Co-ordinated expression of the components of iron transport (mycobactin, exochelin and envelope proteins) in Mycobacterium neoaurum

Abstract Mycobacterium neoaurum was grown with a range of iron concentrations from 0.01 to 4.0 μg/ml. Synthesis of the extracellular siderophore, exochelin, the intracellular iron storage compound, mycobactin and the iron-repressible envelope proteins were co-ordinately expressed. All three components of the iron transport system were synthesized when low amounts of iron (0.01 to 0.2 μg/ml) were added to the medium and were repressed when the iron concentration was increased to 0.5 μg/ml and above. These results re-inforce the conclusion that the iron-regulated proteins do fulfil an essential function in iron metabolism.     

Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris , opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70-90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30-45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.     

The Effect of Monensin on Pure and Mixed Cultures of Rumen Bacteria

The antibiotic monensin was added to pure cultures of Bacteroides ruminicola, Selenomonas ruminantium, Anaerovibrio lipolytica and Megasphaera elsdenii. These organisms, representing succinate- and propionate-producing rumen bacteria, were not affected by monensin up to 10 μg/ml. Methanobacterium ruminantium was slightly inhibited by monensin, Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis were inhibited to differing extents by monensin at concentrations between 0.1 and 10 μg/ml. Bacteroides succinogenes was inhibited at first by monensin at >0.5 μg/ml but after a prolonged lag phase adapted to grow in the presence of monensin at concentrations below 5 μg/ml.
Monensin (1 μg/ml) almost completely stopped the digestion of chopped straw and dewaxed cotton fibres by rumen contents incubated in vitro. The digestion of grass and powdered filter paper was not significantly reduced under these conditions, but when the concentration of monensin was increased to between 3 and 5 μg/ml, the digestion of these substrates was reduced.     

Evaluation of radioprotective activities of Rhodiola imbricata Edgew – A high altitude plant

The present study reports the radioprotective properties of a hydro-alcoholic rhizome extract of Rhodiola imbricata (code named REC-7004), a plant native to the high-altitude Himalayas. The radioprotective effect, along with its relevant superoxide ion scavenging, metal chelation, antioxidant, anti-lipid peroxidation and anti-hemolytic activities was evaluated under both in vitro and in vivo conditions. Chemical analysis showed the presence of high content of polyphenolics (0.971 ± 0.01 mg% of quercetin). Absorption spectra analysis revealed constituents that absorb in the range of 220–290 nm, while high-performance liquid chromatography (HPLC) analysis confirmed the presence of four major peaks with retention times of 4.780, 5.767, 6.397 and 7.577 min. REC-7004 was found to lower lipid oxidation significantly (p < 0.05) at concentrations viz., 8 and 80 g/ml respectively as compared to reduced glutathione, although the optimally protective dose was 80 g/ml, which showed 59.5% inhibition of induction of linoleic acid degradation within first 24 h. The metal chelation activity of REC-7004 was found to increase concomitantly from 1 to 50 g/ml. REC-7004 (10–50 g/ml) exhibited significant metal chelation activity (p < 0.05), as compared to control, and maximum percentage inhibition (30%) of formation of iron-2,2-bi-pyridyl complex was observed at 50 g/ml, which correlated well with quercetin (34.9%), taken as standard. The reducing power of REC-7004 increased in a dose-dependent manner. The absorption unit value of REC-7004 was significantly lower (0.0183± 0.0033) as compared to butylated hydroxy toluene, a standard antioxidant (0.230± 0.091), confirming its high reducing ability. Superoxide ion scavenging ability of REC-7004 exhibited a dose-dependent increase (1–100 g/ml) and was significantly higher (p < 0.05) than that of quercetin at lower concentrations (1–10 g/ml), while at 100 g/ml, both quercetin and REC-7004 scavenged over 90% superoxide anions. MTT assay in U87 cell line revealed an increase in percent survival of cells at doses between 25 and 125 g/ml in case of drug + radiation group. In vivo evaluation of radio-protective efficacy in mice revealed that intraperitoneal administration of REC-7004 (maximally effective dose: 400 mg/kg b.w.) 30 min prior to lethal (10 Gy) total-body -irradiation rendered 83.3% survival. The ability of REC-7004 to inhibit lipid peroxidation induced by iron/ascorbate, radiation (250 Gy) and their combination [i.e., iron/ascorbate and radiation (250 Gy)], was also investigated and was found to decrease in a dose-dependent manner (0.05–2 mg/ml). The maximum percent inhibition of formation of MDA-TBA complex at 2 mg/ml in case of iron/ascorbate, radiation (250 Gy) and both i.e., iron/ascorbate with radiation (250 Gy) was 53.78, 63.07, and 51.76% respectively and were found to be comparable to that of quercetin. REC-7004 (1 g/ml) also exhibited significant anti-hemolytic capacity by preventing radiation-induced membrane degeneration of human erythrocytes. In conclusion, Rhodiola renders in vitro and in vivo radioprotection via multifarious mechanisms that act in a synergistic manner.     

