Regulation of enzyme turnover during tissue differentiation. Studies on 6-phosphogluconate dehydrogenase in rabbit mammary gland in organ culture.

(1) Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 in the presence or absence of insulin, prolactin and cortisol. (2) Antiserum to 6-phosphogluconate dehydrogenase was raised in sheep and used to titrate the amount of enzyme activity present in explant extracts. Changes in enzyme activity were found to be due to corresponding changes in amount of the enzyme. The greatest increases in the amount of the enzyme were only brought about by culture of explants in the presence of hormones (insulin, prolactin and cortisol) in Medium 199 which contained glucose. (3) The increases in the amount of the enzyme were similar in explants cultured with hormones in Medium 199 which contained 1.39 mM, 5.55 mM or 55.5 mM glucose. (4) When explants were cultured with hormones in Medium 199 which contained glucose (5.55 mM) for 24 h and then cultured with hormones in Medium 199 which contained glycerol (10.9 mM), a decrease in the amount of the enzyme occurred. In contrast, the culture of explants with hormones in Medium 199 which contained glycerol (10.9 mM) for 24 h followed by transfer of the explants to medium which contained glucose (5.55 mM) resulted in an increase in the amount of the enzyme to reach values which were not different from those found in explants cultured throughout with hormones in Medium 199 which contained glucose.     

国产199培养基制备狂犬病疫苗、乙型脑炎疫苗及麻疹疫苗的质量研究

用国产１９９培养基,以进口１９９培养基和自配维持液为对照,制备狂犬病疫苗、乙型肝脑炎疫苗、麻疹疫苗。从这三种疫苗的毒力、效力、稳定性三个方面,比较国产１９９培养基作为疫苗维持液能否改进疫苗质量。试验结果表明：国产１９９培养基作为疫苗维持液制备这三种疫苗的质量和进口１９９培养基制备疫苗的质量几乎无差别,但优于自配维持液制备疫苗的质量。以国产１９９培养基作为疫苗维持液能提高疫苗的质量     

Chromosomal breakage in Crohn's disease: Anticlastogenic effect of D-penicillamine and L-cysteine

Summary The incidence of chromosome breakage was found to be elevated in 42 patients with Crohn's disease. This phenomenon was much more striking in cultures set up with TCM 199 than in cultures set up with RPMI 1629 rich in L-cysteine. The drug D-penicillamine, a close analog of L-cysteine, gave an apparent therapeutic response in several patients and reduced the chromosome breakage frequency in the lymphocytes of these patients in vitro and in vivo.Work supported by grants of CNRS (ER 149), INSERM (U 173) and GEFLUC     

The infectivity of Plasmodium yoelii in different strains of mice

We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID50) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID50 obtained for CD-1 mice (147 sporozoites) was significantly higher.     

Co-culture of rabbit one-cell embryos with rabbit oviduct epithelial cells

Summary Rabbit 1-cell embryos were co-cultured with rabbit oviduct epithelial cells (ROEC) to determine if ROEC can enhance embryo development in vitro. Primary ROEC were cultured in serum-free media at 39°C in a 5% CO₂:95% air environment. In experiment 1, 1-cell embryos were co-cultured in Ham's F10 with freshly collected or 4-d-old cultures of ROEC seeded in plastic culture wells or on collagen membranes. One-cell embryos cultured without ROEC served as controls. After 65 h in culture, embryos were stained with Hoechst 33342 to determine the number of cells per embryo. Cell numbers were higher (P<0.035) in all co-culture treatments when compared to controls. Optimal development was obtained by co-culture with 4-d-old ROEC grown on plastic (P<0.003). In experiment 2, Ham's F10, Medium 199, and CZB with glucose medium were compared for their ability to support embryo development in the presence or absence of 4-d-old ROEC growth on plastic. Cell number and the percentage of embryos becoming blastocysts were significantly (P<0.002) higher for embryos cultured in Medium 199 compared to the other media tested. In Medium 199, co-culture with ROEC resulted in only a slight, nonsignificant increase in cell number over culture in Medium 199 alone (110 vs. 96 cells). However, the percentage of embryos reaching the blastocyst stage when co-cultured in Medium 199 with ROEC (49%) was nearly twice (P=0.01) that of embryos in Medium 199 without ROEC (26%). In experiment 3, transfer of embryos cultured in Medium 199 with or without ROEC of 24 or 48 h resulted in no significant differences in posttransfer development. These data indicate a beneficial effect of ROEC on blastogenesis and a salvage effect of ROEC on cell proliferation in embryos grown in a less supportive medium such as Ham's F10. This work was supported by a Multicenter Cooperative Program on Non-HumanIn Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, Bethesda, MD, through Cooperative Agreement HD 21939.     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

