The osmium ammine-SO2 staining method for studying the in situ configuration of viral genomes in ultrathin sections of DNA virus infected cells.

The Feulgen-like osmium ammine-SO2 method developed by Cogliati and Gautier (CR Acad Sci Ser D 1973, 276, 3041-3044) stains the DNA at the ultrastructural level. Compared to several other techniques for detecting DNA, this method remains the only one revealing the configuration of the DNA molecules within the cell whatever their compactness. In the present article we summarize the results we obtained with the osmium ammine method in the study of the fate of viral genomes along the infectious cycles in several DNA virus infected cells including adenovirus, herpes simplex virus, simian virus 40 and poxvirus. The results are discussed in relation to the replicative and transcribing activities of viral DNA.

Cytochemical analysis of DNA organization during encapsidation in herpes simplex virus.

The organization of intranuclear Herpes simplex virus DNA in rabbit fibroblast cells infected for 7 hr with HSV type 1 was examined before and during encapsidation by electron microscopic cytochemistry. Most non-encapsidated viral deoxyribonucleoprotein fibers exhibited a non-nucleosomal configuration. Empty capsids within the virus-specific regions of infected nuclei were wrapped with portions of the viral genome which adhered tightly to their surfaces even under conditions that loosened and spread apart other nucleoprotein fibers. During encapsidation, the internal surface of the capsid shell also appeared to bind a part of the viral genome, specifically the outer cage portion, which is detectable in methanol-dehydrated cells. Variations in the amount of DNA within the capsids indicated that the insertion of HSV genome into the capsid is a progressive process. The cage and core cylinder portions of the viral nucleoid appear to form and develop simultaneously. We propose that there may be binding sites on both the external and internal surfaces of the capsid shells which might play a role in the encapsidation process.

Effect of dehydrating agents on DNA organization in herpes viruses.

With routine procedures of Epon- or GMA-embedding and a stain specific for DNA, the nucleoid of mature herpes simplex virus-type 1 (HSV-1) assumes the well-known form of a short, compact, hollow cylinder or torus. A new, more complex organization of DNA filaments in encapsidated HSV-1 was found in infected cells after aldehyde fixation, methanol dehydration, and Lowicryl embedment. We have determined that it is the use of methanol as dehydrating agent that permits visualization of this internal structure. The same new spatial organization of DNA can be seen in Epon and GMA sections when methanol dehydration is used. This organization is lost in a methanol-ethanol sequence of dehydration but can be restored in an ethanol-methanol sequence. Dimethylsulfoxide (DMSO) is the only other agent among several reviewed here which resembles methanol in its effect on HSV-1 DNA. Methanol had the same effect on five subfamilies of the herpes group (HSV-1, HSV-2, CCV, CMV, CTHV) but did not alter the nucleoid ultrastructure in simian virus 40 (SV40) and adenovirus type 5 (Ad 5). Therefore, it may sometimes, but not always, provide additional information about the organization of biological structures.

Differential characteristics of two newly established human breast carcinoma cell lines.

Two human breast carcinoma cell lines, EP and MW, were established in culture from malignant pleural effusions. In addition to producing tumors in antithymocyte serum-immunosuppressed mice, both cell lines showed epithelial characteristics and anchorage-independent growth in soft agar. EP and MW differed in morphology (spindle-shaped versus round), chromosomal mode (hyperdiploid versus near triploid), estrogen receptor content (43.8 versus 5.1 fmol/mg protein), cloning efficiency (0.24 versus 15%), and activities (milliunits/10(6) cells) of creatine phosphokinase (25.7 versus 62.6) and lactate dehydrogenase (346.7 versus 778.5). Electron microscopy revealed that MW cells had more perinuclear filamentous material and more frequent intracytoplasmic vacuole formation than did EP cells. While having no effect on MW cells at the concentrations studied (10(-5) to 10(-11) M), beta-estradiol (10(-7) M) stimulated the growth of EP cells by 106% over the hormone-depleted control. In a variety of systems, EP was consistently the more drug sensitive of the two lines. In vitro, EP was significantly (p less than 0.001) more sensitive to methotrexate, vincristine, and 5-fluorouracil, respectively. In antithymocyte serum-mouse xenografts, EP displayed a greater response to three different dosages of a combination of cyclophosphamide, methotrexate, and 5-fluorouracil. One such dosage (cyclophosphamide, 32.0 mg/kg/day; methotrexate, 13.0 mg/kg/day; 5-fluorouracil, 190.0 mg/kg/day; for 1 day) reduced EP and MW tumor weights to 5.9 and 41% of controls, respectively. These results correlated well with the clinical responses.

Influence of embedding media on DNA structure in herpes simplex virus type 1.

The organization of encapsidated herpes simplex viral DNA in situ was examined by use of the osmium-amine stain specific for DNA. After either formaldehyde or glutaraldehyde fixation the DNA is packaged in a compact toroid without inner structure with Epon or GMA embedment but revealed a complex inner structure with Lowicryl K4M embedment. In the latter there was an inner cylindrical core, 50 X 80 nm, around which were apposed one or more thick filaments of 5-8 nm diameter. Thinner DNA filaments of 3-4 nm diameter form a cage of loose coils around the core with an intervening space of approximately 15 nm. Lowicryl embedding may be considered as a tool to investigate the packaging of viral DNA in virions.

Cytological effects of sulfur and selenium purine analogues on two transplantable hepatomas and on normal renewing cells in mice.

