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PubMed1995

In vitro quantitative assessment of Echinococcus multilocularis metacestode viability after in vivo and in vitro maintenance.

The aim of this study was to apply the enzymatic MTT (3,5 dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide--formazan colorimetry for quantifying the viability of Echinococcus multilocularis whole cysts, after maintenance in vivo or in vitro. The enzymatic activities of young cysts freshly removed from rodents were linearly correlated with the parasite cyst weight. A comparative evaluation of the MTT assay and the in vitro viability assessments showed that the number of animals used for drug-screening purposes would be reduced by 35.8%. In this way, the use of different parasite samples removed from the same host is required, because of their different ages and their subsequent different abilities to reduce MTT. Cysts removed from mice exhibited higher colorimetric values than those removed from jirds. Thus, small entire cysts obtained from mice were maintained in the CMRL 1066 culture medium. Their enzymatic activities were evaluated at different times. The results indicate that, in such conditions, the optimal period of time for testing the effect of drugs against the metacestodes is limited to the 10 days following their transfer from mouse to culture flasks. The MTT assay encourages further studies to improve the viability of the whole cysts in vitro, using other standardizable culture conditions.

Animals↗
PubMed1989

Peroxidative stress and in vitro ageing of endothelial cells increases the monocyte-endothelial cell adherence in a human in vitro system.

Adherence of monocytes to the endothelium is an early event in atherogenesis. Changes in the endothelium are probably involved in this process. We have investigated the influence of peroxidative stress and in vitro ageing of endothelial cells on the subsequent adherence of monocytes using an in vitro system. Treatment of young human umbilical vein endothelial cells (HUVEC) with 60 microM cumene hydroperoxide (Chp) resulted in a 2.1-fold increase in monocyte adherence. This increase was not dependent on de novo synthesis of membrane antigens. No such increase upon Chp treatment was observed using young human umbilical artery endothelial cells (HUAEC) and in vitro aged HUVEC and HUAEC. As compared with "young" HUVEC, a higher spontaneous adherence of monocytes to "young" HUAEC (1.8 times), "aged" HUVEC (2.5 times) and "aged" HUAEC (2.7 times) was observed. Furthermore, fibroblasts showed an about 3 times higher monocyte adherence, whereas an 8 times higher adherence was observed to endothelial extracellular matrix. These data suggest that the spontaneous monocyte adherence to "young", intact endothelium can be increased by peroxidative damaging and by ageing of the endothelial cells. A denudation and subsequent exposure of the extracellular matrix may further stimulate this process. The possible physiological vector for endothelial peroxidation is discussed.

Arteriosclerosis↗
PubMed1988

In vitro culture of sheep oocytes matured and fertilized in vitro.

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.

Journal Article↗
PubMed1995

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro.

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.

Journal Article↗
PubMed1988

Small RNAs of the silkworm, Bombyx mori as revealed by in vitro capping and in vitro transcription.

1. We characterized several small RNAs in various tissues and strains of the silkworm, Bombyx mori by the in vitro capping and in vitro transcription. 2. Three subspecies of 7SL RNA, which is believed to be involved in protein secretion, were detected in the commercial strains. Structural differences among the three molecules were due to the nucleotide substitution at two positions or more. 3. A very AU-rich RNA, which is about 500 bases long, was always present in any tissue of Bombyx mori while 55 base RNA was only observed in a substantial amount in the fat body and culture cells. 4. These small RNAs were also observed among the in vitro transcripts of total Bombyx DNA in the cell-free extracts from the silkgland and fat body.

Animals↗
PubMed1997

In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro.

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions.

Animals↗
PubMed1997

In vitro fertilization and in vitro culture of bovine embryos in the presence of noncytopathic bovine viral diarrhea virus.