Improved conditions for culture of biosynthetically active cockroach corpora allata

Summary Currently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.     

Chromosomal instability in a woman with infertility and two unaffected brothers: a new familial chromosomal breakage syndrome?

Repeated chromosomal analysis of peripheral blood lymphocytes and skin fibroblasts from a woman referred for amenorrhoea, streak gonads, hyperthyroidism, adiposity and elevated α-fetoprotein levels but no other manifestations of known chromosomal breakage syndromes demonstrated an increased spontaneous chromosomal breakage rate (ISCBR). Chromatid and chromosomal breaks were more numerous than sporadic rearrangements and dicentric chromosomes. Exposure of the cells to mitomycin C, diepoxybutane, X-rays or UV irradiation induced an increase in chromosomal and chromatid abnormalities over that in controls. A micronucleus assay demonstrated an increase in the incidence of formation of micronuclei and the population doubling time of the fibroblasts of the proposita was delayed. Chromosomal analysis was performed on lymphocytes of the parents and of five sibs of the proposita. Two brothers had chromosomal abnormalities identical to those of the patient and elevated α-fetoprotein levels, however, without any clinical abnormalities. The parents were affected by only a moderate ISCBR whereas two brothers and one sister were chromosomally normal. The clinical, chromosomal and biochemical findings in this family represent a novel chromosomal instability syndrome. Received: 30 October 1996 / Accepted: 27 March 1997     

Effects of culture media on spontaneous incidence of mitotic index, chromosomal aberrations, micronucleus counts, sister chromatid exchanges and cell cycle kinetics in peripheral blood lymphocytes of male and female donors

The spontaneous incidence of mitotic index (MI), chromosomal aberrations (CA), micronucleus counts (MNC), sister chromatid exchanges (SCE) and cell cycle kinetics (CCK) were studied in human peripheral blood lymphocytes grown in M199 and RPMI-1640 culture media. Lower frequencies of CAs, MNC and SCEs were observed in lymphocytes cultured in medium RPMI-1640. The reduction of the MI and the replicative index in M199 medium showed delayed cell cycle kinetics.     

Stimulation of endothelia cell proliferation by precursors of thymidylate.

Bovine corneal and aortal endothelial cell cultures were established from primary explants and subcultured for at least 40 passages. With both cell lines, exogenous thymidine, folate or folinate markedly increased the proliferation of these cells and decreased their serum requirement in Medium 199. Medium 199 supplemented with thymidine was particularly useful for cell survival at low densities; cones were readily produced when single cells were plated as low as 0.07 cells. cm-2. In contrast to the results of others, neither fibroblast growth factor nor epidermal growth factor were necessary for cell proliferation or survival at low densities.     

Cytoprotective effect of honey against chromosomal breakage in fanconi anemia patients in vitro

BACKGROUND:

Natural honey is widely used all over the world as a complementary and alternative medicine in various disorders including Fanconi anemia (FA). FA is a rare genetic chromosomal instability syndrome caused by impairment of DNA repair and reactive oxygen species (ROS) imbalance. This disease is also related to bone marrow failure and cancer. The aim of this study was to evaluate the cytoprotective effect of honey on mitomycin C (MMC-) induced chromosomal damage in peripheral lymphocytes from FA patients.

MATERIALS AND METHODS:

Treatment of these complications with alkylation agents MMC may enhance chromosomal breakage. We have evaluated the effect of honey on MMC- induced chromosomal breakage in FA blood cells using chromosomal breakage assay. The basal chromosomal breakage count was higher among FA patients than healthy subjects.

