In vitro growth and chromosome constitution of placental cells. I. Spontaneous and elective abortions

Placental cultures from chromosomally normal spontaneous abortions, as well as placental and fetal cultures from chromosomally normal elective abortions, were established, and the growth and cytogenetic constitution of the cultures were compared. Fetal cultures grew well and remained chromosomally stable over the entire 100-day period of observation. In contrast, placental cultures were relatively short-lived, many becoming senescent and dying within the time of observation. This was particularly marked in cultures established from spontaneous abortions. Five of the 18 cultures established from spontaneous abortions, but only 1 of the 24 cultures from elective abortions, developed chromosomally abnormal clones. The emergence of such clones was unrelated to the senescence of the cultures.     

Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex

Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary acidic protein, and the glutamate transporters EAAT1 and EAAT2. The cultures prepared from 7-day-old animals were additionally characterized metabolically using (13)C-labeled glucose and glutamate as well as (15)N-labeled glutamate as substrates. All types of cultures exhibited pronounced immunostaining of the astrocyte marker proteins. The metabolic pattern of the cultures from 7-day-old animals of the labeled substrates was comparable to that seen previously in astrocyte cultures prepared from new-born mouse brain showing pronounced glycolytic and oxidative metabolism of glucose. Glutamate was metabolized both via the GS pathway and oxidatively via the tricarboxylic acid cycle as expected. Additionally, glutamate underwent pronounced transamination to aspartate and alanine and the intracellular pools of alanine and pyruvate exhibited compartmentation. Altogether the results show that cultures prepared from cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes.     

Effect of ethanol on activity of the plasma-membrane ATPase in, and accumulation of glycine by, Saccharomyces cerevisiae

The pH optimum of the ATPase activity in plasma membranes from Saccharomyces cerevisiae NCYC 431 from 8 h cultures was around 6.5 and that in membranes from organisms from 16 h cultures near 6.0. The Km[ATP] of the enzyme was virtually unaffected by the age of the culture from which organisms were harvested, although the Vmax of the enzyme in membranes from organisms from 8 h cultures was higher than that for organisms from 16 h cultures. Ethanol non-competitively inhibited ATPase activity in membranes, although the inhibition constant for the enzyme from organisms from 8 h cultures was lower than that from organisms from 16 h cultures. Glycine accumulation by the general amino acid permease was non-competitively inhibited by ethanol. Inhibition constants were virtually the same for glycine uptake by deenergized organisms from 8 h and 16 h cultures, but under energized conditions the value was greater for organisms from 16 h rather than 8 h cultures. The data indicate that inhibition of plasma-membrane ATPase activity by ethanol could account, at least in part, for inhibition of glycine accumulation by ethanol.     

Resistance of Salmonellae Isolated in 1959 and 1960 to Tetracyclines and Chloramphenicol

The resistance of cultures of Salmonella typhimurium to tetracyclines and chloramphenicol has been examined periodically. Although none of 200 cultures isolated prior to 1948 was resistant to the antibiotics, 5% of 100 cultures from man and 9% of 100 cultures from fowls which were isolated in 1956 and 1957 were resistant to tetracyclines. Among 158 cultures isolated from man and 100 cultures isolated from fowls in 1959 and 1960, 13.9 and 29%, respectively, were resistant to tetracyclines. In the last series, cultures resistant to chloramphenicol were found for the first time. Among 150 cultures of other Salmonella serotypes from man and 137 similar cultures isolated from fowls in 1959 and 1960, 5.3 and 8%, respectively, were found resistant to tetracyclines. There is no obvious explanation for the higher percentage of resistant strains occurring in S. typhimurium than in other serotypes.     

Trisporic Acid Biosynthesis in Separate Plus and Minus Cultures of Blakeslea trispora: Identification by Mucor Assay of Two Mating-Type-Specific Components

Separate plus and minus cultures of Blakeslea trispora synthesize small amounts of trisporic acids under specific conditions. These amounts are expressed as a percentage of the trisporic acids (50 mg/liter of medium) synthesized by mixed plus-minus cultures in 5 days. Plus cultures, without additives from minus cultures, synthesize 0.1% trisporic acids. Plus cultures synthesize 0.4% trisporic acids when stimulated by M-factor, a mating-type-specific component synthesized by minus cultures. Minus cultures, without additives from plus cultures, do not synthesize even 0.0001% trisporic acids. Minus cultures synthesize 1% trisporic acids when stimulated by P-factor, a mating-type-specific component synthesized by plus cultures. Minus cultures synthesize M-factor when stimulated by pi, a component synthesized by plus cultures. We speculate that (i) minus cultures synthesize a component, mu, which stimulates P-factor synthesis in plus cultures, and (ii) both M-factor and P-factor are precursors of trisporic acids.     

The size of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TSC) differs from that in fibroblasts

Summary Cell cultures were derived from angiofibromas of three patients with tuberous sclerosis (TSC), from the unaffected skin of these patients, and from the skin of five healthy donors. The length distributions of the small polydisperse circular DNA (spcDNA) fraction of these cell cultures were then analyzed. Nearly half the spcDNA molecules from the angiofibroma cultures were longer than 0.4 m, whereas only about 7% exceeded this threshold in the spcDNA preparations from the skin fibroblast cultures. The percentage of the larger size class of spcDNA showed an increase at higher numbers of in vitro passages in all three types of cultures, but this effect was much more conspicuous in the angiofibroma-derived cultures than in those from the skin fibroblasts. An age-dependent increase in the overall amount of spcDNA was only seen in the angiofibroma-derived cultures. Our earlier finding of elevated amounts of spcDNA in angiofibroma cultures was confirmed in cultures from an additional TSC patient.     

