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PubMed2006

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas.

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.

Animals↗

PubMed2005

Segregation of microsatellite alleles in gynogenetic diploid Pacific abalone (Haliotis discus hannai).

Inheritance of 9 microsatellite loci was examined in 3 families of gynogenetic Pacific abalone Haliotis discus hannai produced by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division. The proportion of heterozygous progeny was used to estimate marker-centromere (M-C) distances. All loci conformed to Mendelian segregation in the control crosses when null alleles were accounted for. The absence of paternal alleles confirmed the gynogenetic origin of the offspring and indicated 100% success for 3 families. Estimated recombinant frequencies ranged from 0.10 to 0.60, which is lower than those observed in other gynogenetic diploid animals. The mean recombination frequency was 0.22, corresponding to a fixation index of 0.78 in one generation. This is 3.12 times the increase in homozygosity expected after one generation of sib mating (0.25), suggesting meiotic gynogenesis may be an effective means of rapid inbreeding in the abalone. M-C map distances for the 9 loci varied between 5 and 30 cM under the assumption of complete interference. The information about M-C distances will be useful for future gene mapping in H. discus hannai.

Animals↗

PubMed2006

Contrast between extensive variation of 28S rDNA and stability of 5S rDNA and telomeric repeats in the diploid-polyploid Squalius alburnoides complex and in its maternal ancestor Squalius pyrenaicus (Teleostei, Cyprinidae).

The diploid-polyploid Squalius alburnoides complex resulted from interspecific hybridization. The chromosomal mapping of 28S and 5S ribosomal genes and of (TTAGGG)n telomeric repeats was performed on specimens from the complex and from the sympatric bisexual species S. pyrenaicus (the complex maternal ancestor) as part of an investigation of the evolutionary relationships between genomic constitutions and the consequences of the ongoing polyploidization process in terms of chromosome reshaping. Contrasting results were obtained. While results with 5S rDNA and telomeric probes gave an impression of genomic stability, the variability detected with 28S rDNA probe suggested quite the opposite. The 5S rDNA probe mapped constantly to three chromosomes per haploid genome with apparently conserved locations in morphologically similar chromosomes; conversely, prominent intra- and inter-individual variations of 28S rDNA and of syntenic sites with 5S rDNA were detected with regard to number, size and location. Hypotheses for the causes of such polymorphisms are discussed. The terminal position of most 28S rDNA sites and the absence of detectable interstitial telomeric sequences suggest a mechanism that does not involve major chromosomal rearrangements. These fishes share similar patterns for the studied cytogenetic markers which may be taken as evidence of an apparent stability that may be hiding extensive and subtle genome variations that are possibly related to an ongoing evolutionary process of genome tetraploidization and speciation.

Alleles↗

PubMed2006

Preferential reduction of the alpha-2-6-sialylation from cell surface N-glycans of human diploid fibroblastic cells by in vitro aging.

Human diploid fibroblastic cell line, TIG-3, has a finite life span of about 80 population doubling levels (PDL), and is used for in vitro aging studies. Young cells (PDL 23) grew to higher cell densities at a higher growth rate than aged cells (PDL 77). When the electrophoretic mobility of cells was determined, the negative surface charge of the aged cells decreased significantly when compared to that of young cells. Lectin blot analysis of membrane glycoproteins showed that the alpha-2-6-sialylation but not the alpha-2-3-sialylation of N-glycans decreases markedly in the aged cells when compared to the young cells. In support of this observation, the cDNA microarray assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the gene expression of the alpha-2,6-sialyltransferase I (ST6Gal I), which transfers sialic acid to galactose residues of N-glycans, decreases in the aged cells. These results indicate that the concordant decrease of the alpha-2,6-sialylation of N-glycans with the ST6Gal I gene expression is induced in TIG-3 cells by in vitro aging.

Cell Line, Tumor↗

PubMed2006

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts.

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 microM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 microM AG126 (an inhibitor of ERK1/2), but not by 25 microM SP600125 (an inhibitor of JNK1/2) or 5 microM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1-->S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1-->S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.

Benzo(a)pyrene↗

PubMed2006

Age- and cell cycle-dependent changes in EPC-1/PEDF promoter activity in human diploid fibroblast-like (HDF) cells.

