(Ca-2+ + Mg-2+)-Activated ATPase in the plasma membrane mouse liver cells.

1. Purified plasma membranes from dissociated adult mouse liver cells posses a (Ca-2+ + Mg-2+)-stimulated ATPase (EC 3.6.1.3) activity. 2. Enzyme activity is at a maximum with the addition of 0.3 mM Ca-2+ and 3 mM Mg-2+. 3. Using medium devoid of alkali metal ions (Ca-2+ + Mg-2+)-ATPase enzyme activity was observed with Km1 = 0.35 - 10-3 M at a substrate concentration of 1 mM or less and an apparent Km2 = 0.88 - 10-3 M at higher substrate concentrations. 4. In the presence of Na+ and 4 mM ATP, an increase in activity was seen, suggesting the presence of a (Ca-2+ + Mg-2+ + Na+)-activated ATPase. 5. In the presence of both Na+ and K+ the (Ca-2+ + Mg-2+)-dependent enzyme activity was further increased, indicating that a (Ca-2+ +Mg-2+ + K+)-stimulated ATPase may also be present.

Proliferative activity of vervet monkey bone marrow-derived adherent cells.

Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and ñ = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

Transfer of label from 3H-glucose in Digitaria eriantha leaves to the rust fungus Puccinia digitariae Pole Evans.

Digitaria eriantha pentzii was fed 3H-glucose prior to inoculation with uredospores of Puccinia digitariae Pole Evans. Twenty-one hours after inoculation, uptake of label from 3H-glucose by the primary infection structures of P. digitariae was demonstrated employing autoradiography. These results indicate that an exchange of nutrients between host and pathogen occurs very early on in the infection process, during the formation of the primary infection structures. Despite contrary reports that obligate parasites receive no nutrition before establishment of haustoria, this study supports the work of Andrews (Can J Bot 53:1103, 1975), who demonstrated uptake of 3H-glucose label from lettuce cotyledons into the primary and secondary infection vesicles, appressoria, and germ tubes of Bremia lactucae.

Effect of in vivo exposure to benzene on the characteristics of bone marrow adherent cells.

The effect of benzene on the adherent cell population, cultured from the bone marrow of exposed mice was investigated in the presence and absence of hydrocortisone. The adherent CFUs from exposed animals did not differ either in numbers or self-replicate ability to those derived from shown exposed animals. Adherent layers from mice exposed to 100 or 400 pp-benzene were devoid of fat cells regardless of the presence or absence of hydrocortisone. Hydrocortisone was shown to influence the proportion of acid phosphatase-positive cells derived from benzene-exposed animals. Those results suggest that benzene exposure may influence the bone marrow stromal cells.

Comparison of the infectivity of the laboratory strain AD169 and a clinical isolate of human cytomegalovirus to human smooth muscle cells.

The results displayed by human cytomegalovirus (HCMV) IE/E antigen expression and virus release into the supernatant at various times post infection with a clinical isolate (C3/p5) and AD169 laboratory strain of HCMV, illustrated differences in the biology of these viruses on the various cell lines. While AD169 strain was shown to infect fibroblasts efficiently, it showed a low infectivity profile to smooth muscle cells, whereas C3/p5 displayed comparable infectivity characteristics on both cell lines. Neither virus demonstrated propensity for infecting endothelial cells, although passaging of the C3/p5 for additional 11 passages in endothelial cells provided virus capable of infecting endothelial cells efficiently. These results show that HCMV is capable of infecting smooth muscle cells which could be of relevance to the proposed role of HCMV in atherogenesis and provides further evidence on the adaptation of AD169 to fibroblasts.

Increased cytomegalovirus infection of human fibroblast and endothelial cells by electroporation.

Electroporation of fibroblasts and endothelial cells in the presence of HCMV caused an increase in the infection of these cells by the virus. This method could be applied to cells that are difficult to infect by ordinary laboratory methods and also in cases when synchronized infection of the cells by the virus is needed.

Estimation of total DNA in crude extracts of plant leaf tissue using 4',6-diamidino-2-phenylindole (DAPI) fluorometry.