The effects of 50% lethal doses of three purine analogues, 6-methylmercaptopurine riboside ( MMPR ), 6-thioguanosine (TGR), and 6- selenoguanosine ( SeGR ), on mitotic activity in a slow-growing ( SS1H ) and a fast-growing ( BH3 ) transplantable hepatocellular adenoma in C3H/ StW and BUB mice, respectively, were analyzed statistically. No significant difference in response was found between the two benign hepatomas. MMPR alone effectively reduced mitotic activity in the tumors and did so as efficiently on the first day of treatment as on subsequent days of daily i.p. administrations for up to 10 days. TGR alone and SeGR alone were ineffective in reducing the mitotic index significantly below that of controls. When either TGR or SeGR was injected simultaneously with MMPR , the effect on tumor mitosis resembled that of MMPR alone. The reactions of normal cells of the hosts to these agents were analyzed quantitatively in duodenal epithelium with respect to mitotic activity and to the number of cells present in the crypts. Differences between the two strains of mice were small and, for the most part, not significant. MMPR produced a slight but not significant reduction in duodenal mitotic activity and cell number. TGR alone induced significant decreases in both after 3 and 5 days of treatment. SeGR alone had no effect on the duodena . The effects of a combination of SeGR with MMPR on the duodena differed only slightly from MMPR or SeGR alone, but TGR plus MMPR produced greater inhibition of mitosis than did either administered alone. Our results suggest that MMPR may be a promising chemotherapeutic agent against some types of solid hepatocellular tumors in vivo because it can inhibit mitosis in these tumors effectively, rapidly, and continuously, while its inhibitory effect on normal replicating cells of the host intestine occurs more slowly and only with long-sustained treatment.

Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells.

Small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 small nuclear RNAs were detected by ultrastructural immunocytochemistry in the nuclei of isolated rat hepatocytes using Fab fragments of anti-Sm and anti-RNP autoantibodies. Their localization was carried out in normal cells and in cells treated with two drugs, the adenosine analog DRB and CdCl2, which alter the number and distribution of nuclear RNP components. It was found that more precise determination of the distribution of these small RNAs could be obtained by using two complementary procedures in parallel rather than either one alone. They consisted of an indirect immunoperoxidase labeling carried out before embedment and an indirect immunogold labeling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that snRNPs are associated with all extranucleolar perichromatin fibrils and granules and interchromatin fibrils, which confirms that they occur in structures involved in the synthesis and processing of hnRNA. The snRNPs are not associated with nucleolar perichromatin granules induced by DRB, which confirms that there may be two kinds of perichromatin granules. The snRNPs are also associated with the still enigmatic interchromatin granules which apparently do not contain hnRNA but at least in DRB-treated cells, also contain ribosomal RNA.

Effect of quinacrine on nuclear structure and RNA synthesis in cultured rat hepatocytes.

The effects of quinacrine, an antimetabolite which intercalates into DNA, on the ultrastructure of interphase nuclei and on RNA turnover were studied in primary cultures of rat hepatocytes. Procedures included ultrastructural cytochemical staining for ribonucleoprotein and DNA, autoradiography, and measurement of labeled uridine uptake and incorporation. Addition to the culture medium of a nontoxic dose (10 microM for 30 min) reduces the net accumulation of labeled uridine in RNA. This involves first heterogeneous RNA and then ribosomal RNA since their structural precursors, interchromatin fibrils and nucleolar fibrils, respectively, diminish in that order. Intranucleolar chromatin retracts, and perinucleolar chromatin becomes unusually condensed. A toxic dose (50 microM for 30 min) produces greater inhibition of tritiated uridine incorporation in RNA. This precedes and is not due to a drop in uridine uptake into the cells. Toxic doses produce unusually large clusters of interchromatin granules which are embedded in an unusual dense material which stains positively for ribonucleoprotein. Three regions of the chromatin are altered. (a) Perinuclear condensed chromatin retracts from the nuclear envelope, remaining attached by short DNA-containing bridges. (b) The normally dispersed nucleoplasmic chromatin condenses into a stainable network which retracts centrifugally. (c) Perinucleolar chromatin becomes a network of small highly condensed masses or bands interconnected by fibrils which are either decondensed or stretched. These alterations in chromatin structure probably form the basis of quinacrine-impaired nuclear metabolism.

Detection of simultaneous antibody synthesis in plasma cells and specialized lymphocytes in rabbit lymph nodes.

Antibody to horseradish peroxidase was localized by electron microscopic immunocytochemistry in cells of the popliteal lymph nodes of the rabbit after a single injection of antigen with complete Freund's adjuvant and after a second antigen administration. Synthesis of antibody, chiefly of 7S type, occurred simultaneously in two types of cells: large, clear, fixed, typical plasma cells, and small, dense, circulating cells which exhibit morphological characteristics of both small lymphocytes and plasma cells. We call the latter "lymphoplasmacytes" and propose that they arise from small lymphocytes. They secrete antibody by clasmatosis and continue to develop an elaborate endoplasmic reticulum after specific antibody synthesis ceases. In the presence of an additional antigenic stimulation, a second cycle of antibody synthesis may begin around the nucleus in the same cell, with antibody accumulating in the perinuclear space sometimes even before the previously synthesized antibody has been entirely secreted at the cell periphery. On this basis, we propose that the lymphoplasmacyte is a memory cell and that memory and antibody synthesis are two different activities of the same cell. The appearance of a small amount of 19S antibody may be correlated with the presence of a small number of antibody-containing, large lymphocytes.

Ultrastructural localization of antibody in differentiating plasma cells.

Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Ultrathin frozen sections. I. Methods and ultrastructural preservation.

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25-4% glutaraldehyde for 1-4 hr, are washed overnight in buffer at 3 degrees C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70 degrees C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35 degrees C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.