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PubMed1980

Human X chromosomes: synchrony of DNA replication in diploid and triploid fibroblasts with multiple active or inactive X chromosomes.

We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical stain to measure X-linked glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2) cellulose acetate electrophoresis of G6PD activity in cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of 47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.

Aneuploidy↗

PubMed1976

Concentration-dependent mutation of diploid human lymphoblasts by methylnitronitrosoguanidine: the importance of phenotypic lag.

The mutation of diploid human lymphoblasts by methylnitronitrosoguanidine (MNNG) was measured over the range of 0--45 ng of MNNG/ml of of medium. We found a 12-day lag in the phenotypic expression of 6-thioguanine resistance; the occurrence of this lag was independent of MNNG concentration. We hypothesize that the unexpectedly long lag period reflects a requirement for the loss of previously existing molecules of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) after mutation at the HGPRT locus.

Cell Survival↗

PubMed1976

Human diploid fibroblast mutants with altered RNA polymerase II.

Clones resistant to the cytotoxic action of alpha-amanitin have been isolated from a strain of fetal human lung diploid fibroblasts. Resistant clones were recovered at a frequencey of 5 X 10(-8) after single-step selections following mutagenesis with the mutagen ethyl methane sulfonate. Following propagation in drug-free medium, the clones retained the selected phenotype and in both growth and plating experiments showed a 10-50-fold higher resistance than wild-type cells to the cytotoxicity of 0.25 microgram/ml alpha-amanitin. The alpha-amanitin sensitivity of RNA polymerase II purified from the mutant cells suggests the presence of two forms of the enzyme, one similar to that found in wild-type cells and a second form with increased resistance to alpha-amanitin inhibition. These results are consistent with previous evidence that alpha-amanitin resistance behaves as a codominant marker in mammalian cells.

Amanitins↗

PubMed1981

Requirement of the human chromosome 11 long arm for replication of herpes simplex virus type 1 in nonpermissive Chinese hamster x human diploid fibroblast hybrids.

Somatic cell hybrids between Chinese hamster (CH) lung cells (V79/380-6), nonpermissive for productive infection by herpes simplex type 1 (HSV-1), and permissive human diploid cells support productive HSV-1 infection as long as they retain human chromosome 11. Human chromosome 3 has been reported to complement nonpermissivity in (CH) Don cells (1). Intraspecies hybrids between Don/a3 and V79/380-6 cells, however, did not support HSV-1 replication, indicating lack of complementation. The block in both nonpermissive CH cell lines was determined to involve a step beyond replication of the parental viral DNA. In cell hybrids between nonpermissive Don/a23 cells and human fibroblasts containing a t(11;15) (p11;p12) translocation, HSV-1 production was dependent solely on the presence of either human chromosome 11 or the der(11) (p11 leads to qter) translocation product containing the long arm of chromosome 11. Chromosome 3 was excluded by a discordancy rate of 59%. We conclude that the long arm of human chromosome 11 carries one or more genes coding for host functions necessary for the production of progeny HSV-1 DNA.

Animals↗

PubMed1986

High rate of insufficient antibody titers after single-day immunization with human diploid-cell-strain vaccine against rabies.

We examined the possibility of simplifying the currently recommended immunization schedule against rabies. Four groups of 11 healthy volunteers were each immunized with either one, two, four, or eight doses of 0.1 ml human diploid cell vaccine (HDCV) intradermally on 1 single day. Antibody titers in serum were determined using a rapid fluorescent focus inhibition test. Geometric mean antibody titers 10, 30 and 90 days after immunization were all greater than 0.5 IU/ml, which is considered protective. A dose-proportional increase in the geometric mean antibody titer was observed for all four groups. However, at each dose level, at least 1 of the 11 volunteers on day 30 and 2 of the 11 volunteers on day 90 had insufficient antibody titers less than 0.5 IU/ml. Single-day immunization against rabies with HDCV vaccine cannot be recommended because of the unacceptably high failure rate.

