Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “In Vitro”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources
PubMed1982

Normal development following in vitro fertilization in the cow.

A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.

Animals↗
PubMed1992

Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo.

The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.

Animals↗
PubMed1998

Oocyte competence for in vitro maturation is associated with histone H1 kinase activity and is influenced by estrous cycle stage in the mare.

The in vitro maturation rate of equine oocytes remains low, regardless of culture conditions. Our objective was to determine the reasons for failure of equine oocytes to resume meiosis during in vitro maturation and to ascertain the influence of the estrous cycle stage on meiotic competence. In 10 cyclic mares, 7 ultrasound-guided follicular punctures were performed alternately during the follicular phase (group DF; n = 3 punctures), at the end of the follicular phase (group EF; n = 2), and during the luteal phase (group DL; n = 2). We evaluated the competence of the oocytes for in vitro maturation and measured their maturation-promoting factor activity by histone H1 kinase assay. Puncturing once at the end of the follicular phase and once during the luteal phase, or three times during the follicular phase, yielded about 11 cumulus-oocyte complexes per 22 days. The maturation rate was different between the groups, 51% in group EF, 34% in group DL (p < 0.05), and 15% in group DF (p < 0.01), and it increased with an increase in follicular diameter (p < 0.05). After in vitro culture, the H1 kinase activity was lower in oocytes that remained in germinal vesicle or dense chromatin stages than in oocytes that reached metaphase I or metaphase II (p < 0.05). The H1 kinase activity was not different between oocytes in germinal vesicle stage after in vitro maturation and immature oocytes that were not cultured in vitro, and was higher in preovulatory oocytes that reached metaphase II in vivo than in the oocytes that reached metaphase II after in vitro maturation (p < 0.001). This is the first report on kinase activity in the equine oocyte.

Animals↗
PubMed2000

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo.

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.

Animals↗
PubMed1996

Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.

We measured intracellular pH (pHi) in single vascular smooth muscle cells (VSM) cultured from rabbit abdominal aorta, using 2',7'-biscarboxyethyl-5(6)carboxyfluorescein (BCECF) on a microscope-based fluorimetric system. We previously found substantial errors introduced by using high K+/nigericin to calibrate intracellular BCECF (1). We also previously demonstrated that the necessary correction (pHcor) to the high K+/nigericin-calibrated pHi was linearly dependent on pHi, increasing with increasing pHi (2). When the nigericin calibration data were corrected using this pHcor, the new corrected calibration was similar to the result of calibrating BCECF in vitro (higher Rmax, lower Rmin, and lower pK). Therefore, in this study the possibility is considered that in vitro calibrations might provide better estimates of pHi. Our best estimate for the absolute level of pHi derives from a null method for bracketing steady-state pHi. In VSM cells, using only in vitro calibrations to estimate steady-state pHi leads to less error (only approximately 0.08 different from null estimates) than using nigericin calibrations alone (approximately 0.2 different from null estimates). Unlike high K+/nigericin calibrations, the error, pHcor, introduced by using an in vitro calibration is pHi independent. Using high K+/nigericin or in vitro calibrations, along with the respective pHcor on the same experimental days in the same cells, gave the same estimate of pHi whether in the steady state, in acid-loaded cells, or in alkali-loaded cells. Similarly, when appropriately corrected, both methods gave indistinguishable calibration curves. Thus, the two methods are entirely equivalent from the standpoint of accuracy. Because nigericin is toxic, expensive, and complicated in its use, we suggest that in vitro calibrations, along with simple null determinations to assess the small, constant correction factor, be used to calibrate intracellular BCECF.-Boyarsky, G., Hanssen, C., Clyne, L. A. Superiority of in vitro over in vivo calibrations of BCECF in vascular smooth muscle cells.

Animals↗
PubMed2002

Persistent chimerism despite antidonor MHC in vitro responses in miniature swine following allogeneic hematopoietic cell transplantation.

