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New models of chronic synovitis in rabbits induced by mycoplasmas: microbiological, histopathological, and immunological observations on rabbits injected with Mycoplasma arthritidis and Mycoplasma pulmonis.

A dose-dependent chronic synovitis was induced in rabbit knees after the intra-articular injection of both Mycoplasma arthritidis and Mycoplasma pulmonis. The inflammation progressed from an initial acute phase at 1 week characterized by edema, infiltration of the synovium with monocytes and heterophils, and desquamation of lining cells, to a more chronic phase at 1 and 3 months, in which villus hyperplasia, lymph "nodules," mononuclear cell infiltration, fibroplasia, and collagen deposition were prominent. With one exception, mycoplasmas could no longer be cultivated from the joints 1 month postinoculation. Both mycoplasma species evoked a humoral antibody response that was more marked in synovial fluids than in peripheral blood. A cell-mediated immune reaction, as evidence by enhanced uptake by [3H]thymidine by sensitized blood, spleen, or node lymphocytes in the presence of homologous antigen, was detected only in rabbits injected with M. pulmonis. Lymphocytes taken from arthritic rabbits were no more cytotoxic toward synovial cells derived from normal or arthritic rabbits than were normal lymphocytes. The models of synovitis described in this study offer a convenient probe for determining the mechanisms of mycoplasma-induced inflammation, since they require only a single injection of the initiating agent and, in addition, utilize an animal host large enough for detailed investigation into the nature of mycoplasma/synovium interactions.

Animals↗

Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines.

Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

Animals↗

Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine.

Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.

Amino Acid Sequence↗

The putative E5 open reading frame of cottontail rabbit papillomavirus is dispensable for papilloma formation in domestic rabbits.

In the cottontail rabbit papillomavirus (CRPV)-rabbit system, recombinant CRPV DNA can induce papillomas. This investigation was undertaken to evaluate whether the E5 open reading frame (ORF) of CRPV is required for papilloma formation. The CRPV genome we utilized, CRPV-WA, was sequenced in the E5 region and was found to contain one deletion, two insertions, and one transition mutation compared with CRPV-KS, the CRPV genome that has been fully sequenced. Despite these differences, an intact E5 ORF is preserved, supporting the notion that this gene may serve a biological function. One frameshift and two in-frame mutations were constructed in the small region of the 5' end of the E5 ORF that follows the E2 stop codon and precedes the L2 ORF. Several hundred rabbit skin sites were inoculated with each DNA preparation with a jet injector to test the ability of three CRPV E5 mutant DNAs to induce papillomas. In vivo results showed that each of the mutants induced papillomas, and biochemical analysis demonstrated that the E5 mutations present in DNA inocula were retained in the papillomas. The frequency of papilloma formation, however, was generally lower with each of the CRPV E5 mutants than with wild-type CRPV DNA, particularly so for the E5 frameshift mutant, suggesting that although the recognized E5 ORF is not required in domestic rabbits for the induction of papillomas by CRPV DNA, it may facilitate their formation.

Amino Acid Sequence↗

Inhibitory effect of syphilitic rabbit serum on DNA synthesis in rabbit cells in vitro.

A previously described toxic factor associated with Treponema pallidum (Nichols) and found in extracts of syphilitic rabbit testes has now also been detected in syphilitic rabbit serum. The toxic factor, which inhibits DNA synthesis in baby rabbit genital organ (BRGO) cells in vitro, is present in rabbit serum up to 30 days after infection with T pallidum.

Animals↗

Complete sequence of rabbit kidney aminopeptidase N and mRNA localization in rabbit kidney by in situ hybridization.

We have isolated and sequenced a full-length cDNA for rabbit kidney aminopeptidase N (APN). The 3-kilobase cDNA contains 12 nucleotides of the 5' noncoding region, a 2898 nucleotide long open reading frame, and 113 nucleotides of the 3' untranslated region. The open reading frame encodes a type II membrane protein of 966 amino acid residues composed of a 10 residue NH2-terminal cytosolic domain, a 23 residue transmembrane domain, and a large 933 residue ectodomain that contains the active site. Rabbit APN has eight potential N-glycosylation sites and seven cysteine residues, one of which is located in the transmembrane domain. Computer analysis showed that the enzymes from human, rat, and rabbit were highly conserved, except for the stalk region immediately downstream from the transmembrane domain. Using in situ hybridization techniques we showed that in rabbit kidney, APN mRNA is present in proximal tubules but not in glomeruli, which corresponds to the localization of the protein observed by immunohistochemistry. Taken together, our results strongly suggest that the expression of APN in kidney is modulated at mRNA levels and not at translational and (or) posttranslational levels.

Amino Acid Sequence↗

Quantitative study of alpha-adrenergic receptors in circular and longitudinal muscle of isthmus of rabbit fallopian tube and in rabbit aorta.