The changes in gene expression during senescence are very interesting. Early population doubling cDNA-1 (EPC-1, also known as pigment epithelial derived factor, PEDF) is one of the genes whose expression decreases dramatically during cellular aging. We examined whether or not EPC-1/PEDF promoter activity was affected by the cellular ageing using human diploid lung fibroblast cells in culture. Here we showed the promoter/enhancer region of EPC-1/PEDF existed at more than 1760 bp upstream from the transcriptional initiation site of the gene, and was regulated by both aging and cell cycle. These findings suggest that the expression of the EPC-1/PEDF gene is, at least in part, regulated transcriptionally in the cells. The analysis of the promoter region of the EPC-1/PEDF gene in this paper suggests the age- and cell cycle-dependent expression of specific transcriptional factor(s).

Cell Cycle↗

PubMed2000

Formation of 8-oxo-2'-deoxyguanosine in the DNA of human diploid fibroblasts by treatment with linoleic acid hydroperoxide and ferric ion.

Lipid peroxides are suggested to be related to the occurrence of a variety of diseases including cancer and atherosclerosis. We examined whether lipid peroxides cause oxidative damage to DNA in intact cells. Linoleic acid hydroperoxide (LOOH) and ferric chloride were used at concentrations at which separate treatment had no effect on the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in DNA or the survival rate of cultured human diploid fibroblasts, TIG-7. The amount of 8-oxodG in the cellular DNA increased significantly when TIG-7 cells were treated concurrently with LOOH and ferric chloride. In a LOOH concentration-dependent manner 8-oxodG was formed. However, no significant induction of the activities of superoxide dismutases, catalase, or glutathione peroxidase was observed under these conditions. The formation of 8-oxodG by lipid hydroperoxides seems to be due to the generation of reactive species other than superoxide radicals and hydrogen peroxide. These results indicate that some species formed during the reaction of lipid hydroperoxides with ferric ion can cause oxidative damage to DNA.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1991

Confirmed rabies exposure during pregnancy: treatment with human rabies immune globulin and human diploid cell vaccine.

A review of the literature shows 24 cases of pregnant human exposure to rabies virus through confirmed rabid animal bites. Historically, these patients received passive immunization with equine rabies immunoglobulin and/or purified vero cell vaccine or duck embryo vaccine. With the recent development of human-derived rabies vaccines, we report an additional case of human gestational rabies exposure, which was treated with human rabies immune globulin and human diploid cell vaccine.

Adult↗

PubMed1986

Biochemical and morphological studies of human diploid and fluoride-resistant fibroblasts in vitro.

Fluoride-resistant (FR 30) lines were produced from human diploid fibroblast (NDU-1) cells by progressively increasing the concentration of fluoride (F) in the growth medium up to 1.58 mM. This concentration completely inhibited the growth of the original cells. The resistant cells had decreased incorporation of [14C]-leucine and an increased rate of the cell division. The activities of acid phosphatase, alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alpha-hydroxybutyrate dehydrogenase in the FR 30 cells were lower than in the NDU-1 cells. The FR 30 cells had irregular shapes and sizes; the amount of undeveloped rough endoplasmic reticulum and the number of lysosomes were increased. These biochemical and morphological changes in FR 30 cells suggest that their metabolic activities were depressed by F, although they have some degree of F resistance.

Cell Division↗

PubMed1978

Transport of L-methionine in human diploid fibroblast strain WI38.

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.

Amino Acids↗

PubMed1975

Stimulated phosphorylation of non-histone phosphoproteins in SV-40 transformed WI-38 human diploid fibroblasts.

Because the phosphorylation of non-histone proteins has been suggested to play a role in the regulation of eukaryotic gene transcription, we have compared the phosphorylation of these proteins in normal and SV-40 transformed WI-38 human diploid fibroblasts. The rate of phosphorylation was found to be roughly ten-fold higher in the transformed cells, and this striking difference could not be accounted for by either an increased rate of phosphate transport or by the synthesis of new species of non-histone proteins which subsequently become phosphorylated. To our knowledge this is the most dramatic alteration in non-histone protein phosphorylation ever described, and therefore may have important implications for our understanding of malignant transformation.

Biological Transport↗

PubMed1984

Transcriptional control of human diploid fibroblast collagen synthesis by gamma-interferon.

Recombinant gamma-interferon (rec gamma-IFN) caused potent inhibition of collagen synthesis by cultured confluent human diploid fibroblasts in a dose-dependent manner. Gel electrophoresis of the newly synthesized proteins from the culture media of rec gamma-IFN-treated fibroblasts demonstrated a selective depression of procollagen without a significant change in non-collagenous proteins. Dot blot hybridization to a Type I procollagen cDNA probe showed that the inhibition of collagen production was accompanied by a decrease in the levels of collagen mRNA. These results indicate that rec gamma-IFN is capable of exerting transcriptional modulation of collagen biosynthesis and suggest that it may play an important role in regulation of normal and pathologic fibrogenesis.

