Showing papers on "In vivo published in 1970"

Reduced plaque formation by the chloromethyl analogue of victamine C.

Abstract: T H E C H L O R O M E T H Y L A N A L O G U E of Victamine C,* a cationic surface-active agent, reduces the formation of dental calculus in humans. 1 2 This chemical prevents crystallization of calcium phosphate in calculus smears in vitro, 3 but the mechanism whereby it inhibits calculus formation in vivo is not known. The following study was conducted to determine whether the chloromethyl analogue of Victamine C reduces the formation of dental plaque considered a precursor of calculus. 4 6

N-Hydroxy-2-acetylaminofluorene Sulfotransferase: Its Probable Role in Carcinogenesis and in Protein-(methion-S-yl) Binding in Rat Liver

Abstract: Summary 1- and 3-Methylmercapto-2-acetylaminofluorene (1- and 3-methylmercapto-AAF) were identified as degradation products of hepatic protein-bound methionyl derivatives in rats given AAF or N-hydroxy-AAF. The same derivatives were shown previously to be products of the in vitro reaction of free or peptide-bound methionine with various synthetic esters of N-hydroxy-AAF, including the very reactive O-sulfonate (AAF-N-sulfate). The amounts of o-methylmercapto-AAF obtained from liver protein were proportional to the dose of N-hydroxy-AAF given i.p. and appear to be a measure of the amount of ester-like metabolites of the carcinogen formed in vivo. 1- and 3-Methylmercapto-2-aminofluorene were not obtained as degradation products of the liver proteins of rats or other animals given N-hydroxy-AAF or of rats given N-hydroxy-2-aminofluorene. The soluble proteins of rat liver contain sulfotransferase(s) which synthesize AAF-N-sulfate from N-hydroxy-AAF and 3′-phosphoadenosine 5′-phosphosulfate; magnesium and manganous ions stimulate the system. Under a variety of conditions, the N-hydroxy-AAF sulfotransferase activity in rat liver, as assayed in vitro, paralleled the amounts of o-methylmercapto-AAF which could be released from the livers of similarly treated rats after administration of N-hydroxy-AAF and the susceptibility to liver tumor induction by N-hydroxy-AAF. Adult male rat liver, which is highly susceptible to carcinogenesis with N-hydroxy-AAF, contained considerably more N-hydroxy-AAF sulfotransferase activity and formed considerably more protein-bound 1- and 3-(methion-S-yl)-AAF in vivo than did the livers of female rats or of male mice, hamsters, rabbits, or guinea pigs. Female rats and the latter species are relatively resistant to hepatic carcinogenesis with N-hydroxy-AAF. Similarly, the increased hepatic carcinogenicity of 2-aminofluorene derivatives in female gonadectomized rats given testosterone and the marked inhibition of hepatocarcinogenesis with N-hydroxy-AAF in hypophysectomized or thyroidectomized male rats were mirrored by corresponding changes in hepatic sulfotransferase activity for N-hydroxy-AAF and the formation of protein-bound methionyl-AAF derivatives in vivo. The results strongly suggest that AAF-N-sulfate is at least one of the ultimate reactive and carcinogenic metabolites of AAF and N-hydroxy-AAF in rat liver. Syntheses of the following new compounds are described: N-methoxy-AAF, potassium 4-acetylaminobiphenyl-N-sulfate, 3-methylmercapto-4-acetylaminophenyl, and 3-methylmercapto-4-aminobiphenyl hydrochloride.

The plaque=inhibiting capacity of 11 antibacterial compounds.

Abstract: The plaque inhibiting effect in vivo of 11 antibacterial agents was compared with their antibacterial activity against salivary bacteria in vitro. The in vivo effect was tested for 4 days in a human model with a supplement of sucrose in the diet. The antibacterial activity of the compounds, which were chosen from different main groups of disinfectants (alcohols, iodophores, dyes, quaternary ammonium bases, amidines, guanidines) were tested in four in vitro systems. No correlation was evident between the in vivo and in vitro effects. Chlorhexidine gluconate and -acetate proved most effective in vivo, whereas several other substances equally or more effective against salivary bacteria in vitro, exhibited no effect in vivo. It is concluded that other factors than the antibacterial properties are important in plaque inhibition in vivo.