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

It has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.     

The patterns on chondrogenesis of cells from facial primordia of chick embryos in micromass culture

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.     

Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.     

Occurrence of collagen-degrading microorganisms in associations of mesophilic heterotrophic bacteria from various soils

A total of 437 bacterial cultures was isolated from various soils and sewage water that were tested for the ability to decompose reconstituted collagen. This activity was found in 6.6% of the cultures isolated from sewage water, 15% of the cultures from organic horizons of the spruce growth soil, 30% of the cultures from the meadow soil, 29% of the cultures from the vegetable field soil and in 37% of those isolated from garden soil. The capability to produce collagenolytic enzymes does not appear to be rare among soil bacteria.     

The morphological and functional characteristics of human aortic endothelium. II. Cellular polymorphism and the incorporation of 3H-thymidine in a culture]

The content of multinuclear endothelial cells and the ability of cells to incorporate 3H-thymidine were studied in primary cultures isolated from zones of low (LP) and high (HP) probability of atherosclerosis of adult human aortas. It was found that the percentage of multinuclear EC was at mean 2-fold higher in cultures from HP zones compared to LP zones of the same vessels. In primary cultures and in the first passage cultures only small mononuclear EC were able to incorporate 3H-thymidine. A significant decrease in the thymidine index (TI) was found only in cultures from HP zones of atherosclerotic aortas. In cultures of EC from the LP zones of these aortas the TI was as high as in cultures from the LP and HP zones from grossly normal vessels.     

Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine ( Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non-embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non-embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean ( Vicia faba ) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS-PAGE and non-denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light-yellow non-embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.     

Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

In Vitro Culture of Pimpinella anisum L. (anise)

A simple in vitro protocol has been developed for large scale multiplication of plants from various explants of Pimpinella anisum L., a medicinally important plant belonging to family Apiaceae. Browning of cultures was observed during the maintenance. Frequent subculture at an interval of about 15–17 days was essential for obtaining embryogenic callus cultures and preventing browning of cultures. High frequency of multiple shoot formation was achieved from callus cultures derived from shoot apices, root and stem explants, and also from seed-derived calli. Somatic embryogenesis was observed in callus cultures derived from seeds and shoot apices. Complete plants developed from these embryoids. Direct regeneration of plantlets from shoot apices was also observed. Roots formation occurred in all the cultures. The requirement for exogenous auxin and cytokinin for differentiation was found to be varying in different tissues.     

Effects of Arene-type Hydrocarbon Air Pollutants on Bacillus megaterium

Effects of certain common carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbon organic air pollutants on Bacillus megaterium cultures were noted. Depending on the medium used, either growth suppression or induction of atypical cell forms was observed in cultures grown in the presence of a carcinogen. By contrast, no such alterations were apparent in cultures grown in media supplemented with a noncarcinogen. Both carcinogenic and noncarcinogenic hydrocarbons exerted an enhancing influence, of varying degree, on lipogenesis, glycolysis, and methylene blue reductase activity. A higher than normal level of these reactions, however, was associated with cultures exposed to a carcinogen. In addition, infrared examination of lipids revealed unique spectral characteristics for materials extracted from carcinogen-treated cultures. No difference was noted between materials derived from noncarcinogen-treated cultures and from control cultures.     

Serological identification of enterotoxigenic staphylococci from cheese

Single and double gel-diffusion techniques were employed to examine serologically coagulase-positive staphylococci from cheese for enterotoxigenicity. Supernatant fluid from sac cultures was examined for enterotoxins A and B. The results indicated that 9 of 155 cultures from market cheese and 7 of 77 cultures from food-poisoning cheese produced enterotoxin A, and that none of the cultures produced detectable levels of enterotoxin B. Results of serological tests were confirmed by intravenous injection of cats.     

Differential rates of cytokine production and apoptosis in venipuncture and finger-stab derived blood cultures

The collection of finger-stab (FS) blood is a convenient and non-invasive method of rapidly acquiring human blood and is becoming increasingly popular for use in human biomonitoring studies. This study compared whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures derived from venipuncture (VP) and FS blood, to determine whether they respond similarly under culture conditions. The rates of spontaneous- and radiation-induced apoptosis and pro-inflammatory cytokine production were monitored over 72 h in each of four culture conditions. In non-irradiated WB cultures, the spontaneous rate of apoptosis was significantly lower in cultures from FS-derived blood than from VP-derived blood. However, FS- and VP-derived cultures responded similarly to radiation-induced apoptosis. PBMC cultures, regardless of the source, were the most responsive to radiation. When the levels of pro-inflammatory cytokines were measured, a significant time-dependent increase in TNF-alpha, IL-6 and IL-1beta production was observed in FS-derived cultures, but not in VP-derived cultures. While VP and FS blood cultures were found to respond similarly to radiation-induced apoptosis, there was a significant difference in the rate of spontaneous apoptosis in non-irradiated WB cultures and in the in situ production of pro-inflammatory cytokines between VP- and FS-derived blood cultures.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.     

Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture.

Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.