A method of estimating total double-stranded DNA in crude extracts of citrus leaf tissue by evaluating the enhancement of fluorescence intensity of 4',6-diamidino-2-phenylindole (DAPI) was assessed. For pure citrus DNA and citrus leaf tissue crude extract each in the presence of 100 ng/ml DAPI, excitation spectral response curves converged at excitation wavelength of 360 nm. At this excitation wavelength, maximum fluorescence intensity occurred across a range of emission wavelengths from 445 nm to 460 nm. The appropriate excitation and emission wavelengths were shown to be 360 nm and 450 nm, respectively. Fluorescence intensity increased linearly with DNA concentration and non-DNA components of the tissue homogenates had negligible effect on fluorescence at these wavelengths. Sodium dodecyl sulfate (SDS) in the incubation solution resulted in some suppression of DAPI-DNA fluorescence and produced a non-linear response to changing DNA concentration. The method should be applicable to DNA quantitation from crude tissue extracts of any plant species.

Production of a macrophage chemotactic factor by cultured jaagsiekte tumour cells.

The increase of alveolar macrophages in jaagsiekte sheep lungs is not caused by excessive surfactant production but is due to a chemotactic factor secreted by the tumor cells. This factor has a molecular mass in the region of 13 kilodaltons, is stable at 56 degrees C but labile at 100 degrees C and, being sensitive to proteases, indicates that it is a small protein molecule.

Demonstration of growth-inhibitory as well as growth-stimulatory factors in medium conditioned by lung lavage cells stimulated with a chemotactic factor secreted by jaagsiekte tumour cells.

Both growth-inhibitory and growth-stimulatory factors were detected in vitro in medium from chemotactically stimulated cultures of lung lavage cells. The macrophage component of the lavage cells was found to produce a growth stimulatory factor that was replaced by a growth inhibitory factor following chemotactic factor stimulation.

The localization of a Mason-Pfizer monkey virus-related antigen in jaagsiekte tumour tissue and cell lines.

Mason-Pfizer monkey virus-related antigen was detected in 3 out of 5 jaagsiekte lungs examined using a direct immunoperoxidase staining technique with anti-MPMV p27 serum. Most of the antigen was localized in the alveolar lumina of the lesions. The reaction was further characterised on immune blots and found to involve a protein with a molecular mass of 29 000 daltons (JSRV p29). JSRV p29 antigen was also detected in 2 jaagsiekte cell lines.

The morphology and morphogenesis of jaagsiekte retrovirus (JSRV).

Jaagsiekte retrovirus ( JSRV ) was recently shown to be the aetiological agent of jaasiekte (ovine pulmonary adenomatosis). The morphogenesis of JSRV was studied in jaagsiekte tumour tissue. Intracytoplasmic particles, often associated with centrioles, were found in tumour cells. JSRV budded from tumour cells with a complete core which appeared to mature during the budding process. Extracellular particles were found in the alveolar lumen. Immature extracellular particles were rare. Mature extracellular JSRV was membrane-bound and had a slightly eccentric nucleoid with an electron-dense perinucleoidal space. In negatively stained preparations of JSRV the envelope was covered with spikes. JSRV is morphologically distinct from all known retroviruses.

The possible involvement of immunosuppression caused by a lentivirus in the aetiology of jaagsiekte and pasteurellosis in sheep.

A South African isolate of ovine lentivirus was shown to cause a mild immunosuppression in sheep, reflected by a reduced delayed hypersensitivity reaction. This effect, measured in terms of skin swelling after intradermal inoculation with tuberculin, showed a positive linear relationship with the latency period before the appearance of jaagsiekte symptoms in animals co-infected with JSRV, as well as with the activity of monocytes. In a parallel study, increased susceptibility of lentivirus-infected sheep to infection with Pasteurella haemolytica was demonstrated. It is concluded that the lentivirus may play an enhancing role in both viral and bacterial infections of sheep by compromising the host's cellular immune response.

