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At least 325 records · Page 18Linked to original sources
PubMed1987

Spermatozoal nuclear chromatin decondensation in vitro: a test for sperm immaturity. Comparison with results of human in vitro fertilisation.

Human sperm immaturity was tested by nuclear chromatin decondensation (NCD) in 1% sodium dodecylsulphate (SDS) of spermatozoa used in 74 consecutive IVF treatments. NCD was significantly higher in the presence of 2 mmol/l dithiothreitol (DTT) and after washing sperm from semen with 6 mmol/l ethylenediaminetetraacetic acid (EDTA). NCD was significantly less in insemination suspensions prepared by the swim-up technique than in the original semen. NCD with DTT was inversely correlated with sperm motility and motility index but there were no significant relationships between NCD and other semen analysis variables. There was no significant correlation between any NCD test and the proportion of oocytes fertilised in vitro. The only factors being significantly correlated with the fertilisation rate were proportion of sperm with normal morphology (Kendall correlation, tau = 0.36, P less than 0.001) and sperm concentration (tau = 0.14, P less than 0.05). Logistic regression analysis of fertilisation rates showed that only percentage normal sperm morphology was significant. It is concluded that NCD does not provide additional useful clinical information about sperm fertilising ability in vitro.

Cell Nucleus↗
PubMed1983

[Determination of in vivo and in vitro toxin activity. I. A new method of determining in vitro activity].

The potency of 5 toxins of different microbial species has been studied by common in vivo methods and by the in vitro method based on measuring the relative test-microbe growth inhibiting units 50. The possibility of using this method for the determination of toxicity in vitro with a view of studying the potency of diphtheria and gas-gangrene exotoxins, as well as that of Pseudomonas aeruginosa exotoxin, is substantiated.

Animals↗
PubMed1995

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Animals↗
PubMed1993

In vitro fertilization in domestic and non-domestic cats including sequences of early nuclear events, development in vitro, cryopreservation and successful intra- and interspecies embryo transfer.

The domestic cat may be used as a model for developing assisted reproduction techniques including in vitro fertilization (IVF), embryo culture, cryopreservation and embryo transfer (ET) for application to threatened and endangered species of non-domestic cats. Interoestrous domestic cats were injected with a total of 1.0-6.0 mg follicle-stimulating hormone (FSH) daily for 4 days and with 100 iu human chorionic gonadotrophin (hCG) on day 5. Follicular oocytes recovered at 26 +/- 1 h after hCG were co-incubated for 4-6 h at 38 degrees C in 5% CO2 with spermatozoa (1-2 x 10(5) motile spermatozoa ml-1) collected by artificial vagina. To determine the timing of sperm penetration and early fertilization events in vitro, oocytes were fixed and examined at intervals from 0.5 to 10 h after sperm exposure. The penetration rate of metaphase II (MII) oocytes at 0.5-3 h was equivalent to that at 3-6 h (95 versus 96%). Second polar body extrusion, pronuclear formation and apposition were observed at 2, 6-8 and 10 h, respectively. Simple (Tyrode's) and complex (F-10, M-199 and CMRL-1066) culture media with 10% fetal calf serum were compared for their ability to support development to the morula or blastocyst stage during culture periods of 96-168 h after IVF. The average number of cells per embryo was similar (P > 0.05) in the various media at each interval except that CMRL-1066 reduced (P < 0.05) development at 96 h if it was used before the two-cell stage. In F-10, neither the presence of intact cumulus cells nor changing to fresh F-10 medium at 48 h affected development at 96 h. Blastocyst development at 168 h was similar in both F-10 (18%) and Tyrode's (26%). To determine developmental ability in vivo, IVF-derived embryos (n = 586) were transferred at 96 or 120 h to recipients (n = 49) that had undergone synchronous oocyte recovery as donors. The percentage of recipients producing kittens after transfer of embryos cultured for 96 or 120 h in F-10 was 31 and 25, respectively, compared with 55% of 120 h recipients receiving embryos cultured in M-199 or Tyrode's. Overall, more pregnancies occurred following transfer of > or = 12 embryos (11/26) than if < 12 embryos were transferred (6/23).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Experimental studies on in vitro maturation of porcine oocytes. I. Effects of medium, energy source and protein source on in vitro maturation rate.

Effects of culture medium, energy and protein source on in vitro maturation of cumulus-intact porcine oocytes were examined. The bicarbonate buffered Whittens medium was superior to the PB1 medium buffered with phosphate salts in supporting in vitro maturation. However, modification of energy source of the PB1 medium by reducing Na-pyruvate concentration and adding lactate salts gave also high maturation rate. The supplementation of Whittens medium with 1 g and 3 g BSA/l as a protein source significantly increased incidence of maturation and improved handling of oocytes. There was no significant difference in the ability of low concentration (1 g/l) and high concentration (3 g/l) of BSA in culture medium to support maturation.

