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PubMed1987

Spontaneous and cyclophosphamide-induced sister-chromatid exchanges in diploid and endoreduplicated tetraploid metaphases of preimplantation mouse embryos.

Endoreduplicated tetraploid metaphases could for the first time be induced in preimplantation mouse embryos by culture in the suboptimum medium MEM. In such endomitoses sister-chromatid exchange (SCE) frequency was approximately the same during the first and the second cell cycle. However, when morulae and blastocysts were cultured in the presence of cyclophosphamide metabolites SCE frequency was increased predominantly during the second cell cycle. Compared to diploid metaphases a decreased SCE frequency was found under both conditions of endomitoses induction, which may be related to DNA-repair processes.

Animals↗

PubMed1985

Trypanosomes of subgenus Trypanozoon are diploid for housekeeping genes.

The ploidy of trypanosomes has until now remained undetermined, although isoenzyme studies and direct measurements of DNA content and complexity suggest diploidy. Direct cytogenetic analysis is not possible, because the chromosomes do not condense at any stage of the cell cycle. We now present evidence from analysis of restriction site polymorphisms in and around three glycolytic enzyme genes (phosphoglycerate kinase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase) and the tubulin gene cluster, that trypanosomes of subgenus Trypanozoon are diploid for these housekeeping genes. This result is still compatible with the single copy nature of variant surface glycoprotein (VSG) genes in Trypanozoon, if different VSG genes are present in corresponding positions on paired chromosomes. Using pulse field gradient gel electrophoresis, we show that the genes for the three glycolytic enzymes are all located in very large DNA molecules, but the gene for triosephosphate isomerase is in another fraction from the genes for the other two enzymes. Since all three enzymes are located in glycosomes, which are trypanosome microbodies, the genes for glycosomal enzymes are not all clustered in one chromosomal segment of the trypanosome genome.

Animals↗

PubMed1989

Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid.

The hypothesis that metacyclic trypanosomes are haploid has been tested genetically. Five cloned stocks of Trypanosoma brucei (each having four known isoenzyme markers and six known restriction fragment length polymorphisms) have been independently transmitted through tsetse flies. Fifteen individual metacyclic organisms were taken from flies with mature cyclical infections and used to establish fresh clones. All the sub-clones from all the flies proved to be identical to the starting (parental) stocks, with respect to all the markers examined, including those markers which were heterozygous in the parental stocks. We conclude that metacyclic trypanosomes are diploid, and are not the product of an obligatory meiosis.

Animals↗

PubMed1993

Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro.

Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.

Alternative Splicing↗

PubMed1982

Adaptive enhancement of cystine and glutamate uptake in human diploid fibroblasts in culture.

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2-3-fold increase, and the increased rate of uptake was accompanied by an increase in the Vmax and unchanged Km. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in th uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that te transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.

Adult↗

PubMed1983

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts.

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation (t1/2 approximately equal to 10-15 min for monoacetylated histone H4) compared to the slower rate (t1/2 approximately equal to 140-200 min for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.

Acetylation↗

PubMed1996

Protein kinase C inhibits the CAK-CDK2 cyclin-dependent kinase cascade and G1/S cell cycle progression in human diploid fibroblasts.

Serum stimulation of human diploid fibroblast IMR-90 cells leads to phosphorylation of p33CDK2 at Thr160 and activation of CDK2 kinase, a necessary event for G1/S transition. We report that serum stimulation causes a gradual, sustained increase in the activity of CDK-activating kinase (CAK) that phosphorylates CDK2 at Thr160, which starts by 5 h after serum stimulation and reaches the maximal plateau level at around the G1/S boundary. In this cell type addition of phorbol-12, 13-dibutyrate 5 h but not 16 h after serum stimulation completely inhibits CDK2 kinase activation and DNA synthesis. Phorbol ester treatment does not reduce the protein level of p33CDK2, but does inhibit serum-stimulated increases in the CAK activity and CDK2 phosphorylation at Thr160. The suppression of the CAK activity by the phorbol ester is accompanied by decreases in the message levels of both CDK7 and cyclin H, the catalytic and the positive regulatory subunit of CAK, respectively. These results indicate that in IMR-90 cells activation of protein kinase C in the late G1 phase causes cell cycle arrest before the G1/S boundary at least in part through downregulation of CAK and CAK-mediated CDK2 phosphorylation and activation.