Cleavage rate of mouse embryos in vivo and in vitro

Abstract: Mouse embryos (Q strain) developing in vivo from the 2-cell to the blastocyst stage showed a constant cell doubling time of about 10 h. Embryos cultured in vitro over the same period showed a rate of cleavage which was initially almost as great as in the reproductive tract, but subsequently declined to give a doubling time of about 24 h. Addition of oestrogen to the culture medium increased the diameter of blastocysts but did not increase cell number.

The role of nonlymphoid accessory cells in the immune response to different antigens.

Abstract: Tissue culture techniques were combined with cell separation procedures to investigate the cellular requirements for a response to antigen, leading to the production of antibody-forming cells. Mouse spleen was dissociated, and the cells were separated into various groups on the basis of density, size, and active adherence. The ability of fractions to initiate a response in vivo, on transfer to an irradiated recipient, was compared to the response in vitro; and this ability was correlated with the presence or absence of phagocytic cells. Two different antigens were studied, sheep erythrocytes (SRC) and polymerized bacterial flagellin (POL). Density distribution analysis of spleen showed a wide density range of cells responding to both antigens in vivo. The same fractions responded to POL in vitro as in vivo. By contrast, only the light density regions responded in vitro to SRC. Response occurred in regions of overlap between lymphocytes and phagocytic macrophages. Separation by active adherence on columns of large glass beads gave a preparation containing large, medium, and small lymphocytes but no detectable phagocytic macrophages and very low levels of phagocytic polymorphs. This lymphocyte preparation responded to both antigens in vivo. In vitro it gave a full response to POL, but no response to SRC. Addition of a small quantity of the adherent fraction, enriched for phagocytic cells, restored response to SRC. The use of strain-specific antisera in a mixed culture containing a C57 phagocytic fraction and CBA lymphocytes showed that the lymphocyte fraction contributed the precursors of the final antibody-forming cells. The accessory cells from C57 spleen banded in the light regions of the density gradient where phagocytic macrophages were found. Irradiated spleen cells also activated the lymphocyte preparation, suggesting that the irradiated host provided the accessory cells for the in vivo response to SRC. Small lymphocytes were purified from spleen by the small glass bead size filtration technique. This sample of small lymphocytes responded less well to POL than the total lymphocyte population, but it responded as well in vitro as in vivo. The small lymphocyte preparation responded in vivo to SRC but not in vitro. Addition of a small quantity of the phagocyte-rich fraction from adherence columns restored the in vitro response to SRC. The results indicated that phagocytic cells are not required in the initiation of an immune response to POL. By contrast some accessory cell, possibly a phagocytic macrophage, is required for a response to SRC. The basis for this marked difference is discussed.

Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo.

Abstract: Exposure of normal human skin in vivo to ultraviolet irradiation at wavelengths shorter than 320 nanometers stimtulated an unscheduled DNA synthesis in all of the cell layers of the epidermis and in the upper dermnial fibrocytes. The skin of patients with xeroderma pigmentosum did not show this response. correlation of these findings with previous tissue culture studies suggests that the defect in repair of the damaged DNA in xeroderma cells occurs in vivo as well as in vitro.

Differential Toxicity on Normal and Transformed Cells in vitro and Inhibition of Tumour Development in vivo by Concanavalin A

Abstract: THE carbohydrate binding protein concanavalin A (Con A)1–3 can agglutinate cells transformed by a variety of carcinogenic agents, but it only agglutinates normal cells after they have been treated with trypsin4. This agglutination was reversed by competition with α-methyl-D-glucopyranoside (α-MG). These results indicated that transformed cells contain exposed binding sites that interact with Con A, whereas most such sites on normal cells are in a cryptic form4,5. These experiments were undertaken to determine whether treatment with Con A can result in cell toxicity; and if so, whether the difference in structure of the surface membrane between normal and transformed cells can result in a differential toxic effect, and can be used to inhibit tumour development in vivo.