BACKGROUND: T-cell chimerism predominates in miniature swine receiving hematopoietic-cell transplantation without myelosuppressive conditioning. Several chimeric recipients have become hyporesponsive to donor-major histocompatibility complex (MHC) in vitro and accepted donor-matched renal transplants without immunosuppression. However, some retained antidonor in vitro responses and subsequently rejected donor renal allografts despite the persistence of peripheral blood chimerism. In this study, we characterize the donor cells in both "tolerant" and "nontolerant" chimeric miniature swine. METHODS: Peripheral blood chimerism was determined by flow cytometry. In vitro antidonor responsiveness was determined by mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Donor cells were separated from chimeras by immunomagnetic bead separation and used as stimulators or targets in CML assays. Phenotypic analysis of donor cells in chimeras was performed using flow cytometry. RESULTS: Peripheral blood chimerism stabilized beyond 100 days and was made up almost entirely of T cells. PBMC from nontolerant chimeras could be stimulated in vitro to kill donor cells isolated from the mixed chimera itself. In contrast, PBMC from tolerant chimeras hyporesponsive to donor-type cells could not be stimulated in vitro to kill their own sorted donor cells. CONCLUSIONS: The in vivo persistence of donor T cells in mixed chimeric animals with in vitro antidonor responsiveness is not caused by an inability of these cells to be killed but rather by the poor stimulating capacity of these donor T cells. The nature of donor T cells that persist in the face of in vitro antidonor responses, has important implications for the induction of transplant tolerance by way of the generation of mixed chimerism.

Animals↗
PubMed1979

Labelling index of human squamous cell carcinomas. Comparison of in vivo and in vitro labelling methods.

In vivo and in vitro labelling has been compared in sixteen human solid squamous cell carcinomas (ENT). The median in vivo/in vitro LI ratio was 1-2, but for two-thirds of the patients it was only 1-1, suggesting a slight LI underestimation in vitro. Two factors can possibly explain the divergences: heterogeneity from one biopsy to another in the same tumour, and lower mean grain count in the deep cell layers of the in vitro labelled tumour samples. Therefore, the fact we did not find a good agreement between in vivo and in vitro data for one-third of the tumours points out that one must be cautious in considering in vitro LI as a valid result for a given patient. However, even if the in vitro LI leads to a certain underestimate, it can provide useful data.

Autoradiography↗
PubMed2004

A comparative expression analysis of gene transcripts in post-fertilization developmental stages of bovine embryos produced in vitro or in vivo.

This study was carried out to examine the temporal variation in the relative abundance of transcripts during the post-fertilization stages of bovine embryos derived from in vitro or in vivo culture. For this purpose, cumulus-oocyte complexes obtained from ovaries from slaughterhouses were matured, fertilized and cultured in vitro. The in vitro culture was carried out using CR1 medium. In vivo embryos were derived from superovulated and artificially inseminated Simmental heifers and cows. Embryos were recovered from both systems approximately at day 1 (two-cell), day 2 (four-cell), day 3 (eight-cell), day 4 (16-cell), day 5 (morula) and day 7 (blastocyst) after insemination. Relative abundance of target transcripts was performed using real-time PCR. The transcripts studied were the nucleosome assembly protein, mRNA for alivin 1, Pleckstrin homology Sec7 coiled domain, polyadenylate binding protein, NADH dehydrogenase subunit 2, high-mobility group transcription factor, cytokine-like nuclear factor, NY-REN-58 antigen mRNA, mRNA for KIAA1764 and one novel transcript. These transcripts were derived from our previous study conducted using mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) between in vitro-produced bovine eight-, 16-cell, morula and blastocyst-stage embryos. The results of the present study have demonstrated the temporal variation in the relative abundance of these transcripts between in vitro and in vivo culture systems. Such variation in the relative abundance of transcripts during the post-fertilization developmental stages of in vitro and in vivo embryos may reflect the effect of the in vitro culture condition on the transcriptional activity thereby on the developmental competence of the resulting embryos.

Animals↗
PubMed2002

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.

Animals↗
PubMed1987

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Animals↗
PubMed1975

Correlation of structural changes at different levels of the jejunal villus with positive net water transport in vivo and in vitro.