Periodic Rotation of Chromosomes During the Mitotic Divisions in Secondary Spermatogonia of Newt, Cynops Pyrrhogaster

Mitosis was frequently observed in the secondary spermatogonia of newt in in vitro cultures. From prometaphase to mid-anaphase, the whole set of the chromosomes rotated alternately clockwise and counterclockwise generally in the same plane as the bottom of a plastic dish. The axis of rotation was almost always perpendicular to the bottom of a dish, passing through the central part of the cell. This rotation of chromosomes was so fast that we could discern it directly by a phase contrast microscope. It was a rhythmic and regular motion with almost the constant frequency and magnitude. The average period of each cycle during metaphase varied from cell to cell and between 70 to 20 seconds (0.9–3.0 rotations/min) and the average angle traversed during each motion also varied and between 10 to 90 degrees at 25°C. By marking the cell surface with iron particles, it was demonstrated that the inner part of the cell actively rotated and not the cell as a whole. Colcemid at the concentration of 1.0 μg/ml reversibly arrested the chromosomal rotation and karyokinesis. Cytochalasin B (4.0 μg/ml) also reversibly disturbed the rotation though the karyokinesis continued. These results suggest that the rotation of chromosomes as a set may be mediated by filamentous organelles such as microtubules in the mitotic spindle and cytoplasmic microfilaments.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

Type IV collagen modulates angiogenesis and neovessel survival in the rat aorta model

Summary Type IV collagen is a major basement membrane component that has been implicated in the regulation of angiogenesis. The purpose of this study was to evaluate the effect of type IV collagen on the angiogenic response of native endothelial cells in three-dimensional vascular organ culture. Rings of rat aorta were cultured under serum-free conditions in gels of type I collagen with or without type IV collagen. In the absence of type IV collagen, aortic rings generated neovessels, which proliferated until day 9 and gradually regressed during the second and third weeks of culture. Type IV collagen promoted neovessel elongation and survival in a dose-dependent manner. Microvascular length increased by 43, 57, and 119% over control values in cultures treated with 3, 30, and 300 μg/ml type IV collagen, respectively. When used at high concentrations (300 μg/ml) type IV collagen stabilized the neovascular outgrowths and prevented vascular regression. Type IV collagen also promoted the formation of neovessels, but significant stimulatory effects were observed only at an intermediate concentration (30 μg/ml) and were no longer significant at the high concentration (300 μg/ml). The observation that type IV collagen has dose-dependent effects on vascular elongation, proliferation, and stabilization, supports the concept that the developing basement membrane of neovessles acts as a solid-phase regulator of angiogenesis, whose function varies depending on the concentration of its molecular components.     

Chromium-reducing and plant growth-promoting Mesorhizobium improves chickpea growth in chromium-amended soil

Mesorhizobium strain RC3, isolated from chickpea nodules, tolerated chromium up to 500 μg/ml and reduced it by 90% at pH 7 after 120 h. It produced plant growth-promoting substances, both in the presence and absence of chromium. Strain RC3 produced 35 μg indole acetic acid/ml in Luria Bertani broth with 100 mg tryptophan/ml, which decreased with an increase in chromium concentration. Chromium application to soil at 136 mg/kg was toxic to chickpea plants but when RC3 at 136 mg/kg was also added, it increased the dry matter accumulation, number of nodules, seed yield and grain protein by 71, 86, 36 and 16%, respectively, compared to non-inoculated plants. Nitrogen in roots and shoots were increased by 46 and 40%, respectively, at 136 mg Cr/kg. The bio-inoculant decreased the uptake of chromium by 14, 34 and 29% in roots, shoots and grains, respectively.     

Response of plateau-phase C3H 10T1/2 cells to radiation and concurrent administration of bleomycin

The mode and extent of interaction between bleomycin and radiation were assessed in contact-inhibited cultures of C3H 10T1/2 cells, which in confluent monolayers display a low turnover rate and behave more like late-responding normal tissues in vivo with respect to response to fractionated radiotherapy (i.e., having a low alpha/beta value). Plateau-phase C3H 10T1/2 cultures were exposed to gamma rays delivered in 1, 2, 5, or 10 fractions. The radiation doses administered ranged from 2 Gy in one exposure to 26 Gy in 10 fractions. Half of the cultures were also treated with 1 micrograms/ml of bleomycin for 5 days during which radiation was also given. It was found that 1 micrograms/ml of bleomycin sterilized approximately 40% of the C3H 10T1/2 cells in the cultures. The radiation dose-survival curves of various fractionation schedules (1, 2, 5, and 10 fractions) plus bleomycin were displaced downward (i.e., to lower survival levels) but not modified in shape. The alpha/beta ratios, parameters of the linear-quadratic model of cell survival, were 2.6 (2.2-3.1) and 2.4 (1.8-3.1) Gy for radiation only and radiation plus bleomycin, respectively. This observation indicates that the effect of combining irradiation and bleomycin on C3H 10T1/2 cells in monolayers was additive.     

RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is discussed.     