In vitro embryo production has been used extensively in research and is now offered as a commercial service, yet the hazards of introducing specific infectious agents into in vitro embryo production systems have not been completely defined. The introduction of noncytopathic bovine viral diarrhea virus (BVDV) is a special concern. One objective of this study was to determine if noncytopathic BVDV-infected uterine tubal cells in IVF and IVC systems affected the rate of cleavage and development. An additional objective was to determine if either degenerated ova or embryos produced in the presence of the infected cells had virus associated with them after washing. Follicular oocytes (n = 645) collected from slaughterhouse ovaries were matured and fertilized in vitro, and presumptive zygotes were cultured for 7 d. Primary cultures of uterine tubal cells for use during IVF and IVC were divided into 2 groups. One-half of the cultures was infected with noncytopathic BVDV while the other half was not exposed to the virus. Approximately equal groups of mature oocytes were inseminated, and the presumptive zygotes were cultured with infected or noninfected uterine tubal cells. After 7 d in IVC, zona pellucida-intact (ZP-I) morulae and blastocysts and degenerated ova were washed and assayed for the presence of infectious virus. Infections of uterine tubal cells were not apparent and did not reduce rates of cleavage and development (P > 0.05; Chi-square test for heterogeneity). After washing, BVDV was isolated at a significantly higher rate from groups of virus-exposed degenerated ova (79%) than from individual virus-exposed morulae and blastocysts (37%; P = 0.0002; Mantel-Haenszel summary, Chi-square).

Journal Article↗
PubMed1980

"Self-reactive" T cells. III. In vitro restimulation of T cells, "responding" in vivo or in vitro to syngeneic lymphoid cells.

Polyclonally activated lymphoblasts, transferred to syngeneic recipient mice, elicited a host T-cell-mediated "response" in vivo. These T cells, which "responded" in vivo to syngeneic lymphoblasts (i.e. in vivo primed "responder" T-cell population), acquired the capacity to "stimulate" a "response" of syngeneic T cells in vitro in a syngeneic one-way mixed lymphocyte culture, S-MLC (i.e. in vitro primed "'responder" T-cell population). We now describe the presence of memory and specificity in these two types of "self-reactive""responder" T-cell populations. This is investigated in in vitro "restimulation" experiments with mitomycin-blocked syngeneic and allogeneic lymphoid cells of various origin. "Self-reactive" T cells could be restimulated repeatedly (over many weeks) with mitomycin-blocked syngeneic lymphoid-cell populations, but not with mitomycin-blocked allogeneic normal spleen cells. "Self-reactive" T cells "responded" to syngeneic large activated lymphoblasts, as well as to syngeneic small resting lymphocytes. We found no "responder" T-cell reactivity specific for the mitogen that induced syngeneic "stimulator" cell populations. Both populations of "self-reactive" T cells displayed reactivity to mitogen-induced allogeneic lymphoblasts.

Animals↗
PubMed2003

Structural and morphological diversity of (1-->3)-beta-D-glucans synthesized in vitro by enzymes from Saprolegnia monoïca. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus).

Detergent extracts of microsomal fractions from Saprolegnia monoïca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-d-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and (13)C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoïca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization () exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their was approximately 5000. Of all the in vitro (1-->3)-beta-d-glucans, the one from R. fruticosus had the shortest length and the smallest. Crystallographic and spectroscopic data showed that the three in vitro samples consisted of triple helices of (1-->3)-beta-d-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoïca synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-d-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-d-glucans, and in relation with the possible existence of several (1-->3)-beta-d-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-d-glucans.

Crystallography, X-Ray↗
PubMed1999

A single-fiber in vitro motility assay. In vitro sliding velocity of F-actin vs. unloaded shortening velocity in skinned muscle fibers.

We describe an approach that allows us to form a micro in vitro motility assay with as little myosin as can be retrieved from a short (approximately 10 mm) segment of a single skinned skeletal muscle fiber (diameter some 100 microm). Myosin is directly extracted from the single fiber segment by a high ionic strength solution in the presence of MgATP, and the extracted myosin is immediately applied to a miniaturized flow cell that has been pretreated with BSA. The observed sliding velocities of fluorescently labeled F-actin are essentially identical with those reported in the literature. Since at the single fiber level most muscle fibers contain only a single myosin heavy chain isoform this approach allows us to determine without additional purification steps, the sliding velocity driven by myosins with different heavy chain isoforms. In addition, this approach can be used to directly correlate under identical experimental conditions unloaded shortening velocity measured in segments of skinned muscle fibers with the in vitro sliding velocity of fluorescently labeled F-actin by extraction of myosin from the same skinned fibers. Such direct correlation was performed with different myosin heavy chain isoforms as well as at different temperatures and ionic strengths. Under all conditions studied, unloaded shortening velocity was 4- to 8-fold faster than sliding velocity in the motility assay even at high temperature (22 degrees C) and ionic strengths >50 mM. This suggests that sliding velocity in the motility assay is limited by additional factors beyond those thought to limit velocity of unloaded shortening in muscle fibers. One such factor might be unspecific ionic interactions between F-actin and the substrate in the motility assay resulting in somewhat higher sensitivity for ionic strength of sliding velocity in the motility assay. This might become of special relevance when using in vitro sliding velocity in assessing functional consequences of mutations involving charged residues of actin or myosin.