RESULTS:

The addition of MMC alone gave a significantly higher of chromosomal breakage in FA patients than control group (P < 0.0001). Pre- treatment with honey significantly inhibited breakage induced by MMC in FA patients by its antioxidant effect.

CONCLUSION:

Honey can prevent MMC- induced chromosomal breakage by its antioxidant effect.     

Spontaneous and induced chromosome instability in patients with fragile X syndrome

Spontaneous and degranol- and dimatiph-induced chromosomal instability in the lymphocyte culture of patients with fra-X syndrome was investigated. The cultures contained TC 199 and 5% FC serum. It was found that the frequency of spontaneous chromosomal aberrations (CA) was 7.3% in cells from patients with fra(X), 3.9% in patients with MR of unknown origin, and 1.3% in normal individuals. Spontaneous break-points in the patients with fra(X) were localized in 1p, 2q, 3p, 6q, 7q, 16 q more often than in normal individuals. No significant difference was found in SCEs. The cells of patients with fra(X) were not sensitive to the induction of CA by degranol. It was found that chromosomal telomeric changes (CTC) were mutagen-independent, remaining at the spontaneous level: in the patients with fra(X) CTC were 10.5% (9.5% fra-Xq27, and 1% autosomal telomeric changes); in normal individuals CTC were 0.1%.     

A comparison of different nutrient media and supplementation with dexamethasone for mouse colon organ culture

Several complex nutrient media were compared for their effectiveness in maintaining viable and functional mouse colon mucosa in organ culture. The order of superiority for preserving survival of normal tissues for 14 days was: Williams' Medium E > Morgan's 199 > CMRL-1066 > Waymouth's MB 752/1 > Eagle's MEM > Trowell's T8. The ₃H-thymidine labeling index was highest in colon explants maintained in Morgan's 199 > Williams' Medium E > Waymouth's MB 752/1 > CMRL-1066 > Eagle's MEM. However, the very high labeling produced by Morgan's 199 medium was abnormal in comparison to in vivo levels. Supplementation with 1.0 µM dexamethasone almost always improved crypt survival and maintained normal DNA synthetic activity.Supported by National Cancer Institute contract No. 1-CP-75952 and grant No. CA-29602.     

Differentiation of Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary

Striated muscle fibers appeared in monolayer cultures of rat anterior pituitary cells maintained in αMinimum Essential Medium (αMEM). As muscle differentiation in cultures of pituitary cells under ordinary conditions has not hitherto been reported, an in vitro study was undertaken to determine what factor(s) is responsible for this myogenesis. When dispersed anterior pituitary cells were culrured in three different media, αMEM, Medium 199 and Dulbecco's Modified Eagle Medium (DMEM), only αMEM induced a high incidence of striated muscles. The nature of the serum (fetal calf, calf and horse) and its concentration (1–10%) did not affect myogenesis.
In monolayers in αMEM, the sequence of differentiation of striated muscle was as follows: 1) Elongated cells, resembling myoblasts appeared; 2) these cells fused; and finally 3) cross striations appeared. Rhythmic contraction was most intense in striated muscle fibers, but it was also obsrved in myotubes without cross striations and even in myogenic cells before fusion. The possible origin of muscles in these pituitary cultures is discussed.     

Culture and morphological observations on Trypanosoma melphagium.

The cultural characteristics of Trypanosoma melophagium of sheep were studied. Aspects investigated were size of the inoculum and population growth in Modified Monophasic Medium for Trypanosomes (MMMT), population growth in Medium 199 with 10% inactivated calf serum containing 5, 10, and 15% hemolyzed defibrinated rabbit blood (199-CS-5, 199--CS-10, 199-CS-15) at 27 degrees C, effects on population growth of temperature and hydrogen ion concentration in MMMT, and morphology and morphometrics of the developmental stages found under different experimental conditions. The best growth occurred in medium MMMT at 30 degrees, C, pH 7.25. Temperature seemed to be a critical factor for differentiation of epimastigotes to trypomastigotes. Statistically significant differences were found between the trypomastigotes in MMMT and 199--cs-5 at 37 degrees C on day 4 of incubation for the following measurements: PK (distance from posterior end to kinetoplast), KN (from kinetoplast to middle of nucleus), PN (from posterior end to middle of nucleus), and nuclear and kinetoplastic indices. The trypomastigotes formed in both media were much smaller in size than the blood forms reported by Hoare (1972).     