In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 μg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted.
It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.     

Response of Campylobacter jejuni to combinations of ferrous sulphate and cadmium chloride

On Mueller Hinton (MH) agar, Campylobacter jejuni showed 20.0 and 30.9mm zones of inhibition surrounding discs impregnated with 2.5 and 20 μg CdCl₂ respectively. The minimal inhibitory concentration (MIC) ranged from 0.64 to 3.2 μg CdCl₂/ml of MH agar for four C. jejuni strains. In the presence of 23 μg FeSO₄/ml of MH the MIC increased to a range of 1.5–5.4 μg CdCl₂/ml of MH. Moreover, the numbers of colonies present on MH supplemented with FeSO₄ were greater than on MH without iron. The growth response of C. jejuni in the presence of 0.025% (w/v) FeSO₄ in MH broth was increased about 10000 fold in three of four strains when compared with the growth in unsupplemented MH broth. Zones of inhibition surrounding 20 μg discs of CdCl₂ were 50.6 and 24.4 mm on MH and Campy-BAP media respectively, with cells grown on MH. These results suggest that the blood-containing medium 'neutralized' the biocidal influence of the CdCl₂. In comparison, C. jejuni inoculum from fluid thioglycollate (FT) medium showed smaller zones of inhibition. These decreased from 34.9 mm on MH agar to 19.6 mm on Campy-BAP agar, suggesting that components in the FT growth medium ameliorated the toxic influence of CdCl₂. Atomic absorption spectroscopy analysis indicated mean values (mg/100 g dry weight) of selected metals bound by C. jejuni as: Cu, 10.4; Mg, 146; Na, 2385; Fe, 45.1; Zn, 13.0; and K, 172.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Yersinia intermedia: A new species of enterobacteriaceae composed of rhamnose-positive,melibiose-positive,raffinose-positive strains (formerly calledYersinia enterocolitica orYersinia enterocolitica-like)

The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate (50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency, when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR⁺, HCR⁻, and irradiated HCR⁻ strains. These drugs increased mutation frequency and lethality of irradiated HCR⁺ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine. Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR⁺ cells. Our findings suggest that these chemicals selectively interfere with excision-repair.     

Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8-100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated.     

The toxicity of a number of different bactericides to Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] and to the tomato plant, Lycopersicon esculentum

Twenty-seven proprietary products and pure chemicals were tested in vitro against cells of Clavibacter michiganense subsp. michiganense (Smith 1910) Jensen 1934 comb. nov. [basonym Corynebacterium michiganense pv. michiganense (AL)] (the cause of bacterial canker of tomato) and also for their phytotoxicity to tomato plants. The most bactericidal of these, with a minimum cidal concentration (MCC) range of > 10-< 100 μg/ml, were a phenolic product called Applied 3–78, two quaternary ammonium compounds (benzalkonium chloride and cetrimide), and a silver colloid compound. Of these, only Applied 3–78 was not phytotoxic at values of 10 μg/ml or less, although it was phytotoxic at 10000 μg/ml. Copper oxychloride and sodium hypochlorite were amongst the group with a middle range of bactericidal properties, their MCC range being from > 1000 to < 10000 μg/ml. They were phytotoxic at 1000 μg/ml or less. When organic matter, a dead yeast suspension, was added to Applied 3–78, Kohrsolin and Panacide, only the activity of Applied 3–78 was relatively unchanged. The MCC ranges were: Applied 3–78, >80–< 100 μg/ml; Kohrsolin, > 800-< 1000 μg/ml; and Panacide, > 1000 μg/ml. Phytotoxicity tests on 10 different tomato cultivars confirmed that Applied 3–78 was the least phytotoxic of these three products. Field trials on tomato crops showed that when Applied 3–78 was sprayed on the plants once, and Kohrsolin was either sprayed on or they were drenched with it once at 1000 μg/ml, no phytotoxicity symptoms developed.     

Long-term culture of epithelial cells from the normal rat colon

Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures. Supported by National Cancer Institute Contract N01-CP-75914.     

Effect of liver cell coat acid mucopolysaccharide on the appearance of density-dependent inhibition in hepatoma cell growth.

Yoshida ascites hepatoma 66 (AH 66) cells grown in monolayer cultures show a lack of density-dependent inhibition of growth. When acid mucopolysaccharide (AMPS) isolated from rat liver cell coats is added to growing cultures at concentrations of 50–100 μg/ml, AH 66 cell cultures became markedly inhibited and exhibited density-dependent inhibition of growth at a cell density of 19 × 10⁴ cells/cm². Inhibition reached 84% below control levels. Inhibition is a density-related phenomenon since cells at densities below 19×10⁴ cells/cm² do not exhibit inhibition of growth. AH 66 cells inhibited in the plateau state are capable of resuming growth when AMPS is removed from the cultures. When AMPS is added at a concentration of 0.5 μg/ml, growth of tumor cells is promoted. Promotion reaches 78% above control levels. It is suggested that AMPS may play an important part in the regulation of cellular proliferation.