Actins↗
PubMed1989

Use of an in vitro model for the assessment of muscle damage from intramuscular injections: in vitro-in vivo correlation and predictability with mixed solvent systems.

The potential of binary mixtures of propylene glycol-water, ethanol-water, and polyethylene glycol 400-water to cause skeletal muscle damage (myotoxicity) following intramuscular injection was examined with an in vitro model using the isolated rat muscle. At moderate concentrations (20-40%, v/v) of the organic cosolvent, the order of myotoxicity was propylene glycol greater than ethanol much greater than polyethylene glycol 400. The in vitro results were then compared with in vivo toxicity in rabbits after injection of normal saline, 40% (v/v) polyethylene glycol 400, 40% (v/v) propylene glycol, indocyanine green in normal saline, and indocyanine green in 40% (v/v) propylene glycol. Employing the area under the creatine kinase activity curve from 0 to 72 hr as the index of skeletal muscle damage, an excellent in vitro-in vivo correlation was observed. The basic myotoxicity relationships obtained from the binary cosolvent systems were then used to examine the myotoxicity of ternary organic cosolvent mixtures. Several mixed solvent systems with the same theoretical molar solubilization power for a model compound, diazepam, were selected to determine (1) if myotoxicity can be reduced by changing the composition of the ternary mixtures and (2) if myotoxicity of the individual components is additive. For the solvent systems containing propylene glycol, ethanol, and water, the total myotoxicity equaled the sum of the individual myotoxicity of each component. In contrast, for the solvent systems containing polyethylene glycol 400, the total myotoxicity was only half of the sum of individual toxicities. These results suggest that polyethylene glycol 400 in mixed cosolvent systems might have a protective effect on the myotoxicity generated by intramuscular injections.

Animals↗
PubMed2003

Prediction of in vivo tissue distribution from in vitro data. 2. Influence of albumin diffusion from tissue pieces during an in vitro incubation on estimated tissue-to-unbound plasma partition coefficients (Kpu).

PURPOSE: To determine the extent of albumin diffusion from tissue pieces into medium during in vitro incubations, to develop and assess the utility of mathematical models describing this effect on the estimation of tissue-to-unbound plasma partition coefficients (Kpu) of drug substances and to derive factors to correct for associated errors. METHODS: Twelve separate tissues were obtained from rats sacrificed by cervical dislocation, 48 h after an intravenous dose of 125I-human albumin, and tissue pieces incubated to determine the efflux of albumin into media over 2 to 4 h. A mathematical model was developed to predict and correct for the effect of albumin diffusion on the measured Kpu values of drugs. RESULTS: The model predicted that the effect of albumin diffusion from tissue pieces during in vitro incubation (ranging from 14 to 59% remaining in tissue) on Kpu values was generally minimal, except for compounds that are highly plasma bound and have a low measured Kpu. Under these circumstances, the measured Kpu substantially underestimates the true value. An equation was derived from readily available or measurable parameters to correct for this underestimation. CONCLUSIONS: Albumin diffuses from tissue pieces into protein free media during in vitro incubations until equilibrium is reached, defined by the albumin Kpu. Model predictions indicated that for the majority of compounds albumin diffusion would have a minimal effect on the measured Kpu value and that a correction factor could be calculated to account for any deviation.

Albumins↗
PubMedDate not supplied

In vitro infection of aortic endothelial cells by caprine arthritis encephalitis virus enhances in vitro transmigration of peripheral blood leukocytes and modulates their phenotypic expression.

Infection of goats by caprine arthritis-encephalitis virus (CAEV) provides a convenient example of the infiltration of various tissues by leukocytes following a natural lentiviral infection. This event is important in determining organ susceptibility and local immunity. Caprine vascular endothelial cells are susceptible to infection by CAEV in vitro, so we have investigated the consequences of this infection on the transmigration of uninfected leukocytes in an in vitro model. After in vitro infection by CAEV or stimulation by TNFalpha, the endothelial cells allowed the passage of tenfold more leukocytes from uninfected donors than did the uninfected endothelial cells. The transmigrating leukocytes were enriched in CD8+ lymphocytes, and the leukocytes appeared to have been activated during transmigration, as demonstrated by their expression of IL2R, MHC class II antigens and gamma-delta T-lymphocyte markers. CD4+, CD8+ and B-lymphocytes all proliferated in culture after transmigration. These results suggest that any possible infection or specific stimulation of endothelia in an infected animal could profoundly influence the choice of target organs and could activate the cells involved in local mucosal immune responses.