Animals↗
PubMed1993

Human plasma alpha 2-macroglobulin promotes in vitro oxidative stress cracking of Pellethane 2363-80A: in vivo and in vitro correlations.

It is hypothesized in this study that the phenomenon of environmental stress cracking (ESC) in polyetherurethane is caused by a synergistic action of biological components in the body fluids, oxidative agents, and stress. An in vitro system is designed to mimic the in vivo system; human plasma contains certain biological components that can act as a stress cracking promoter, while H2O2 (Co) solution provides an oxidative reaction comparable to that observed in the respiratory burst of adherent macrophages and foreign-body giant cells. It is demonstrated that the phenomenon of in vivo stress cracking in Pellethane 2363-80A is duplicated by an in vitro system that involves a pretreatment of prestressed specimens with human plasma at 37 degrees C for 7 days followed by oxidation in 10% hydrogen peroxide with 0.10M cobalt chloride at 50 degrees C for 10 days. The pretreatment with plasma has a synergistic effect with the oxidation by H2O2 (Co) treatment to produce ESC. A plasma component responsible for promoting stress cracking in Pellethane polyurethane is identified to be alpha 2-macroglobulin (alpha 2M).

Animals↗
PubMed1998

In vivo and in vitro phosphorylation of the alpha 7/PRS1 subunit of Saccharomyces cerevisiae 20 S proteasome: in vitro phosphorylation by protein kinase CK2 is absolutely dependent on polylysine.

In this paper, we show that the Saccharomyces cerevisiae 20 S proteasome subunit 1 (PRS1), recently renamed as alpha 7, is the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome component. In vitro phosphorylation occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasome from mammalian ones which are phosphorylated by CK2 in the absence of polylysine. A peptide reproducing the long acidic C-terminal tail of alpha 7/PRS1, where consensus CK2 phosphorylation sites are located, was also phosphorylated by the CK2 holoenzyme in a polylysine-dependent manner, suggesting that this region contains structural features responsible for this particular behavior.

Amino Acid Sequence↗
PubMed1996

In vitro effects of gamma-hexachlorocyclohexane on in vitro biosynthesis and metabolism of steroids in goldfish Carassius auratus.

Testicular and ovarian fragments of Carassius auratus, taken during the reproductively active prespawning phase (June) of its annual reproductive cycle, were incubated with different concentrations (0 mg/ml, 0 ppm; 0.001 mg/ml, 1 ppm; 0.01 mg/ml, 10 ppm; and 0.02 mg/ml, 20 ppm) of gamma-hexachlorocyclohexane (gamma-HCH) in the presence of either exogenous precursor [3H]-17-hydroxyprogesterone ([3H]17-P) or carp hypophyseal homogenate. The free (unconjugated) and conjugated metabolites (glucuronides and sulfates) of [3H]-17-P [androstenedione (AD), androstenetrione, 17-hydroxyprogesterone, testosterone (T), 11-deoxycortisol (S), 17, 20alpha-dihydroxy-4-pregnen-3-one (17,20alphaP), 17, 20beta-dihydroxy-4-pregnen-3-one (17,20betaP), 7alpha-pregnanetetrols (7alpha-P), and other polar metabolites] were separated by thin-layer chromatography and high-performance liquid chromatography. The endogenous production of unconjugated (free) steroids T, 17,20betaP, S, and 11-ketotestosterone (11-KT) in response to gamma-HCH were measured by radioimmunoassay. Among the in vitro metabolism of [3H]-17-P, in males, free steroids of AD, T, 17,20alphaP, S, and polar-free steroids were increased with the decreased yield of 11-KT. Percentage yield of testosterone glucuronide (TG) was increased with highly significant decreased yields of polar glucuronide steroids. The sulfate steroids of 17, 20alphaP, 17,20betaP, S, and 11-KT remain unchanged. In females, the decreased percentage of yield of AD and S and elevated T were noticed. The yield of TG was increased with decreased yield of 7alpha-P glucuronides. The percentage yield of AD sulfate and sulfate steroids of 17,20alphaP, 17,20betaP, and S were noted to be increased, but the yield for S sulfate was very high. Endogenous production of T was increased in both sexes in the presence of gamma-HCH, but 11-KT in male and S in female were depressed. 17, 20betaP was stimulated at some concentrations in both sexes but levels were very low. Results indicate that gamma-HCH in vitro perturbed the steroid biosynthesis during this phase thereby affecting reproductive physiology.

17-alpha-Hydroxyprogesterone↗
PubMed2002

Effects of dietary phytoestrogens in vivo and in vitro in rainbow trout and Siberian sturgeon: interests and limits of the in vitro studies of interspecies differences.