Antibodies↗

PubMed1988

Role of active oxygen species in metal-induced DNA strand breakage in human diploid fibroblasts.

The ability of 6 metal salts to induce DNA damage in human diploid fibroblasts was examined. Cadmium, magnesium, manganese, chromium(VI), zinc and selenite were all shown to induce DNA strand breaks as measured by two independent assays. DNA strand breaks were repaired within 2-4 h after removal of metal and this repair appeared not to be sensitive to "long-patch" repair inhibitors. With the exception of selenite, metal-induced DNA damage appeared to be mediated via the formation of active oxygen species since oxygen scavengers when administered simultaneously with the metal, antagonized strand break formation. Selenite-induced DNA damage (as previously reported) was dependent on the formation of a selenite-glutathione conjugant and was not affected by oxygen radical scavengers. Scavenger treatment did not enhance cloning ability of metal-treated cells suggesting that DNA strand breaks may not be important in metal-induced cytotoxicity.

Cells, Cultured↗

PubMed1984

Pre-exposure rabies prophylaxis: efficacy of a new packaging and delivery system for intradermal administration of human diploid cell vaccine.

The efficacy of a newly designed syringe prepacked with human diploid cell vaccine in a sufficient quantity to deliver individual 0.1 ml doses intradermally was tested by injecting 40 veterinary students with a single dose on each of days 0, 7 and 28. A second group of 20 students received the ordinary series of three 1.0 ml intramuscular vaccine doses by needle and syringe. All participants in both groups developed neutralizing antibodies to rabies above the suggested minimum of 0.5 international units per ml by day 49. The geometric mean titres were somewhat lower in the group receiving the 0.1 ml doses compared to the group given 1.0 ml doses; however, this was considered of no clinical significance since everyone achieved a titre above the suggested minimum. There was little difficulty in reconstituting the vaccine prepacked in the lumen of the syringe and the syringe was easy to manipulate.

Adult↗

PubMed1986

Immune complex-like disease in 23 persons following a booster dose of rabies human diploid cell vaccine.

Following a routine 0.1 ml booster dose of Merieux rabies human diploid cell vaccine (HDCV), administered intradermally, 23(10.2%) of 226 persons had signs and symptoms compatible with an immune complex-like disease. The disease had its onset from 3-13 days after the injection, lasted 1-5 days, and consisted primarily of urticaria (78.3%), macular rash (65.2%), angiooedema (39.1%), and arthralgia (17.4%). None of the cases were considered severe, and all recovered with no sequelae. There were significant differences in attack rates between men (78.3% of all cases) and women, and between those receiving vaccinations on different days. Similar reactions have been reported following intramuscular booster doses of HDCV. Since the Merieux HDCV is used worldwide, physicians administering HDCV must be aware of these adverse reactions and warn patients. Appropriate therapy should be instituted as warranted by severity of reactions.

Adult↗

PubMed1986

Rhesus diploid rabies vaccine (adsorbed): neurological safety in guinea pigs and Lewis rats.

Rabies vaccine produced in rhesus diploid cells (RDRV) and adsorbed on aluminium phosphate was evaluated for its neurological safety in guinea pigs and Lewis rats. The vaccine (as well as aluminium phosphate itself) in combination with myelin basic protein did not induce experimental allergic encephalomyelitis (EAE) when injected into either species. RDRV combined with complete Freund's adjuvant still failed to induce any signs of EAE. In contrast, basic protein combined with complete Freund's adjuvant induced severe EAE in both guinea pigs and rats. No experimental evidence was obtained to indicate adverse neuroimmunological activity of RDRV.

Animals↗

PubMed1987

Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man.