A chromosomal method to distinguish between X- and Y-bearing spermatozoa of the bull in zona-free hamster ova

The interspecific in-vitro fertilization system using zona-free hamster ova was adopted to distinguish chromosomally between X- and Y-bearing bull spermatozoa. Frozen semen samples were thawed and washed with modified BWW medium including 0.3% BSA (pH 7.2) to remove cryoprotective medium. Motile spermatozoa were recovered from the semen by the 'swim-up' method. These spermatozoa were treated with ionophore A23187 to facilitate capacitation. Adequate capacitation of spermatozoa was found by their movement patterns and the insemination was performed at the optimum time. The fertilized ova were cultured in Medium TC199 with 10% FBS including podophyllotoxin and vinblastine until they reached first cleavage metaphase. Chromosome slides were prepared by our gradual fixation-air drying method. The success rate of the method was 56% of the number corresponding to the zona-free ova used. Altogether 1116 spermatozoa from 4 different bulls were successfully analysed and the ratio of X- and Y-bearing spermatozoa was 542:574 (P greater than 0.3).     

Effects of incubation temperature and bicarbonate on maturation of pig oocytes in vitro

Pig oocytes cultured at 39 degrees C had a higher percentage of polar body formation than did those cultured at 37 degrees C. A culture medium based on Medium 199 with Earle's salts and supplemented with 15% serum from a young castrated boar was just as good as the same formulation containing additional pyruvate, lactate and insulin, and superior to a modified Krebs-Ringer bicarbonate medium. When the bicarbonate buffer system (Earle's salts) of Medium 199 was replaced with a phosphate buffer system (Hank's salts), the rate of polar body formation was decreased. When the Hank's based medium was supplemented with a bicarbonate buffer system, polar body formation was restored to the level in Earle's based medium. This suggests that CO2/bicarbonate may be important for the normal maturation of pig oocytes.     

Improved development of rabbit one-cell embryos to the hatching blastocyst stage by culture in a defined, protein-free culture medium

In Exp. 1, Medium 199 and Medium RD (RPMI-1640 and Dulbecco's MEM, 1:1 v/v) were compared in a 2 x 2 factorial design by supplementing each with 15 mg bovine serum albumin (BSA)/ml of 1 mg polyvinyl alcohol (PVA)/ml. All media contained 5 micrograms insulin/ml, 5 micrograms transferrin/ml, 5 ng selenium/ml (ITS), and 10 ng epidermal growth factor (EGF)/ml. One-cell embryos were cultured at 39 degrees C with 5% CO2 in air for 65 h and then stained with Hoechst 33342 to determine blastomere number. Embryos in Medium 199 developed poorly (P less than 0.001) when PVA was used instead of BSA (30 vs 76 cells/embryo), but developed rapidly in Medium RD with PVA or BSA (118 and 121 cells). Similar results were obtained in Exp. 2 in BSA- and PVA-free medium. In Exp. 3, the development of 1-cell embryos after 65 h in unsupplemented (protein-free) Medium RD (68% blastocysts, 117 cells) did not differ (P greater than 0.37) from that obtained using Medium RD with insulin, ITS or EGF alone. Culture in protein-free Medium RD for 96 h resulted in 82% of the 1-cell embryos forming blastocysts and 40% hatching through the zona pellucida. In a preliminary test of viability, 1-cell embryos cultured in this medium for 48 or 65 h and transferred to synchronous recipients resulted in 5/18 (28%) and 3/24 (12%) Day-15 viable fetuses. Cell counts of approximately 120 per blastocyst after culturing 1-cell embryos for 65 h in Medium RD indicated that cell division was more rapid than that obtained with all other media tested previously in this laboratory. This is the first report of rabbit embryo development from the 1-cell to the hatching blastocyst stage in a defined protein-free culture medium.