The affinity of phenylephrine (pD2 value) and KB value for phentolamine for the alpha-adrenergic receptors of circular and longitudinal muscle of isthmus of rabbit fallopian tube and rabbit aorta have been estimated. The affinity of both phenylephrine and phentolamine was significantly more for the alpha adrenoceptors of the longitudinal muscle of isthmus of rabbit fallopian tube and rabbit aorta than circular muscle of isthmus of fallopian tube. The results of this investigation suggest that alpha adrenoceptors of circular muscle of isthmus are atypical. If this holds good for the human fallopian tube, the development of selective alpha-adrenergic-receptor agonists and antagonists may be of potential practical application for the control of fertility.

Animals↗

Enhanced diltiazem deacetylase activity in pre-term and full-term rabbits compared with adult rabbits.

The in vitro metabolic activity of the esterase responsible for the hydrolysis of diltiazem (DTZ) to its deacetylated metabolite (M1) was determined in an age-dependent fashion using the rabbit as an animal model. The presence of the enzyme in several tissues (liver, lung, small intestine, and brain) and in whole blood from pre-term and full-term fetuses, full-term newborns, yound and adult rabbits was examined. To this end, DTZ was spiked to 10,000-g tissue homogenates and whole blood to yield a final concentration of 1 microgram/ml. Serial samples were withdrawn from the incubation medium up to 240 min and assayed for DTZ and M1 concentration. In all tissues examined there was a net production of M1. Chemical breakdown and stability studies confirmed the metabolic origin of the M1 formed throughout the incubation. In pre-term fetuses (25 days of gestation) the brain was found to be the most active tissue in eliminating DTZ (brain > liver > lung > small intestine). This trend changed in young and adult rabbits (lung = brain > liver > small intestine). Although an important age-dependent DTZ deacetylase activity was observed in blood, it was not included in the comparison between organs because of the unequal composition of the incubation medium. In conclusion, results showed that fetuses and newborn rabbits have a similar, and in some instances higher, DTZ deacetylase activity to that in adults (p < 0.05). In vitro findings were further confirmed by in vivo experiments.

Aging↗

Effect of hypercholesterolemia on vascular reactivity in the rabbit. I. Endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits.

We studied the effects of hypercholesterolemia on vascular responsiveness in different arteries isolated from rabbits: control groups of rabbits and groups receiving the atherogenic diet consisted of eight animals each. In the arteries, 16 weeks of cholesterol-rich (0.3%) diet evoked intimal lesions which were more pronounced than those noted after 8 weeks of hypercholesterolemia; the aortic arch was affected significantly more by the lesions than the abdominal aorta and the pulmonary artery. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxant factor. Contractions caused by acetylcholine and prostaglandin F2 alpha were not altered by the hypercholesterolemia; those evoked by serotonin were moderately augmented only in the aortic arch of hypercholesterolemic rabbits. As the degree of intimal lesion formation increased, the contractions to norepinephrine and clonidine were progressively inhibited. The endothelium-independent relaxations to nitroglycerin were inhibited in only the most severely affected arteries; the endothelium-dependent relaxations to acetylcholine and adenosine triphosphate were progressively inhibited as the degree of fatty streak formation augmented. Thus, in the aortic arch, the relaxations to 3 X 10(-6) M acetylcholine, expressed as percent of the initial contraction, decreased from 86.7 +/- 3.3% in control tissues to 16.3 +/- 8.6% in the 16-week hypercholesterolemic vessels; in the abdominal aortas these relaxations averaged 93.5 +/- 2.2% in control vessels and 72.0 +/- 6.9% in the hypercholesterolemic tissues. The acetylcholine-induced release of endothelium-derived relaxant factor from the abdominal aorta was not significantly affected by the hypercholesterolemia. We conclude from these studies that in arteries obtained from hypercholesterolemic rabbits: the contractions caused by serotonergic mechanisms tend to be augmented, while those to alpha-adrenergic activation are decreased, the endothelium-independent relaxations are modified only in the more severely affected arteries, and the endothelium-dependent relaxations are progressively inhibited as the degree of fatty streak formation augments, probably because a step subsequent to the release of endothelium-derived relaxant factor is altered.

Acetylcholine↗

Clearance of phosphatidylcholine and cholesterol from liposomes, liposomes loaded with metaproterenol, and rabbit surfactant from adult rabbit lungs.

