Stability of fluorescein labeled dextrans in vivo and in vitro.

Fluorescein thiocarbamoyl dextrans (FITC-dextrans) were studied with respect to their potential use as tracer substances in circulatory research. It was found that the preparations did not contain any detectable free fluorescein. FITC-dextrans could conveniently be determined by fluorometry in the concentration range of 0.05–30 μg/ml. Gel filtration experiments demonstrated that the fluorescein label was evenly distributed in the molecular weight range of the dextran molecules, and the carbamoyl-dextran linkage was found to be perfectly stable both in in vitro and in vivo conditions. Intravenous infusion of FITC-dextran into rabbits resulted in plasma disappearance curves similar to those obtained with corresponding unlabeled dextran preparations. Consequently, FITC-dextrans should prove valuable as tracers for the study of many aspects of circulation.     

In vivo and in vitro binding of 1,2-dibromoethane and 1,2-dichloroethane to macromolecules in rat and mouse organs

Summary The comparative interaction of equimolar amounts of 1,2-dichloroethane and 1,2-dibromoethane with rat and mouse nucleic acids was studied in both in vivo (liver, lung, kidney and stomach) and in vitro (liver microsomal and/or cytosolic fractions) systems. In vivo, liver and kidney DNA showed the highest labeling, whereas the binding to lung DNA was barely detectable. Dibromoethane was more highly reactive than dichloroethane in both species. With dichloroethane, mouse DNA labeling was higher than rat DNA labeling whatever the organ considered: the opposite was seen for the bioactivation of dibromoethane. RNA and protein labelings were higher than DNA labeling, with no particular pattern in terms of organ or species involvement. In vitro, in addition to a low chemical reactivity towards nucleic acids shown by haloethanes per se, both compounds were bioactivated by either liver microsomes and cytosolic fractions to reactive forms capable of binding to DNA and polynucleotides. UV irradiation did not photoactivate dibromoethane and dichloroethane. The in vitro interaction with DNA mediated by enzymatic fractions was PB-inducible (one order of magnitude, using rat microsomes). In vitro bioactivation of haloethanes was mainly performed by microsomes in the case of dichloroethane and by cytosolic fractions in the case of dibromoethane. When microsomes plus cytosol were used, rat enzymes were more efficient than mouse enzymes in inducing a dibromoethane-DNA interaction: the opposite situation occurred for dichloroethane-DNA interaction, and this is in agreement with the in vivo pattern. In the presence of both metabolic pathways, addition or synergism occurred. Dibromoethane was always more reactive than dichloroethane. An indication of the presence of a microsomal GSH transferase was achieved for the activation of dibromoethane. No preferential binding in vitro to a specific polynucleotide was found. Polynucleotide labeling was higher than (or equal to) DNA binding. The labeling of microsomal RNA and proteins and of cytosolic proteins was many times lower than that of DNA or polynucleotides. The in vivo and in vitro data reported above give an unequivocal indication of the relative reactivity of the haloethanes examined with liver macromolecules from the two species and agree, on the whole, with the relative genotoxicity (DNA repair induction ability, mutagenicity and carcinogenicity) of the chemicals.Abbreviations CBI covalent binding index - EDTA ethylenediaminetetraacetate - GSH glutathione, reduced form - NADP nicotinamide adenine dinucleotide phosphate - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - PB phenobarbitone - poly(A) polyadenylic acid - poly (C) polycytidylic acid - poly (G) polyguanylic acid - poly (U) polyuridylic acid - poly (G) polyguanylic acid - poly (U) polyuridylic acid - POPOP 1,4-bis[2-(5-phenyloxazolyl)]-benzene - PPO diphenyloxazole - Tris tris(hydroxymethyl)aminomethane - UV ultraviolet Supported by a grant from Ministero della Sanità, Rome, Piano di ricerca nel campo delle malattie sociali, no. 500.4/RSC/135/L/1208     

Uptake of chloroquine by Onchocerca volvulus in vivo and in vitro.