Studies in vivo and in vitro on an abnormality in the metabolism of C3 in a patient with increased susceptibility to infection

Abstract: In a patient with increased susceptibility to infection, lowered serum C3 concentration, and continuously circulating C3b, it was shown that purified 125I-labeled C3 was converted to labeled C3b shortly after intravenous administration. The fractional catabolic rate of C3 was approximately five times normal at 10% of the plasma pool per hr. The synthesis rate and pool distribution of C3 were normal. Despite this evidence of C3 instability in vivo, no accelerated inactivation of C3 was found in vitro. Similarly, no free proteolytic activity could be detected in the patient's serum, and serum concentrations of known protease inhibitors were normal. Complement-mediated functions, which were markedly deficient in the patient's serum, could be restored partially or completely by the addition of a 5-6S heat-labile beta pseudoglobulin from normal serum. The C3 proinactivator, which has these physicochemical characteristics, was also shown to be either absent or nonfunctional in the patient's serum. An unidentified 6S beta pseudoglobulin to which a monospecific antiserum was available was not detectable in the patient's serum. This last protein appeared not to be a complement component, nor was it the C3 inactivator or proinactivator. Finally, the substance or substances necessary for the conversion of C3b to C3c were missing from the patient's serum. The administration of 500 ml of normal plasma to the patient corrected all of his abnormalities partially or completely for as long as 17 days. The changes in C3 were dramatic; serum concentration rose from 8 to 70 mg/100 ml, and C3b could no longer be detected. A second metabolic study during this normalization period showed a decrease in fractional catabolic rate toward normal. The patient's histamine excretion was constantly elevated but increased further after a warm shower and after receiving normal plasma; at both times he had urticaria. These observations were consistent with the endogenous production of C3a and the resulting histamine release from mast cells. The inactivating mechanism for C3a was apparently intact in the patient's serum. The difference in the electrophoretic mobilities of C3b and C3c was shown as well as the electrophoretic heterogeneity of C3c. Suggestive evidence was also presented that the form of C3 with an activated combining site for red cells, previously postulated by others, is a transient C3 conversion product with an electrophoretic mobility slower than that of C3 on agarose electrophoresis.

Density distribution analysis of in vivo and in vitro colony forming cells in bone marrow.

Abstract: The technique of buoyant density separation in gradients of Bovine Serum Albumin has been used to separate hemopoietic cell populations in mouse bone marrow that form in vivo spleen colonies and in vitro colonies of granulocytes and macrophages in an agar culture system. The density distribution profiles showed a number of reproducible density subpopulations of both in vivo and in vitro colony forming cells (C.F.C.'s). The mean density of in vitro C.F.C.'s exceeded that of the in vivo but overlap of the density profiles of the two populations was evident. Density-related differences in seeding efficiency of in vivo C.F.C.'s were observed. Freund's adjuvant treatment increased marrow and spleen in vitro C.F.C. populations. Marrow density profiles obtained three and seven days after adjuvant showed a progressive increase in in vitro C.F.C.'s in a restricted density region with no associated elevation of in vivo activity. The antimitotic agent, vinblastine, revealed differences in mitotic activity between the two cell populations, reducing the in vitro C.F.C. population to .07% and the in vivo to 5% of normal in 24 hours. Density separation of vinblastine-treated marrow produced density regions devoid of in vitro activity but containing in vivoin vivo C.F.C.'s which, upon transfer to irradiated recipients, regenerated both in vivo and in vitro density distribution profiles.

On the characteristics of actinomycin D resistance in L5178Y cells.

Abstract: Summary A subline of the L5178Y murine leukemia was selected for resistance to actinomycin D by drug administration in vivo . The resistant line, L5178Y/D, had impaired capacity for uptake of actinomycin D in vivo and in culture but not under nongrowing conditions. Administration of the drug in vivo (50 μg/kg) or in culture (0.1 μg/ml) inhibited uridine incorporation into RNA by L5178Y but not by L5178Y/D. An altered cell surface glycoprotein fraction was found in L5178Y/D. Enzymatic studies showed that levels of glycoprotein transferases catalyzing formation of amino acid-sugar and sugar-sugar bonds were generally higher, while specific activities of glycosidases were generally lower in L5178Y/D. Alterations in membrane composition and conformation in the drug-resistant subline could account for the observed changes in actinomycin D permeability.