Experiments were done for indentification and localization of certain structural changes at different levels of jejunal villus of the hamster during positive and negative water transport across the intestine in vivo and in vitro. Positive transport occurred when the mucosal surface of the intestine was bathed (in vitro experiments) or perfused (in vivo experiments) with isotonic Krebs-Ringer bicarbonate solution containing 10 mM glucose, and negative water transport was achieved by rendering this solution hypertonic with 150 mM mannitol. Results indicate that during positive net water transport the intestine in vivo transported more fluid and exhibited a more conspicuous dilatation of the lateral intercellular spaces (L.I.S.) than did the in vitro preparation. Dilatation of the L.I.S. in both preparations was present only in the apical part of the villus, suggesting that this is the principal site of water absorption. When the mucosal solution was made hypertonic with mannitol, the L.I.S. in the in vivo intestine totally collapsed, whereas in the in vitro intestine these spaces remained open very slightly. These morphological changes correspond well with our finding that in the presence of the hypertonic mucosal solution there was a greater net negative water transport in vivo than in vitro. Incubation of the intestine in the isotonic mucosal solution produced subnuclear swelling of the mid-villus epithelial cells, and this morphological change was associated with an increase in the water content of the tissue. Perfusion of the in vivo intestine with the isotonic solution produced neither the swellings nor the increase in water content of the tissue. In the presence of hypertonic mucosal solution there was a water loss from the tissue both in vivo and in vitro, and these swellings were not observed. These results are discussed in relation to intestinal sugar transport and to the maturity of the epithelial cells, and it is concluded that transport studies on in vitro preparations may provide valid information on a qualitative basis, if not on a strictly quantitative basis.

Animals↗
PubMed1996

Angiotensin II formation in dog heart is mediated by different pathways in vivo and in vitro.

Angiotensin-converting enzyme (ACE) inhibitors (I) have beneficial effects that are presumably mediated by decreased angiotensin II (ANG II) production. However, in vitro assays in human heart extracts have demonstrated that > 75% of ANG II-forming enzyme activity was not inhibited by captopril (Cap) and therefore did not appear to be related to ACE but was inhibited by chymostatin, suggesting that it was predominantly chymase-like activity. Previous work in our laboratory has demonstrated a similar relative contribution of ACE and chymase-like activity toward ANG II formation in vitro in dog heart tissue extracts. Accordingly, we compared Cap-inhibitable ANG II formation in vitro in heart tissue of five adult mongrel dogs to the in vivo Cap-inhibitable, ANG II-forming activity across the myocardial bed in four openchest, adult mongrel dogs. In vitro studies demonstrated that only 6 +/- 2% of ANG II formation was inhibited by Cap from heart tissue extracts of the left ventricular midwall. In in vivo studies, ANG I (0.5 nmol/min) followed by ANG I plus the ACE inhibitor Cap (0.1 mumol/min) was infused into the left anterior descending artery, and ANG II was assayed in the proximal aorta and coronary sinus. The arterial-venous (A-V) difference of ANG II across the myocardial circulation increased significantly during ANG I infusion (-13.4 +/- 23.5 to 142.8 +/- 71.4 pg/ml; P < 0.03). Subsequent coinfusion of Cap with ANG I significantly decreased the myocardial A-V difference of ANG II by 60 +/- 18% (P < 0.05). Thus, in contrast to the in vitro situation, ANG II formation in vivo is inhibited significantly by Cap in the normal dog heart. This comparison of in vivo and in vitro conversion of ANG I to ANG II by ACE and chymase-like activity suggests that in vitro assays may underestimate the functional contribution of ACE to intracardiac ANG II formation.

Angiotensin I↗
PubMed1985

Effects of in vitro corticosteroids on B cell activation, proliferation, and differentiation.

The present study demonstrates the graded effect of in vitro corticosteroids (CSs) on the different phases of B cell activation, proliferation, and differentiation. Early events such as activation and proliferation of high-dose anti-mu or Staphylococcus aureus-stimulated B cells are profoundly suppressed by the presence of in vitro CSs. The suppressed proliferative response may be mediated by a direct effect on B cells and/or modulation of accessory cell function. Later events in the B cell cycle such as the proliferative response to B cell growth factor after either in vivo or in vitro activation are less sensitive to the suppressive effects of in vitro CSs. The final events in the B cell cycle; namely, the differentiation to the immunoglobulin-producing state, is not suppressed by in vitro CSs. Indeed, depending on the systems employed, there is either no effect or enhancement of immunoglobulin secretion by the presence of in vitro CSs. The graded effect of in vitro CSs on the discrete phases of the B cell activation, proliferation, and differentiation cycle provide new insights into the complex nature of CS-induced modulation of human B cell responses.