Patients infected with Onchocerca volvulus in the Cayapa River focus in north-east Ecuador were given 500 mg chloroquine diphosphate (CQ) orally prior to nodulectomy. The concentrations of CQ were determined in parasite fragments and host tissue dissected from the nodules, in skin overlying the nodules, and in plasma at 3, 4, 7, and 24 hours after dosing. Onchocerca volvulus took up CQ rapidly, in some cases accumulating the drug to concentrations of over 600 pmol mg-1 worm tissue by three hours, and maintaining similar concentrations through 24 hours. These amounts were markedly higher than peak concentrations in plasma (3.16 pmol microliters-1) and in host tissues (78 pmol mgm-1) and skin (up to 93 pmol mg-1). In vitro uptake of CQ by females of O. volvulus was greater under alkaline conditions (pH 8.4) than at pH 6.8 and 7.4. Uptake reached equilibrium after one to two hours, with final concentrations being approximately 10 times lower than those reached in vivo. Inhibitory effects of chloroquine and its major metabolite desethylchloroquine on the motility of O. volvulus and other filariae have been observed previously in vitro; whether or not the drug had adverse effects on adult parasites in vivo was not determined in these experiments. However, the results illustrate the accessibility of O. volvulus to blood borne agents in vivo, and the potential importance of pharmacodynamic characteristics in the search for new macrofilaricidal agents.     

Factors affecting the inhibition by alloxan,and effect of streptozotocin on phosphate transport in isolated mouse mitochondria

Summary The swelling technique evidenced inhibition of P_i transport in isolated mouse liver mitochondria by 1 mM or higher concentration of alloxan, while no effect was found with streptozotocin. Complete or partial protection against alloxan inhibition was found under the following conditions: preincubation at 4 °C, or pretreatment with P_i, glucagon, succinate, malate or pyruvate. Complete protection was observed in isolated mitochondria from mice injected with glucagon. No protection was seen under the following conditions: preincubation at 37 °C, addition of microsomes, or pretreatment with insulin or glucagon in the presence of glucagon antibodies. So-called light and heavy mitochondria were as sensitive to alloxan as those obtained with the routine technique. Alloxanic acid had no effect, and addition of cysteine or glutathione abolished the inhibition by alloxan. Alloxan inhibited P_i transport also in isolated lung mitochondria. The findings suggest a direct action of alloxan on mitochondrial P_i transport which is affected by the energetic state. A relationship seems to exist between protection against alloxan toxicityin vivo and protection under the present experimental conditions.     

Uptake of polypeptide fragments of proteins by rat intestine in vitro and in vivo

Minute amounts of intact proteins were previously shown to be taken up from the intestine into the systemic circulation of mature animals; fragments were not detected. In this study, we sought evidence for uptake of fragments in both in vitro and in vivo experiments. Polypeptide fragments produced by pepsin digestion of bovine serum albumin (ranging in molecular weight from approximately 6000 to 25,000) were labeled with 125I. Everted jejunal gut sacs prepared from rat intestine were incubated with labeled fragments. After incubation, fluid exposed to the serosal surface was applied to a Sephadex G-50 gel permeation column. Radioactivity was detected in fractions corresponding to the elution position of the fragments. Transfer of fragments from the mucosal to the serosal surface was temperature-dependent. In in vivo studies, labeled fragments were infused into the jejunum of rats. Blood samples obtained from a mesenteric vein or the portal vein contained labeled fragments. After infusion of unlabeled fragments, nanogram amounts of immunoreactive fragments were detected by radioimmunoassay of mesenteric and portal venous blood. Thus, polypeptide fragments of a potential food protein were capable of being transferred across the mucosa in vitro and in vivo. Failure to detect fragments in the systemic circulation most likely results from their rapid clearance.     

In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria.