Clinical reactions and neutralizing antibody responses to six pre-exposure regimens of purified chick embryo cell culture rabies vaccine (PCECV) and human diploid cell strain rabies vaccine (HDCSV) were studied in 177 volunteers. Antibody kinetics, height of the response and persistence of antibody over two years were virtually identical after PCECV and HDCSV. An antibody response was detected in all subjects on day 14 when the highest titres were found after two intramuscular (i.m.) 1.0 ml doses of a schedule of immunization on days 0, 7 and 21. In comparison, a schedule of immunization on days 0, 28 and 56 ultimately evoked the highest titres 21 days after the final injection, but antibody persisted equally well over two years with either schedule. Neutralizing antibody titres were lower after intradermal (i.d.) vaccination with 0.1 ml compared to 1.0 ml i.m. on days 0, 7 and 21, but when given on days 0, 28 and 56 the responses were comparable. Three subjects with a personal or family history of atopy developed urticarial lesions after PCECV. Both vaccines were otherwise well tolerated.

Adolescent↗

PubMed1989

Comparative study of the safety and protective value, in pre-exposure use, of rabies vaccine cultivated on human diploid cells (HDCV) and of the new vaccine grown on Vero cells.

The object of the study is to compare the tolerance and the efficacy of a new inactivated rabies vaccine grown on Vero cells (PVRV), with the vaccine cultivated on human diploid cells (HDCV), using the schedule recommended by WHO for pre-exposure on days 0, 7 and 21. Of students exposed to a risk of rabies at Alfort Veterinary School, 144 volunteers received either HDCV or PVRV vaccine. No student received a booster between the first blood sample before immunization on day 0 and the last antibody titration 21 months after the beginning of immunization. No serious side-effect occurred with either vaccine, although some vaccinees complained of redness, induration or local pain and, exceptionally, of fever. The study indicates the excellent immunogenicity of both HDCV and PVRV vaccines in all vaccinees. The geometric mean of antibody titres shows a higher titre in the PVRV group and a rapid decline in immune response with both vaccines, four months after the first injection, followed by a stabilization of the antibody level throughout the rest of the study. It should be noted, however, that very few individuals were found to be seronegative 21 months after the first injection.

Adult↗

PubMed1989

Human diploid cell rabies vaccine purified by zonal centrifugation: a controlled study of antibody response and side effects following primary and booster pre-exposure immunizations.

Systemic allergic reactions following booster immunizations have complicated rabies pre-exposure prophylaxis with the human diploid cell rabies vaccine licensed in the US (conventional HDCV). We conducted two studies comparing an HDCV purified by zonal centrifugation to conventional HDCV. In a study of primary pre-exposure immunization, volunteers received one of four regimens: three 1.0-ml intramuscular (i.m.) or 0.1-ml intradermal (i.d.) doses of conventional or purified HDCV over 28 days. Although volunteers vaccinated i.m. had significantly greater rabies neutralizing antibody titres (VNA) 49 days, 91 days and 26 months after immunization began than volunteers vaccinated i.d. (p less than 0.005-p less than 0.05), there were no significant differences between vaccines. In a study of booster immunizations, 77 volunteers immunized with conventional HDCV 2 years earlier received a 0.1-ml i.d. booster with either conventional or purified HDCV. VNA was significantly greater with the conventional HDCV on days 7 and 28 after booster, but not on day 365. A moderate or severe reaction was reported by 5 (13%) of the 40 persons who received boosters with conventional HDCV, versus none of 37 who received the purified HDCV (p = 0.03). Purified HDCV appears to be preferable to conventional HDCV for booster vaccination.

Antibodies, Viral↗

PubMed1992

Longevity of rabies antibody titre in recipients of human diploid cell rabies vaccine.

Sera from individuals across the USA and Peace Corps Volunteers (PCV) were assayed for neutralizing antibody to human diploid cell rabies vaccine (HDCV). A pairwise comparison between intramuscular (i.m.) and intradermal (i.d.) administration, PCV and non-PCV, revealed statistically significant differences between i.m. and i.d. for PCV and non-PCV, and between PCV and non-PCV for i.d. and i.m. administration. Survival analysis and frequency tables employing chi 2 tests determined that the percentage probability of maintaining an adequate titre from 1.5-2.0 years after primary vaccination was 99% in non-PCV i.m., 93% in non-PCV i.d., 88% in PCV i.m., and 64% in PCV i.d.