The effect of dialysates and ultrafiltrates of plasma of saline-loaded dogs on toad bladder sodium transport

Abstract: In order to obtain direct evidence for the existence of a natriuretic hormone, dialysates and ultrafiltrates of plasma of dogs expanded with saline were tested for effects on sodium transport by the toad urinary bladder. Dialysate was obtained by dialysis of blood in vivo in a clinical dialyzer and by dialysis in vitro of small volumes of blood using a miniature model of the clinical dialyzer. Ultrafiltrates were prepared using selective molecular filters which permit passage of substances on the basis of molecular weight and three dimensional configuration. Dialysates and ultrafiltrates of hydropenic dogs caused a change in toad bladder potential difference of + 1% and in short circuit current of - 5%. In contrast, dialysates and ultrafiltrates from expanded dogs caused a change in potential difference of - 23% and in short circuit current of - 32%, a highly significant difference. Onset of reduction of short circuit current occurred within 3-5 min, reaching a maximum in 10-20 min. The effect was rapidly reversible, was specific for the serosal surface of the bladder, and could not be explained on the basis of nonspecific alterations in ionic composition or by dilutional effects. Ultrafiltrates of jugular vein plasma caused significantly more reduction of short circuit current than ultrafiltrates of femoral vein plasma. The data indicate the presence in plasma of saline-loaded dogs of a dialyzable inhibitor of toad bladder sodium transport. Ultrafiltrate studies using membranes of appropriate selectivity suggest the factor has a molecular weight of less than 3000.

A Probable Role for Protein Synthesis in Intestinal Epithelial Cell Damage Induced in Vivo by Cytosine Arabinoside, Nitrogen Mustard, or X-irradiation

Abstract: Cell death induced in vivo in the continuously dividing intestinal crypt epithelial cells by 1-β-d-arabinofuranosylcytosine, nitrogen mustard, or X-irradiation can be prevented by two agents which inhibit protein synthesis: cycloheximide and tenuazonic acid Both pretreatment and posttreatment (up to 45 min), in doses sufficient to produce an 80% drop in the rate of protein synthesis, are effective Protection against hydroxyurea-induced cell death is similar but more variable Our data suggest that, in this system, the active metabolic response of the cell (protein synthesis), to injurious agents, rather than mere passive attrition, is necessary for cell death While protective to the intestinal crypt epithelium, inhibitors of protein synthesis enhance damage to lymphoid cells It is suggested that these agents may be useful adjuncts to the X-ray and chemical treatment of lymphomas

Some Properties of Angiotensin Converting Enzyme in the Lung in vivo

Abstract: THE lung has many important metabolic functions1,2 which include the selective activation or inactivation of endogenous peptides. Bradykinin is inactivated on transit through the pulmonary circulation3,4, probably by at least two endopeptidases5, but other peptides such as angiotensin II (refs. 6–10), polistes kinin11 and eledoisin3,4 pass through without loss.

Cytotoxicity: Specificity after in vitro Sensitization

Abstract: Animals sensitized in vivo against an allogeneic tissue subsequently show accelerated rejection specificially of that or antigenically similar tissues. Lymphocytes sensitized in vitro will destroy target cells isogeneic with the sensitizing cells. Lymphocytes sensitized in vitro can differentiate specifically between different allogeneic target cells—as occurs in vivo.

DL-[2-14C]p-chlorophenylalanine as an inhibitor of tryptophan 5-hydroxylase.

Abstract: — The distribution in vivo of dl-[2-14C]p-chlorophenylalanine (p-CP) in regions and subcellular fractions of the rat brain was determined. The half-lives of p-CP and its metabolite p-chlorophenylpyruvic acid (p-CPPA) in plasma and brain were correlated with the development of inhibition of cerebral tryptophan 5-hydroxylase (EC 1.99.1.4). There was active transamination in vivo of p-CP and p-CPPA in the brain. Transport of indolealkylamino acids into brain was impaired by p-CP. Inhibition of tryptophan 5-hydroxylase could not be reversed by administration of large doses of l-tryptophan, l-tyrosine, or l-phenylalanine. After administration of [2-14C]p-CP in vivo, appreciable radioactivity was bound to cerebral proteins, including those with tryptophan 5-hydroxylase activity, as well as to phenylalanine 4-hydroxylase (EC 1.99.1.2) purified from liver. Amino acid analysis of the acid hydrolysate of purified, radioactive hepatic phenylalanine 4-hydroxylase showed over 80 per cent of the radioactivity to be present as p-CP. Neither the inhibition in vivo nor in vitro of tryptophan 5-hydroxylase could be reversed by dialysis; in controls, dialysis resulted in marked loss of enzyme activity. After incubation for 5 min with p-CP in vitro, enzymic activity was inhibited 60 per cent. In vitro, p-CPPA labelled protein much more extensively than p-CP, yet inhibited the enzyme less. Some of the label from p-CPPA was removable by dialysis.