A study of the effects of dietary genistein on trout and sturgeon in vivo showed that sturgeon was sensitive to 20 ppm of genistein, whereas trout was not. To analyze the origin of this interspecies difference in sensitivity, a cell culture technique was developed with hepatocytes from sturgeon and compared to results obtained with hepatocytes from trout in the same system. The hepatocyte culture proved to be useful as bioassay for estrogenicity. Vitellogenin (VTG), assayed by a specific enzyme-linked immunosorbent assay, was used as a biomarker of the estrogenic activity. 17 beta-Estradiol, its glucuronide and sulfate derivatives, and estradiol analogues (ethynylestradiol and diethylstilbestrol) were tested. Nonestrogenic compounds such as androgens, progesterone, and cortisol were tested as negative controls. VTG production was monitored at doses ranging from 1 nM to 10 microM estradiol. Phytoestrogens, from the isoflavone family, were tested individually at increasing doses exhibiting dose response curves for concentrations from 500 nM to 10 microM. With tamoxifen, an antagonist of estrogen receptors, the estrogenic effect was partially reduced. The effect was the same with ICI182,780 in sturgeon, whereas the effect was the opposite in trout. The estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A < daidzein = formononetin < genistein < equol in trout and biochanin A < genistein < daidzein < formononetin < equol in sturgeon. Further, in sturgeon, formononetin was the most potent phytoestrogen in vitro, whereas its activity was weakest in vivo. These data suggest that one must reconsider the relevance of heterologous estrogenic tests and of homologous in vitro tests for estrogenic potency of chemicals.

Animals↗
PubMed2000

Cytotoxic activity of kaempferol glycosides against human leukaemic cell lines in vitro.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control.

Antineoplastic Agents, Phytogenic↗
PubMed1993

Technetium-99m labelled LDL as a tracer for quantitative LDL scintigraphy. I. Tracer purification, in vitro and in vivo long-term stability, in vitro validation and biodistribution.

The goal of the present study was to optimize technetium-99m labelling of low-density lipoprotein (LDL) and to investigate the in vitro and in vivo properties of the tracer to determine whether its application for quantitative scintigraphy of hepatic LDL receptor activity is feasible. LDL labelled with iodine-125 by the iodine monochloride method was used as a reference tracer. Comparison of different assessments of radiochemical purity [trichloro-acetic acid precipitation (%ppTCA), paper chromatography, size-exclusion chromatography and chloroform-methanol extraction] exhibited %ppTCA to be superior as a parameter of tracer quality. In spite of a high radiochemical purity immediately after labelling, modifications of 99mTc labelling of LDL did not overcome the poor long-term stability of the tracer. Subsequent dialysis in phosphate buffer over about 3 h sufficiently increased the long-term stability in vitro and in vivo. The competitive recognition of dialysed 99mTc-LDL and 125I-LDL with native LDL by high-affinity binding sites was demonstrated in human hepatoma cells (HepG2) and human fibroblasts. Biodistribution data of simultaneously injected 99mTc-LDL and 125I-LDL in New Zealand White rabbits showed a high uptake of both tracers in tissues with high LDL receptor activity, yet 99mTc-LDL uptake exceeded 125I-LDL uptake by two- to sevenfold. In contrast to 125I-LDL, 99mTc-LDL showed a higher unspecific uptake into the bone marrow and the spleen, suggesting an additional uptake mechanism probably via the scavenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Aqueous extract of betel-nut of north-east India induces DNA-strand breaks and enhances rate of cell proliferation in vitro. Effects of betel-nut extract in vitro.

An aqueous extract of betel-nut has been found to be able to induce strand breaks in DNA of mouse kidney cells in vitro. It has been also found to be able to enhance the rate of cell division at a dose of 100 micrograms/ml while a higher dose of 250 micrograms/ml was extremely toxic to the cells. Compared with arecoline (10 micrograms/ml), the aqueous extract of betel-nut seems to be a more potent carcinogen to mouse kidney cells in vitro.

Animals↗
PubMed1985

An economical large scale procedure to purify E. coli amplifiable plasmids for DNA sequencing, in vitro transcription and in vitro mutagenesis.

A reproducible and economical procedure for obtaining a large and quantitative yield of highly purified covalently closed circular plasmid DNA is described. The procedure departs in several ways from more commonly used methods. These are a) avoidance of the use of CsCl, ethidium bromide and ultracentrifuge, b) enrichment of the plasmid DNA by selective denaturation of chromosomal DNA with an alkaline-SDS solution, c) enrichment of covalently closed circular plasmid DNA by extraction with acid-phenol, and d) removal of small degraded RNA fragments by molecular sieve chromatography after digestion with RNase A. The plasmid DNA prepared by this new procedure is free of contaminants and has been used for DNA sequencing, in vitro transcription, transformation and in vitro mutagenesis.

Base Sequence↗
PubMed1999

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro.