Adult↗
PubMed1986

Percutaneous absorption of benzo[a]pyrene in the rat: comparison of in vivo and in vitro results.

Percutaneous absorption of 14C-labeled benzo[a]pyrene (BaP) was studied in five-day in vivo and in vitro experiments with female Sprague-Dawley rats following single topical doses of 9-10 micrograms/cm2. The in vivo percutaneous absorption was measured by the presence of 14C radioactivity in urine, feces and tissues. In vitro percutaneous absorption was measured with excised (non-viable) skin in Franz-type diffusion cells. Several modifications of standard diffusion cell techniques which are known to enhance the transport of lipophilic compounds were evaluated. In vitro penetration was determined by directly measuring the level of 14C radioactivity in the receptor fluid. BaP was observed to readily penetrate in vivo with a total of 46.2% (n = 4, SD = 3.4%) of the applied dose being absorbed over five days. In in vitro experiments using an approximately 350 micronthick skin section and normal saline receptor solution, only 2.1% of the applied BaP diffused into the receptor fluid over five days. In in vitro experiments using full-thickness skin and a 6% solution of nonionic surfactant receptor fluid, 28.0% of the applied BaP diffused into the receptor fluid over five days. When both an approximately 350 microns-thick skin section and a 6% surfactant receptor solution were used in vitro, 49.9% (n = 4, SD = 3.1%) of the BaP dose was found in the receptor fluid after five days. The results show that the modified in vitro method is suitable for studying percutaneous absorption of lipophilic compounds such as BaP.

Administration, Cutaneous↗
PubMed2006

A new extract of the plant Calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation.

BACKGROUND: Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS: An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS: The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION: These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Animals↗
PubMed1985

In vitro 3,5,3'-triiodothyronine binding to rat cerebrocortical neuronal and glial nuclei suggests the presence of binding sites unavailable in vivo.

The maximal binding capacity (MBC) of the rat cerebrocortical nuclear T3 receptor, as determined by in vivo saturation techniques, is approximately half that measured in vitro on isolated nuclei or solubilized receptors. To investigate this disparity, the MBC values determined in vivo and in vitro for both rat cerebral cortex and liver were compared, taking into account nuclear receptor loss or inactivation and the presence of endogenous T3. By Scatchard analysis of T3 binding to isolated nuclei in vitro at 37 C, the uncorrected MBC values (mean +/- SEM; n = 3) for the cerebrocortical nuclear T3 receptor in euthyroid and hypothyroid rats were 0.80 +/- 0.14 and 0.66 +/- 0.07 ng T3/mg DNA, respectively, and were not significantly different. The Kd values were also not significantly different (5.6 +/- 0.3 and 5.2 +/- 0.9 X 10(-10) M, respectively). After corrections for incomplete dissociation and receptor inactivation under the in vitro conditions, the overall mean MBC increased by approximately 33% to 0.97 ng T3/mg DNA, or about 3.6 times the in vivo MBC. In addition, cerebrocortical nuclei prelabeled in vivo with +/- 131I]T3 at near-saturating levels and subsequently incubated with [125I]T3 in vitro at concentrations up to 10 times the Kd were shown to bind as much as 4 times more T3 in vitro relative to the amount of endogenous hormone which dissociated, thus exceeding the in vivo MBC by a factor of two. Parallel experiments with isolated liver nuclei did not show the existence of nuclear T3 receptors which were available only in vitro, even when the corrected MBC (0.77 ng T3/mg DNA) was compared with the MBC obtained by the in vivo saturation technique (0.76 ng T3/mg DNA). The experiments with liver nuclei were done at 25 C to reduce the rate of inactivation or loss of nuclear T3 receptors in this tissue. By fractionating isolated cerebrocortical nuclei into neuronal and glial subpopulations on discontinuous sucrose gradients, the high affinity, limited capacity nuclear T3 receptor could only be identified in the neuronal fraction. No consistent specific binding of T3 was observed in glial nuclei that were 80% pure, suggesting that either glial cells in the adult rat are not likely to be direct targets of thyroid hormone or that thyroid hormone may act via nonnuclear receptor-mediated pathways. We conclude that only neurons have specific high affinity, limited capacity nuclear T3 receptors and that as many as half of these receptors may not be accessible to plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