Slipped mispairing between repeated sequences during DNA replication is an important mutagenic event. It is one of several suggested mechanisms thought to be responsible for generating polymorphic regions and large-scale deletions found in mammalian mitochondrial DNA. In the porcine mitochondrial genome, a domain carrying a 10-bp tandemly repeated sequence displays a unique in vivo pattern of repeat copy number polymorphs. Upon passage in Escherichia coli, a recombinant plasmid containing this domain also displays a unique polymorphic pattern that is different from that seen in the animal. To test the hypothesis that these polymorphisms were slippage induced and that the different polymorphic patterns reflected differences in modes of replication, we performed a series of in vitro primer extension reactions. By utilizing either single- or double-stranded templates containing the repeat domain we were able to correlate in vitro generated repeat polymorphism patterns with those seen in the mitochondria or the bacteria, respectively, thus providing experimental evidence that slippage replication is responsible for a major class of mammalian mutations.     

Streptozotocin,but not alloxan,induces DNA repair synthesis in mouse pancreatic islets in vitro

In the present investigation, the abilities of streptozotocin and alloxan to induce DNA repair synthesis in isolated mouse pancreatic islets have been compared using an autoradiographic technique. Streptozotocin exposure in vitro induced a dose-dependent DNA repair synthesis, whereas no such effect was observed after alloxan treatment. The hydroxyl radical scavenger dimethyl urea and the poly(ADP-ribose) synthetase inhibitors nicotinamide and theophylline reduced the streptozotocin-induced DNA repair. The results suggest that the initial events in streptozotocin-induced B cell injury are DNA damage and repair and that alloxan exerts its major cytotoxic effect by a different mechanism.     

In vivo and in vitro effects of alloxan on collagen characteristics of bone, skin and tendon of Swiss mice

Alloxan diabetes in Swiss mice induced decreased levels of percent solubility of collagen in bone, skin and tendon both after 4 days (96 h) and 28 days of treatment. In vitro treatment of tissues with alloxan in physiological saline did not influence the cross-linking of collagen. It is suggested that in alloxan diabetes, metabolites formed in the tissues might accelerate aging of collagen.     

Effect of alloxan on phosphate transport in isolated mouse liver mitochondria: influence of pH, and differentiation between influx and efflux of phosphate

The effect of alloxan on inorganic phosphate (Pi) transport in isolated mouse liver mitochondria was studied by swelling techniques. Mitochondria preincubated with alloxan exhibited inhibition of Pi uptake assessed by NH4-Pi, K+-ionophore and acetate/Pi exchange systems, and also inhibition of Pi efflux assessed by the K+-ionophore and FCCP1-ATP systems. The effect on Pi uptake was pH dependent. Swelling in the FCCP-ATP system in the presence of alloxan and NEM was unaffected by rotenone and cysteine but was blocked by oligomycin, whereas the swelling caused by mersalyl was unaffected by rotenone, blocked by oligomycin, and reversed by cysteine. Alloxan stimulated mitochondrial ATPase activity, this effect being blocked by oligomycin. These findings suggest that alloxan causes an irreversible and pH-dependent inhibition of Pi influx and efflux in isolated mouse liver mitochondria.     

Embryologic development of a mouse sympathetic ganglion in vivo and in vitro.

The morphologic and biochemical development of the embryonic mouse superior cervical ganglion was characterized in vivo and in tissue culture. From 13 days of gestation, when the superior cervical ganglion was first visible, to birth at 19 days, tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] activity increased 100-fold in vivo. Explants of ganglia from 14-day embryos exhibited abundant neurite outgrowth in basal medium without added nerve growth factor (NGF), and increases in tyrosine hydroxylase activity paralleled that observed in vivo. Ganglia from 14-day embryos elaborated neurites and exhibited 3-fold increases in enzyme activity in vitro in the presence of antiserum to NGF (anti-NGF) or NGF + anti-NGF. In direct contrast, ganglia from 18-day fetuses failed to grow without added NGF or in medium containing anti-NGF or NGF + anti-NGF: virtually no axon outgrowth occurred and tyrosine hydroxylase activity decreased by half. These observations suggest that developmental regulatory mechanisms change radically during embryologic and fetal life of mammalian superior cervical ganglion.     

Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

The effect of zinc on mouse melanoma growth in vitro and in vivo

The effect of Zn2+ on mouse melanoma growth in vitro and in vivo was studied. Under in vitro conditions the proliferation of a Cloudman mouse melanoma cell line was inhibited by zinc ions at 10(-4) M, as measured by 3H-thymidine incorporation and optical density of NaOH cell digests. However, in vivo it was not possible to suppress both B16 and Cloudman S91 melanoma growth in mice by the administration of zinc ions. There were no significant differences in tumor growth after subcutaneous inoculation between mice constantly receiving 0.1% zinc acetate or 0.05% zinc sulphate in their drinking water and control groups, nor was it possible to decrease the number of lung metastases by zinc treatment after intravenous inoculation of tumor cells. The increased dietary supply of Zn failed to influence the survival time of mice in both melanoma types studied. Preincubation in vitro of cell suspensions in 10(-3) M zinc acetate prior to injection inhibited melanoma development in vivo. This implies that the in vivo zinc levels did not reach the necessary cytotoxic concentration.     

Uptake of hematoporphyrin derivative by atheromatous plaques: studies in human in vitro and rabbit in vivo

Hematoporphyrin derivative, a photosensitive material used to identify and treat neoplastic tissue in humans, has been found to localize in atheromatous plaques in animals and has recently been found in postmortem human atherosclerotic plaques. It is not known whether human plaques take up hematoporphyrin derivative in vivo. In five patients undergoing surgical vascular procedures, specimens containing atheromatous plaques were removed and immediately incubated in autologous oxygenated blood at 37 degrees C with hematoporphyrin derivative at a clinically relevant concentration for 2 hours. On exposure to ultraviolet light, porphyrin fluorescence was noted throughout each plaque, whereas adjacent plaque-free tissue showed no fluorescence. To compare in vitro with in vivo hematoporphyrin derivative uptake by plaques, the fluorescence of three types of arterial lesions (induced by a high cholesterol diet, catheters or balloon injury) was studied in 16 New Zealand White rabbits. Each lesion fluoresced selectively with the same intensity whether hematoporphyrin derivative exposure was performed in vitro or in vivo. Fluorescence microscopy did not show a difference in the pattern of hematoporphyrin derivative fluorescence between in vitro and in vivo specimens. The results suggest that human atheromatous plaques should take up hematoporphyrin derivative in vivo and are, therefore, potentially suitable for photochemical treatment as a new therapeutic approach to atherosclerosis.     

Variations in DNA methylation during mouse cell differentiation in vivo and in vitro.

Mouse teratocarcinoma cells induced to differentiate in vitro undergo a massive (30%) demethylation of DNA. A similar undermethylation is also observed in the mouse extraembryonic membranes, the yolk sac and placenta. In both cases, the decrease in methyl moieties occurs at a large number of CpG sites spread out over the entire genome, as indicated by a restriction enzyme analysis of several mouse genes including dhfr, beta-major globin, and the H-2K gene family. In contrast to this, the embryo itself appears to undergo methylation de novo during early stages of embryogenesis. Thus, as opposed to somatic cells, events during early mouse development are associated with wide variations in the level of DNA methylation. Although these changes in DNA methylation seem to be an integral part of the differentiation process, its relation to specific gene expression is still unclear.     

Flow cytometry of mouse bone marrow cells cultured in vivo or in vitro

We have developed new methods for counting cyto-(fluoro)-metrically cells retrieved from diffusion chamber and methylcellulose cultures of mouse bone marrow. Total cell counting and DNA distribution measurements were based on detection of the fluorescence from propidium iodide stained nuclei. Staining of cell nuclei with propidium iodide further allowed the separate enumeration of avid phagocytes that had endocytosed fluorescent latex beads. Finally, we quantified peroxidase positive cells by using o-Dianisidine as a substrate for the enzyme, the formation of brown reaction product being enhanced by the presence of lidocaine and dichlorophenol. We found the new techniques precise, reliable and suitable for analysis of cell growth and maturation in culture.