Renal-Clip Hypertension in Rabbits Immunized Against Angiotensin II

Abstract: The rabbit immunized against angiotensin II was shown to be a valid model for the study of renal-clip hypertension. In particular, there was a specific and complete blockade of the pressor effect of high doses of intravenous renin and angiotensin in vivo, even at angiotensin II production rates which far exceeded those associated with renal-clip hypertension. Despite this, four immunized rabbits developed hypertension after renal-artery clipping with contralateral nephrectomy, and in three of these the hypertension was severe. In four other rabbits, there was no evidence of modification of an established hypertension after immunization against angiotensin II. In both groups, the specific absence of pressor response to high doses of renin and angiotensin II after immunization was confirmed. These studies provide strong evidence that angiotensin is not the sole or even the major factor in either the initiation or maintenance of this form of hypertension.

A specific competitive inhibitor of angiotensin II.

Abstract: A peptide analog, [4-phenylalanine, 8-tyrosine]-angiotensin II, was prepared by solid phase peptide synthesis and its structure confirmed. The compound was found to be a potent inhibitor of angiotensin II in vitro (isolated rat uterus strips) and in vivo (rat blood pressure). The analog was found to be highly specific and did not inhibit the action of a number of other peptides and spasmogenic compounds.

In vivo Conversion of 3H-L-Tryptophan into 3H-Serotonin in Brain Areas of Adrenalectomized Rats

Abstract: Rats were adrenalectomized 10 days before we estimated in vivo the conversion index of 3H-tryptophan into radioactive serotonin in brainstem and telediencephalon. We found that the conversion index in the brainstem of adrenalectomized rats is smaller than in the same area of sham-operated rats. Conversely, the conversion index in the telediencephalon was similar in the two groups of rats. The serotonin concentrations were unchanged by adrenalectomy, which suggests that in brainstem the decrease of tryptophan hydroxylase is reflected by the conversion index estimation and not by measurement of serotonin steady-state concentrations.

The effect of prostaglandin E1 on platelet function in vitro and in vivo.

Abstract: Summary Low concentrations of prostaglandin E1 (PGE1) inhibit ADP-induced aggregation in pig and rabbit citrated platelet-rich plasma and in suspensions of washed platelets. Higher concentrations also inhibit the initial change in shape induced by ADP and the release of platelet ATP, ADP and serotonin caused by stimuli such as collagen, thrombin, antigen-antibody complexes and gamma-globulin-coated polystyrene particles. PGE1 is not taken up by platelets and its effects can be removed by resuspending platelets in fresh medium. Immediately following an intra-arterial injection, ADP-induced platelet aggregation is suppressed, but after 5 min the response returns to normal. PGE1 inhibits haemostasis in rabbits when given as a continuous infusion. It is concluded that the effect of PGE1 on haemostasis involves inhibition of the release of ADP from platelets exposed to collagen and thrombin, and inhibition of ADP-induced aggregation.

In Vitro Stimulation of Leucine Incorporation Into Muscle and Cartilage Protein by a Serum Fraction with Sulfation Factor Activity: Differentiation of Elfects from Those of Growth Hormone and Insulin

Abstract: Effects of growth hormone, insulin and a serum fraction with sulfation factor activity on in vitro incorporation of (3H) leucine into TCA-insoluble protein by diaphragm and costal cartilage of hypophysectomized rats were compared. Tissues were incubated in a medium containing 14 amino acids and glucose. Cartilage was very sensitive to growth hormone in vivo but relatively insensitive in vitro. This tissue was also relatively insensitive to insulin in vitro but very sensitive to sulfation factor. Muscle was less sensitive than cartilage to growth hormone in vivo or sulfation factor in vitro. In all muscle studies combined, the maximum effect of growth hormone in vivo and the maximum effect of either sulfation factor or insulin in vitro each exceeded the maximum effect of growth hormone in high concentration in vitro. Sulfation factor in serum of normal rats was sufficient for stimulation of both muscle and cartilage from hypophysectomized rats in vitro, and the effects were not augmented by a high concentr...