In-vivo and in-vitro metabolic clearance of midazolam, a cytochrome P450 3A substrate, by the liver under normal and increased enzyme activity in rats.

The metabolic clearance of midazolam, a cytochrome P450 (CYP) 3A substrate, by the liver under normal and increased enzyme activity in rats was determined in-vivo and in-vitro to elucidate the reproducibility of the in-vivo hepatic extraction ratio of midazolam from the in-vitro study. The hepatic enzyme activity was modified by pretreating rats with a CYP inducer such as dexamethasone and clotrimazole. The in-vivo hepatic extraction ratio (ERh,obs) of midazolam under a steady-state plasma concentration (approx. 3 nmolmL(-1)) in untreated (control) rats was 0.864. This value increased to 0.984 in dexamethasone-pretreated rats and to 0.964 in clotrimazole-pretreated rats. The in-vitro hepatic intrinsic clearance (CL(int,in-vitro)), expressed as mLmin(-1) (mg microsomal protein)(-1), of midazolam was estimated as Vmax (Km)(-1) by in-vitro metabolism studies using liver microsomes. The CL(int,in-vitro) value was converted to the CL(int,cal) value, expressed as mLmin(-1)kg(-1), by considering the microsomal protein content (g liver)(-1) and the microsomal protein content (g liver)(-1)kg(-1). The estimated CL(int,cal) value was then converted to the ERh value (ER(h,cal)) according to the well-stirred, the parallel-tube and the dispersion models. The ERh(h,cal) values obtained by the parallel-tube model were in good agreement with corresponding in-vivo ERh(h,obs) values. In conclusion, it was demonstrated that high hepatic clearances of midazolam under normal and increased CYP3A activity were reasonably predicted from in-vitro metabolism studies using liver microsomes.

Administration, Oral↗
PubMed2004

In vitro delayed senescence of extirpated buds from zooids of the colonial tunicate Botryllus schlosseri.

In the colonial growth of botryllid ascidians, blastogenesis (bud formation) is a cyclical and synchronized developmental process characterized by a weekly rhythm of budding and apoptotic events. Very little is known about this cycle regulation and its control. In this study, the in vitro fate of developing buds and regressing zooids extirpated from Botryllus schlosseri colonies at different blastogenic stages were examined, revealing that stages 'B' to 'D' buds (but not stage 'A' buds) developed new structures under in vitro conditions. These were mainly spheres (up to 1 mm in diameter) and epithelial monolayers around the attached buds. We also found that: (1) when attached spheres and epithelial monolayers appeared, the life expectancy of an isolated bud in vitro reached 50-60 days, five times the life expectancy of intact, in vivo developing zooids; (2) the life expectancy of in vitro buds that remained unattached to the substrates was at least 150 days; (3) after attaching to the substrates, buds obeyed a newly imposed developmental clock dictating up to 35 survival days for spheres and up to 14 days for epithelial monolayers; (4) the prevailing mode of death in vitro was necrotic, in contrast to the apoptotic mode of zooidal deterioration at the takeover phase of blastogenesis; (5) under in vitro conditions, degenerating zooids surprisingly produced epithelial monolayers within 3 weeks of culturing. Monolayers survived for up to 10 additional days, extending the lifespan of the degenerating zooids from a few hours to up to 1 month. We conclude that under in vitro conditions, not only are the underlying colonial growth mechanisms replaced by different developmental pathways, but also the internal colonial-level clocks programming death, are replaced by a new biological mechanism with different timetables.

Aging↗