Cells, Cultured↗

PubMed1987

Histone variant synthesis patterns in confluent populations of human diploid cells of different in vitro ages.

Histone variant synthesis patterns have been used to determine the cell cycle state of cultured cells. H2A and H3 histone variant synthesis patterns from confluent populations of human diploid fibroblast-like cells of different in vitro ages were determined and compared as ratios. In the absence of significant levels of DNA synthesis, there was an age related increase in the H2A ratio while the H3 ratio remained constant. These results indicate that confluent populations of older HDF are either in a different state of the cell cycle than younger cells or that they are synthesizing a different set of histone components while being maintained in the same cell cycle state at confluence. The synthesis and deposition of inappropriate histone components may alter chromatin structure in older cells and inhibit their subsequent progression through the cell cycle.

Arginine↗

PubMed1988

Proteose peptone enhances production of tissue-type plasminogen activator from human diploid fibroblasts.

Proteose peptone markedly enhanced the production of human tissue-type plasminogen activator (t-PA) from confluently cultured cell of human diploid fibroblasts. The cells continued synthesizing and secreting high levels of t-PA under periodic replacement of medium containing proteose peptone for more than one month. The highly increased activity correlated with equally increased levels of t-PA antigen and concomitantly increased levels of t-PA specific mRNA.

Caseins↗

PubMed1991

Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1.

Normal human diploid fibroblasts, TIG-1, which have a replicative life span of about 62 population doublings (PD), tended to senesce after about 50 PD with a gradual decrease in sensitivity to serum. Treatment of TIG-1 cells with the antisense-Rb oligomer, which completely depleted the retinoblastoma susceptibility gene product (RB), extended life span by about 10 PD. Treatment with the antisense-p53 oligomer alone had no effect; however, cotreatment with the antisense-Rb oligomer further potentiated the extension and the increased sensitivity to serum caused by the antisense-Rb oligomer alone, suggesting that p53 and RB function in separate, yet complementary pathways in signal transduction to senescence. The c-fos expression, which is presumed to be regulated negatively by RB, was not stimulated in partially senescent TIG-1 cells by treatment with the antisense-Rb oligomer.

Base Sequence↗

PubMed1985

14C-labeled substrate catabolism by human diploid fibroblasts derived from infants and adults.

Untransformed diploid skin fibroblasts from eight normal adults, aged 24 to 74 years, catabolized several 14C-labeled substrates less effectively than cells from ten normal male infants. 14C-labeled substrate metabolism was quantitated either by measuring the evolution of 14CO2 from the 14C-labeled compounds or the incorporation of 14C into cellular protein via transamination of tricarboxylic acid cycle intermediates derived from the 14C-labeled substrates. With these methods, adult cells catabolized [1-14C]butyrate, [1-14C]octanoate, and 1-[2-14C]leucine at rates 44 to 64% of those found in infant cells. The oxidation of [1,4-14C]succinate and [U-14C]malate was identical in both infant and adult cells, while [2,3-14C]succinate catabolism was mildly decreased in adult cells (65-80% of control). These observations parallel those made in rat tissues and confirm that the same phenomenon occurs in cultured human fibroblasts.

Adult↗

PubMed1984

Deoxyribonucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea and deoxynucleosides.

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to log-phase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18-21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.

Cells, Cultured↗

PubMed1983

Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations.

Both normal and leukemic human lymphoid cell lines were separated into populations corresponding to different positions in the cell cycle by centrifugal elutriation. Each population was analyzed for cell concentration, cell volume, [3H]thymidine incorporation, percentage S phase by autoradiography, and percent G1, S, and G2/M phases by flow cytometry. The smallest cells, collected at the lowest flow rate, were in G1 phase. Cells collected at increasing flow rates progressively increased in volume and represented distinct positions in the cell cycle transition from G1 phase, through S phase, and into G2/M phase. The purity of the G1 population varied according to cell load. One hundred percent of cells were recovered and cells collected in G1- and S-phase populations proliferated in culture with patterns characteristic of synchronized cells. An aneuploidy leukemia cell line, CEM, was separated into near-diploid and near-tetraploid populations by centrifugal elutriation. This method of cell separation provides large numbers of human lymphoid cells at different positions in the cell cycle for investigating the relationship between the cell cycle and various surface membrane and metabolic properties of cells. Aneuploid leukemia and lymphoma cells can be separated by centrifugal elutriation into populations which contain different numbers of chromosomes for comparisons of their biologic properties.

Aneuploidy↗