated at 37"C for 2-3 hr in a phosphate-buffered Krebs-Ringer medium with 12% autologous serum, with or without the alkaloids (vinblastine 2.5 X 10 -5 M, vincristine 1 X 10 -4 M).In other series of experiments, colchicine (final concentration 1-4 X 10-5 M) was added to the fibro-95 on

Electron‐microscopic examination of normal bronchial epithelium and mucous glands has revealed the presence of cells similar in appearance to Kultschitzky (argentaffin) cells in the intestinal canal. The cells, which possess long cytoplasmic processes, are present in small numbers at all levels of the bronchial tree and are found adjacent to the basement membrane. They contain characteristic cytoplasmic granules referred to as neurosecretory‐type granules, which are similar to those present in intestinal argentaffin cells, adrenal medullary cells and sympathetic nerve endings. Eighteen bronchial carcinoid tumors and 22 oat‐cell pulmonary cancers have also been found to contain similar neurosecretory‐type granules in the tumor cell cytoplasm. On the basis of light‐microscopic similarities and the production of similar common patterns of hormonal disturbances and the possession of similar ultramicroscopic features, it is suggested that oat‐cell pulmonary cancer and bronchial carcinoid tumors are closely related. They may be, respectively, the malignant and locally malignant form of tumors derived from Kultschitzky‐type cells normally found throughout the bronchial tree.

A procedure for the isolation and in vitro cultivation of endothelial cells from the microvessels of the newborn human foreskin dermis is described. The epidermis was removed from foreskin tissue using a Castroviejo keratotome (0.1 mm shim). Endothelial cells were released from the dermal vessels by trypsinization of 5 mm2 sections of dermis at 37 degrees C for 40 min. Cells were expressed into Minimal Essential Medium (MEM) containing 10% pooled human serum, collected by centrifugation and plated onto either a plain plastic or a fibronectin treated culture surface. In primary culture the rate of endothelial cell proliferation was dependent upon serum type and concentration being optimal in 50% pooled human serum. High serum concentration in combination with pretreatment of the culture surface with fibronectin was required for maximal proliferation rate, for the cells to achieve confluence and for subcultivation. Primary and subcultured cells were characterized as endothelial by light microscopic, immunofluorescent (Factor VIII associated protein) and ultrastructural (Weibel-Palade body) criteria.

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Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (02) to H202 and 02Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of 02 and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin Sc gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an 02-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H202 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor but positive effect on mean longevity for several strains.