Adolescent↗

PubMed1993

Risk factors for systemic hypersensitivity reactions after booster vaccinations with human diploid cell rabies vaccine: a nationwide prospective study.

To determine the incidence of and risk factors for adverse reactions following the boosters, we conducted a nationwide prospective study of persons receiving pre-exposure booster vaccination with human diploid cell rabies vaccine (HDCV). Persons who had previously received three pre-exposure doses of HDCV and whose rabies neutralizing antibody titres were < or = 1:5 were enrolled in the study if they stated that they intended to receive a booster. Of the 98 persons enrolled in the study, 40 (41%) were in risk groups for whom boosters are not recommended. Three (3%) of 98 developed generalized urticaria or wheezing within 1 day of receiving boosters and three others (3%) developed urticaria 6 to 14 days after the booster. No differences were found between individuals with reactions (either type) and those with no adverse reaction according to age, gender, occupation, history of previous allergies, or time since or route of primary vaccination. Reactions were somewhat more common among persons who received primary vaccinations by the intramuscular route (i.m.) and booster vaccinations by the intradermal route (i.d.) (3/15, 20%) or primary vaccinations i.d. and booster vaccinations i.m. (2/10, 20%), and somewhat less common among persons who received both these vaccinations i.d. (1/52, 2%) or i.m. (0/7). The number of persons who develop allergic reactions may be minimized by administering vaccinations only when vaccination is strictly indicated. The influence of the route of primary and booster vaccinations on the development of reactions deserves further study.

Adult↗

PubMed1995

Efficacy study of a new albumin-free human diploid cell rabies vaccine (Lyssavac-HDC, Berna) in 100 severely rabies-exposed Thai patients.

A newly developed human diploid cell rabies vaccine (Lyssavac-HDC), produced without added serum albumin and with an effort to remove the virus-inactivating beta-propriolactone prior to addition of the gelatin, L-cysteine and potassium phosphate stabilizer, was tested for safety immunogenicity, adverse reactions and efficacy in 100 severely rabies-exposed Thais. All patients also received human rabies immune globulin and vaccine was administered using the conventional 5-dose intramuscular schedule of one dose on days 0, 3, 7, 14 and 28. One hundred percent of a subgroup of 40 subjects, where blood had been collected, had neutralizing antibodies greater than 0.5 IU ml-1 on days 28 and 90 and all had detectable titers on days 7, 14, 28, 90, 180 and 360. All patients could be followed for at least 1 year and remained well. No significant side-effects from this vaccine were noted.

Adolescent↗

PubMed1980

Analysis of intracellular distribution and binding of benzo[alpha]pyrene in human diploid fibroblasts.

Previous work with low passage synchronized human foreskin fibroblast cell populations has indicated that benzo[alpha]pyrene (BP) can induce a carcinogenic event [3]. BP additionally has shown to damage DNA in log-arithmically growing low passage cultures [9]. High passage cells, on the other hand, seem to be refractory to transformation by BP, even though this agent can induce DNA damage, similar to that seen in low passage cells. When low passage cells were treated with BP, the initial binding of the hydrocarbon was primarily to a cytoplasmic protein complex of molecular weight 12,500 while in high passage cells, a major portion of BP was bound to protein complex of molecular weight 200,000. High-pressure liquid chromatography (HPLC) profiles of ethyl acetate extractable fractions from the BP-cytoplasmic protein complexes of low and high passage cells demonstrated that the majority of the BP remained unmetabolized. When nuclei were isolated from low and high passage cells prior to the HPLC analysis, the major component (90%) was again unmetabolized BP. The results suggest selective attachment of BP to different cytoplasmic protein complexes of logarithmically growing human diploid fibroblast cells dependent on the passage level of the cells.

Benzopyrenes↗