Adolescent↗

PubMed1978

Quercetin inhibits hexose transport in a human diploid fibroblast.

The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pK = 7.65) is the inhibitory form of the molecule (K1 = 10micron). Quercetin, unlike phloretin, is rapidly degraded in 0.1 N NaOH; the degradation products are weakly inhibitory to hexose transport.

Biological Transport, Active↗

PubMed1977

Effects of ethidium bromide in diploid and duplication strains of Aspergillus nidulans.

Unstable duplication and diploid strains of Aspergillus nidulans were treated with ethidium bromide, and it was shown that this drug reduces the number of sectors produced by such strains. The mechanisms which could be responsible for the partial stabilization of the strains are discussed and it is suggested that a similar mechanism is responsible for the production of sectors in both strains. It is also suggested that ethidium bromide could be useful for the reduction of instability of industrial strains.

Aspergillus nidulans↗

PubMed1977

Fertile diploid drones in africanized honeybees, Apis mellifera adansonii.

59 diploid drones of Apis mellifera adansonii, 12-37 days old, were tested for the presence of semen after provoked ejaculation; 13 drones ejaculated semen enough to be used in an instrumental insemination, but only three on them (5%) furnished 1 mm3 of semen. The problems referring to the attainment of descendants from the 2n drones are briefly discussed.

Animals↗

PubMed1996

Genetic and physical mapping in Brassica diploid species of a gene cluster defined in Arabidopsis thaliana.

We report the genetic and physical analysis by pulse field gel electrophoresis (PFGE) in three Brassica diploid genomes for a cluster of five genes characterized in a selected segment of 15 kb on chromosome 3 of Arabidopsis thaliana, encoding a Bradyrhizobium CycJ homologue (At1), a rat p67 translation factor homologue (At2), an Em-like (early methionine) protein (At3), chlorophyll synthase (At4) and a yeast Sac1 homologue (A5). The Arabidopsis gene array was found to be conserved on a single linkage group in each of the Brassica genomes. However, partial complexes were found to be duplicated in other chromosome segments on the same or other linkage groups. Some of the At genes, which could not be genetically mapped because of lack of polymorphism, were assigned to their respective linkage groups by physical mapping. The presence of multiple copies of the A. thaliana gene cluster in the three Brassica genomes further establishes their complex nature, which results from extensive duplication and chromosomal rearrangement. In general, genetic distances between the At genes agreed with values expected for the physical distances determined in Brassica.

Aminopeptidases↗

PubMed1981

Ketone bodies, glucose and glutamine as lipogenic precursors in human diploid fibroblasts.

Incorporation of [14C] from acetoacetate, D(-)- and L(+)-3-hydroxybutyrate, glucose, glutamine, acetate and palmitate in cellular lipids were studied in cultures in human diploid fibroblasts (HDF). The results showed that acetoacetate was 2-10 times more effective as a lipogenic precursor than was either D- or L-3-hydroxybutyrate. Its extent of incorporation into lipids was 2- to 8-fold more than the other precursors examined under conditions when the overall rates of nonsaponifiable and saponifiable lipogenesis as measured by 3H2O incorporation were essentially unchanged. Acetoacetate supported both saponifiable and nonsaponifiable lipid synthesis with half-saturation values (Km app.) of 185 microM and 30 microM, respectively. Glucose stimulated acetoacetate incorporation into lipids whereas, conversely, acetoacetate inhibited [14C]glucose incorporation into lipids. The presence of low density lipoproteins (LDL) cholesterol (40 microgram cholesterol/mL) inhibited the incorporation of [14C] from acetoacetate 56% into nonsaponifiable lipids; the inhibition was consistently higher (75%) when [14C]glucose or glutamine were the precursors. The loss of 3-hydroxy-3-methyl-glutaryl CoA (HMG CoA) reductase activity upon addition of LDL-cholesterol was greater than the suppression of [14C] incorporation from acetoacetate or glucose into nonsaponifiable lipids. In the presence of glucose, [14C]acetoacetate was incorporated into 3-beta OH sterols (digitonin precipitable) 7.7 +/- 1.1 times more effectively than was [14C]glucose. The results suggest that HDF would be a suitable model to investigate the effects of various precursors of HMG CoA on the rate of cholesterol biosynthesis.