Animals↗
PubMed1975

In vitro infection of lymphoid cells by thymotropic radiation leukemia virus grown in vitro.

Murine lymphoid cells were infected in vitro with purified leukemogenic radiation leukemia virus (RadLV) produced by virus-induced lymphoblast cell lines. Thymocytes were shown to be highly susceptible to infection by the virus, whereas murine or other fibroblasts were refractory to it. Murine bone marrow and spleen cells were shown to be much less sensitive to infection by this thymotropic RadLV. By comparison, a B-tropic RadLV isolate (RadLV), propagated on a mouse fibroblast cell line, was noninfectious for lymphoid cells but infected fibroblasts. A correlation was shown to exist between in vitro infection of thymocytes, as assayed by immunofluorescence, and in vivo leukemogenicity of the thymotropic RadLV. This constitutes a rapid in vitro test for in vivo leukemogenicity of a natural lymphatic leukemia virus.

Animals↗
PubMed2005

In vitro synthesized small interfering RNAs elicit RNA interference in african trypanosomes: an in vitro and in vivo analysis.

RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific double-stranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.

Adenosine Triphosphate↗
PubMed1978

In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

Amoeba↗
PubMed1967

Gamma globulin and antibody formation in vitro. VII. Effect of x-irradiation on the secondary antibody response in vitro.

The present studies have shown that the influence of X-irradiation on the secondary antibody response in vitro is remarkably similar to its effect on the primary response in vivo. When sensitized tissue was first irradiated and then reexposed to antigen, the duration of the interval between irradiation and antigen addition determined the degree of inhibition of the secondary response obtained. A delay of 12 hr resulted in stronger inhibition than a delay of 6 hr, and an interval of 24 hr before reexposure to antigen caused complete suppression of antibody production to diphtheria toxoid and almost complete suppression when sheep RBC were used as the antigen. Induction of the secondary response in rabbit lymph node tissue in vitro followed by exposure to X-irradiation, revealed that immediate exposure to irradiation after antigen produced stronger inhibition of the subsequent response than irradiation on days 2-3. Irradiation on day 6 had no detectable effect. The effectiveness of the early radiation is probably due to prevention of the proliferation of the antibody-forming cells. BUDR was found to be effective at similar time periods as X-irradiation, whereas colchicine could still stop antibody formation when added late during the secondary response in vitro. It was noted that lymph nodes from some BSA-sensitized rabbits as late as 18 months after sensitization gave a response indistinguishable from a typical secondary response, even when not reexposed to antigen.

Animals↗
PubMed1969

Immune responses in vitro. II. Suppression of the immune response in vitro by specific antibody.

The effects of hyperimmune anti-sheep erythrocyte (SRBC) antibody on the plaque-forming cell (PFC) response to SRBC by mouse spleen cells in vitro were studied. Anti-SRBC antibody specifically suppressed the PFC response against SRBC. The degree of suppression was directly related to the amount of antibody added and was overcome by large amounts of antigen. Suppressive activity was absorbed from the sera by SRBC and could be partially eluted from the antigen by heat. The PFC response in cultures stimulated with antigen-antibody complexes prepared with high concentrations of antibody were suppressed; however, some complexes prepared at lower antibody concentrations stimulated greater responses than SRBC alone. Antibodies collected after four immunizations had greater suppressive ability than those collected after two immunizations. The degree of suppression was as great whether antibody was added at the initiation of the cultures or 24 hr later, suggesting that during the first 24 hr the culture system was antigen-dependent. Incubation of separated lymphoid cells with antibody did not impair their ability to develop a PFC response in vitro. However, if macrophages were incubated with antibody either before or after incubation with SRBC, the subsequent PFC response by lymphoid cells was suppressed. The data are consistent with the conclusion that antibody suppresses the PFC response in vitro by neutralizing the antigenic stimulus at the macrophage-dependent phase of the response.

Animals↗