Demonstration of the chemotactic properties of collagen.

Abstract: SummaryAn in vivo method for measuring chemotaxis has been described using Millipore filters. This method demonstrated that soluble collagen, but not gelatin, was quite chemotactic. Further, the products of collagenolysis produced by cutaneous collagenase, but not by bacterial collagenase, were extraordinarily chemotactic for polymorphonuclear leukocytes within 4 hr after testing at a concentration of about 0.1 μg.

In vitro and in vivo studies of the immune response to sheep erythrocytes using partially purified cell preparations

Abstract: The BSA density-gradient technique for separating mouse spleen cells into partially purified populations has been used to compare the responsiveness of such populations to SRBC using in vivo and in vitro techniques. Two major populations were distinguished, one of which responded very well in vivo with an exponential dose response and poorly in vitro (fraction 3), and another which responded in vivo and in vitro with a linear dose response (fraction 2). A light density, radiation-resistant component was identified which markedly stimulated the response of fraction 3 in vitro, and a density gradient profile was obtained for this cell which did not correspond with a macrophage profile. A high density, radiation-sensitive cell was identified which stimulated the response of PFC precursors in lighter regions of the gradient. The activity of this cell could be replaced using thymus cells. A density profile for the PFC precursor cell was obtained by assaying small numbers of spleen cell fractions in the presence of an excess of the two auxiliary cell types.

The role of ingested hemoglobin in the nutrition of Schistosoma mansoni.

Abstract: This study demonstrates that ingested host hemoglobin is involved in schistosome nutrition. Homologous reticulocytes, labeled with tritiated L-leucine, were injected into infected mice. Four days postinjection the animals were killed and the schistosomes were recovered by perfusion. The harvested worms were examined for radioactivity by direct counting and radioautography. These experiments clearly demonstrate that L-leucine was incorporated and extensively distributed in the tissues of the schistosomes. Previous investigators have concluded that ingested hemoglobin is involved in the amino acid nutrition of schistosomes because material resembling hematin has been observed in the guts of worms (Rogers, 1940) and because an enzyme associated with the worm, "hemoglobin protease," specifically hydrolyzes host hemoglobin (Timms and Bueding, 1959). However, these conclusions are based upon in vitro experiments and microscopic observation of harvested worms; there has been no direct experimental data to support the hypothesis that ingested blood cells are utilized by the parasite. At the outset, in vitro experiments seemed attractive to us because of their relative simplicity and because it has been possible to maintain schistosomes for at least 60 days in artificial media (Ross and Bueding, 1950; Robinson, 1954; Senft and Senft, 1962; and our unpublished experiments), a period entirely satisfactory for the labeling of worms for a radioautographic study. However, in our experience, worms maintained in artificial medium did not consistently evacuate their gut contents nor did they seem to ingest particulate material from the medium, an obstacle in getting radioactive blood cells into the schistosome cecum. We showed that the failure of worms to evacuate their guts in vitro involved more than a lack of stimulation by particulate matter because when latex spheres, approximately the size of red blood cells (8 yt), were presented to engorged worms in artificial medium, neither was the gut evacuated nor were the spheres ingested. Also, we observed that worms incubated in tissue culture medium 199 (Morgan et al., 1950) with 10% serum, or in Received for publication 17 June 1969. the schistosome medium cited above, failed to maintain normal oogenesis and that the size of the mature worms decreased with prolonged incubation even though the medium was changed periodically. This suggests that these media do not completely satisfy the nutritional requirements of the worms. Finally, we felt that the most directly interpretable data would be provided by in vivo experiments and that, at any event, in vitro results would have to be supported later by in vivo studies. Thus we decided to proceed directly with in vivo experiments. The experiments reported herein were designed to ascertain whether or not S. mansoni utilizes host hemoglobin for the synthesis of tissue components. MATERIALS AND METHODS 1. Production of reticulocytosis Ten CFP female mice were injected subcutaneously, once daily for 12 days, with 0.1 ml of 0.25% phenylhydrazine hydrochloride solution, neutralized to pH 7.0 with sodium hydroxide. Blood samples were removed by infraorbital sinus puncture. Thin blood smears were treated with methylene blue reticulocyte reagent and the percentage of reticulocytes was determined by direct