Alterations in the expression of the cytomegalovirus-induced cytopathogenic effect in fibroblasts by aflatoxin B1.

Aflatoxin B1 has been shown both to promote and to alter the expression of the cytopathogenic effect observed when human fibroblasts are challenged with human cytomegalovirus (CMV). Although the cells become round, as is the characteristic effect of this virus on fibroblasts, multinucleate cells are seen to arise from cell fusion within 48 h after virus addition.

Energy-dependent accumulation of Ca2+ by human embryonic lung fibroblasts.

Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions.

Altered polyamine concentrations in cytomegalovirus-infected human cells.

Polyamines have a regulatory effect on DNA and RNA synthesis and their levels are elevated in rapidly growing cells, including lymphoblasts. However, as shown in the current experiments, exposure to cytomegalovirus (CMV) reduces the polyamine levels in these cells, suggesting that the virus interferes with their metabolism. Studies have shown that the activity of ornithine decarboxylase is increased in CMV-infected fibroblasts and that there is an increased conversion of putrescine to spermidine and spermine. Thus it may be expected that the concentration of these molecules would increase in the infected cell. However, the results presented here demonstrate that only the concentrations of putrescine and spermidine are increased, the spermine concentration decreasing with infection.

The effect of arginine deprivation on the cytopathogenic effect and replication of human cytomegalovirus.

Arginine is necessary for the development of the cytopathogenic effect of human cytomegalovirus in human embryonic fibroblasts. It is also required, though in greater concentrations, for the production of infective virions, the requirement being at an early stage of replication. Inhibitor studies suggested that this block in replication caused by arginine deficiency was prior to the formation of viral DNA. Withdrawal of arginine from the medium 24 or 48 hours after infection resulted in a decline in virus production indicating that the continued presence of the amino acid is necessary for constant virus production. Infected cultures deprived of arginine could be stimulated to produce cytopathic effects and infective virions by replacement of the amino acid even eight days after inoculation, demonstrating that the information for cytomegalovirus replication remains intact within the cell. This establishment of latency in vitro may be related to the ability of the virus to establish a similar state in vivo.

Increased ability of human embryonic fibroblasts to accumulate Ca2+ due to cytomegalovirus infection.

Cytomegalovirus infection of fibroblasts enhances the ability of these cells to accumulate Ca2+. This functional alteration precedes the development of the cytopathic effect, and it is proposed that increased intracellular Ca2+ concentration may initiate the rounding phenomenon. Changes in the aggregative behaviour and the antigenicity of the infected cells were seen to accompany this functional alteration.

A scanning electron microscope study of the sequential changes in morphology occuring in human fibroblasts placed in suspension culture.

Trypsinized human embryonic fibroblasts placed in suspension culture demonstrated a distinct sequence of changes in the topography of the underlying surface and in the nature and distribution of processes. After 4 h the typical cell showed a rippled surface from which protruded a few small 'blebs' and several microvilli. This appearance was very similar to that displayed by confluent monolayer cultures of fibroblasts. The fibroblasts survived in suspension for at least 8 h and, as their topography appeared to revert to normal, suspended cells may prove a reasonable system for studying early alterations in the surface appearance and metabolism of fibroblasts following the addition of various agents, including viruses, provided they are allowed 4 h to recover from the trypsin.

Fusion of cytomegalovirus infected fibroblasts to form multinucleate giant cells.

Multinucleate giant cells have been observed to arise by cell fusion in cultures of human embryonic fibroblasts infected with human cytomegalovirus (CMV), strain AD 169. The infected cells develope intranuclear and intracytoplasmic inclusions and began to fuse three to four days postinfection when the majority of the cells were showing signs of a cytopathic effect.

Early alterations in the morphology of cytomegalovirus-infected human fibroblasts.

Cytomegalovirus-infected human fibroblasts developed large numbers of fine microvilli within 90 min after addition of virus. The structure of these microvilli gradually changed as the infection proceeded, although the infected cells were easily detected by their large number of processes. These processes may be involved in the increased transport of ions and nutrients into the infected cell.