3-Hydroxybutyric Acid↗

PubMed1977

Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein.

A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 micrograms per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium.

Adenine↗

PubMed1977

Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts.

The effects of hydrocortisone and ascorbic acid on growth parameters were measured in human diploid skin fibroblasts from fetal and adult donors. In the presence of culture medium containing 10% fetal bovine serum, 0.3 micrometer hydrocortisone produced a 20% increase in the population growth rate and a 50 to 70% increase in the confluent density of fibroblasts from adult donors. Daily addition of 28 micrometer ascorbic acid also stimulated the population growth rate and cell density at confluency. The effects of hydrocortisone and ascorbic acid on the final cell density were additive. The action of hydrocortisone was restricted to cells in log-phase growth, whereas ascorbic acid affected cells in both the log and the post-confluent phases of the growth cycle. In fibroblasts from fetal donors, ascorbic acid was stimulative but hydrocortisone was not. The data suggest that whereas both compounds stimulate cell growth in an additive manner, they do so by different cellular mechanisms.

Adult↗

PubMed1977

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.

Blood↗

PubMed1978

Initiation and characterization of a diploid cell line from larval tissues of Aedes dorsalis (Meigen).

Mosquito cell cultures were initiated from the minced tissues of newly hatched Aedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n = 6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (including Mycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation.

Aedes↗

PubMed1978

gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells.

gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.

Animals↗

PubMedDate not supplied

Extensive chromosome aberrations caused by [3H]thymidine incorporation in a diploid monkey cell line DBS-FRhL-2.

Extensive chromosome aberrations were induced in a diploid monkey cell line designated as DBS-FRhL-2 after exposure to [3H]thymidine ([3H]Tdr) for either 30 or 60 min at a dose of 1 muCi per ml of medium. Cultures exposed to [3H]Tdr for a longer period had significantly larger numbers of aberrations than those exposed for a shorter period. The most common type of aberrations were chromatid breaks. The majority of aberrations were observed in cells which were in contact with [3H]Tdr during S phase, especially the middle S. Cells from cultures of early and late passages exposed to [3H]Tdr were affected to a similar extent when chromosomes were examined. No clear relationship between sites of breakage and intensity of labeling could be established.

Cell Line↗

PubMed1978

Effects of cystine or glutamine restriction on human diploid fibroblasts in culture.

Amino-acid restriction was studied using a strain of human diploid fibroblasts (HDF) isolated from fetal skin. HDF populations were maintained at reduced proliferation rates by lowering cystine or glutamine concentrations of Eagle's MEM. Effects of this treatment on mean cell diameters, total cell protein and specific activity of acid phosphatase were assessed. Threshold levels of cystine required for maintenance were found to be markedly influenced by type and/or batch of serum used irrespective of their free cystine content. A 96-hr exposure of passage 25 cells to medium 0.01 or 0.005 mM in cystine resulted in an increased mean cell diameter and a greater heterogeneity of cell size. This was accompanied by an increase in the total cellular protein. A 48-hr exposure, followed by return to control serial cultivation, induced persistent change in mean cell diameter without affecting the total life span (49 passages over 165 days). The mean size of untreated cells increased with serial passage reaching a size similar to treated cells only at passage 44. Acid-phosphatase specific activities were higher in cells maintained under conditions restrictive in cystine. None of the above changes were observed when glutamine concentrations were reduced to 0.0025 mM, a level required to inhibit division rates. The possible relevance of these findings to the phenomenon of in vitro cell senescence is discussed.

Acid Phosphatase↗

PubMed1983

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells.

Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free "attachment medium" was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation.

Animals↗