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal beta-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of beta-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

Animals↗
PubMed1997

Characterization of in vitro culture of HIV-negative Kaposi's sarcoma-derived cells. In vitro responses to alfa interferon.

We established long-term cultures from skin tumors of nine patients suffering from classical Kaposi's sarcoma (KS). Spindle cells obtained after enzymatic digestion were cultured on gelatin- or fibronectin-coated flasks in DMEM with 15% fetal calf serum, aFGF and heparin. Immunohistochemical staining was positive for MHC class I, laminin, type IV collagen, vimentin, alpha smooth muscle actin (20-40% of cells), caldesmon (20%), calponin (20-40%) and smooth muscle myosin (20-40%), and was negative for common leukocyte antigen, CD4, LFA1, CD34 and cytokeratin. Around 20% of cells up to the third passage in culture expressed the endothelial markers CD36, BMA 120 but were negative for UEA and Fc von Willebrand. Smooth muscle proteins were detected with immunoblotting. Using the polymerase chain reaction, human herpes virus 8 (HHV8) sequences were detected in primary cultures of three out of seven cell lines but were rapidly lost during in vitro passaging. KS-derived cells did not proliferate in serum-free medium, had a normal karyotype and did not grow in soft agar medium. Tumors formed in nude mice injected with KS-derived cells. The tumors were composed of mouse cells and were highly vascularized. Our results suggest that KS-derived cells are heterogeneous: the majority of cells have either a smooth muscle cell or a fibroblastic phenotype. Another minor cell compartment was composed of endothelium-derived cells. KS cells do not possess the characteristics of transformed cells in vitro and may be composed of polyclonal activated cells. Recombinant alpha interferon (rIFN) slightly inhibited the growth of KS-derived cells and increased the expression of MHC class I antigens. While cells were resistant to natural killer (NK) cell-mediated cytotoxicity, they became sensitive to rIFN-primed NK cells. Thus, the antitumor potential of rIFN against KS in vivo could result from immunomodulatory rather than from direct antiproliferative effects.

Animals↗
PubMed1979

Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.

Animals↗
PubMed1977

In vitro induction of cell-mediated immunity to murine leukemia cells. II. cytotoxic activity in vitro and tumor-neutralizing capacity in vivo of anti-leukemia cytotoxic lymphocytes generated in macrocultures.

The main findings of the present study are: (a) highly reactive cytotoxic lymphocytes (CTL) against syngeneic and allogeneic murine leukemia cells were generated in vitro in macro 'one-way' mixed leukocyte-tumor cultures (MLTC). Cultures set up in large tissue culture flasks contained up to 400 X 10(6) normal spleen cells (responder cells) and 20--40 X (10(6) mitomycin C-treated leukemia cells (stimulator cells). Successful sensitization in macrocultures was greatly dependent upon the responder cell density and the responder/stimulator cell ratio. Cytotoxic activity, as measured by the 51Cr-release assay, peaked on day 5--7. (b) Sensitized 'memory' lymphocytes produced in primary MLTC could be restimulated with the original tumor cells to give a more rapid and stronger secondary cytotoxic response. (c) lymphocytes sensitized to allogeneic leukemia cells reacted equally well with sensitizing leukemia cells and with the corresponding normal lymphoid target cells, whereas lymphocytes sensitized to syngeneic leukemia cells did not react with the homologous normal lymphocytes. (d) Cryopreserved normal splenocytes and leukemia cells were as efficient as fresh cells in generating allogeneic and syngeneic CTL. (e) Using a Winn-type tumor neutralization assay, it was shown that both allogeneic and syngeneic splenocytes sensitized in vitro to EL4 leukemia (of C57BL/6 mice) and to YAC leukemia (of A mice) were capable of preventing tumor growth in the syngeneic host, whereas cultured normal splenocytes frequently showed a tumor-enhancing effect. Long-term survivors, remaining after inoculation of leukemia cells and sensitized lymphocytes, also became resistant to a tumor challenge that was up to 10,000 greater than the minimum lethal dose.

Animals↗
PubMed1975

In vitro metabolic activation of chemical mutagens. I. Development of an in vitro mutagenicity assay using liver microsomal enzymes for the activation of dimethylnitrosamine to a mutagen.

Qualitative and quantitative assays were developed to study the in vitro enzymatic activation of dimethylnitrosamine (DMNA) to its mutagenic form. Three different fractions from mouse liver homogenates, including purified microsomes, were employed for the activation, and several parameters of the assays were investigated. Qualitative tests were conducted to measure the ability of hepatic enzymes obtained from six mammalian species to activate DMNA. A comparison between two inbred mouse strains using the in vitro activation assay demonstrated that this technique might be a useful tool in quantitatively measuring differences in genetically influenced levels of DMNA metabolism in individuals animals and their tissues.

Animals↗