Rabbits were given by tracheal instillation liposomes, liposomes carrying metaproterenol sulfate (MPS) and suspended in a MPS solution, rabbit surfactant, or rabbit surfactant suspended in a MPS solution. The lipid suspensions were labeled with [14C]cholesterol and [3H]phosphatidylcholine. The percent recoveries of the labels were measured over 24 h in alveolar wash, lung tissue after alveolar wash, and the total lungs. All clearance curves for both phosphatidylcholine and cholesterol from liposomes were the same in the presence or absence of MPS. Alveolar clearance curves for both labels from rabbit surfactant were the same; however, the surfactant-associated labels were cleared to the lung tissue more rapidly than were the liposome-derived labels. Despite different alveolar clearance curves, all clearance curves for cholesterol from the total lungs were similar at a rate of 20 to 30%/24 h of the injected labeled cholesterol. Phosphatidylcholine was cleared from the total lungs more rapidly, at rates from 35 to 56%/24 h. Although MPS did not change labeled liposomal lipid clearance, the beta-agonist increased surfactant lipid clearance. The different responses to beta-agonist and the different alveolar clearance curves indicated distinct alveolar-to-lung tissue metabolism for liposomal versus surfactant phosphatidylcholine and cholesterol, although overall lung clearance rates were similar.

Animals↗

The effect of sulfonylurea drugs on rabbit myocardial contractility, canine purkinje fiber automaticity, and adenyl cyclase activity from rabbit and human hearts.

Long-term clinical studies have associated tolbutamide therapy with an increased incidence of cardiovascular deaths. The effects of this and other sulfonylurea drugs on contractility and rate of isolated rabbit atria, automaticity of isolated dog Purkinje fibers, and adenyl cyclase activity in particulate preparations of rabbit and human hearts were studied. At concentrations that are attained clinically, tolbutamide (10 mg/100 ml) increased contractility of driven rabbit atria to 124+/-5% of control, acetohexamide (3.9 mg/100 ml) to 140+/-5%, chlorpropamide (8.3 mg/100 ml) to 139+/-6%, and tolazamide (3.1 mg/100 ml) to 119+/-6%. These effects were accentuated in the presence of 2.5 x 10(-4) M theophylline and were not blocked by 1 x 10(-5) M propranolol. Adenyl cyclase was activated by each of these drugs at concentrations below those which increase contractility. The drugs also increased the rate and slope of phase 4 depolarization in spontaneously beating Purkinje fibers, but did not alter the spontaneous rate of isolated rabbit atria. Since inotropic and chronotropic stimulation can be deleterious in some clinical settings, these findings may be of significance in interpretation of cardiovascular mortality data.

Acetohexamide↗

In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol.

Animals↗

Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea.

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410-425) were used to study uptake of [3H]thymidine, [3H]adenine, and [3H]ganciclovir. Transport of [3H]adenine and [3H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34 degrees C. Thymidine uptake by SIRC displayed saturable kinetics (K(m) = 595.9 +/- 80.4 microM, and V(max) = 289.5 +/- 17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [3H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K(m) = 14.4 +/- 2.3 microM, V(max) = 0.4 +/- 0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [3H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [3H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.

Adenine↗

The presence of a cytoplasmic estrogen receptor in sexually mature rabbit epididymides: comparison with the estrogen receptor in immature rabbit epididymal cytosol.

Cytosol prepared from epididymides of castrated adult rabbits contains an estrogen-specific binding component that sediments in the 4--5S region on 0.01 M KCl sucrose gradients. The estrogen-specific binding component is not detectable on sucrose gradients unless the cytosol-[3H]estradiol mixture is first extracted with charcoal to remove uncomplexed and rapidly exchangeable ligand from the sample. The macromolecular bound [3H]estradiol that remains after charcoal extraction dissociates from the receptor with a dissociation half-time of greater than 24 h and is of high affinity (Kd = 6.5 +/- 0.9 X 10(-9) M). The estrogen receptor in the adult rabbit epididymis can also be demonstrated when charcoal-extracted samples are chromatographed on Sephadex G-200. Using this system, the major estrogen-specific binder has an apparent molecular weight of less than 200,000. In contrast, the epididymal estrogen receptor in sexually immature rabbits sediments as an 8S species on low salt gradients and has an apparent molecular weight of 200,000 or greater when assessed by chromatography on Sephadex G-200. The estrogen receptor in both age groups is distributed along the length of the epididymal duct. The highest concentration of the cytoplasmic receptor in both mature and immature rabbits is in the cauda epididymidis. The age-dependent changes in the amount of the receptor suggest that it may be involved in maturational changes in the epididymis. The alterations in the physicochemical properties may reflect physiological changes in the epididymis or in the endocrine status of the animal.

Aging↗

Carbonyl reductase purified from rabbit liver is not the product of a carbonyl reductase gene (RCBR5 or RCBR6) cloned from the rabbit liver cDNA library.

Six peptides were obtained by the digestion of carbonyl reductase purified from rabbit liver. The amino acid sequences of the six peptides were virtually identical to the corresponding regions in amino acid sequences deduced from two cloned carbonyl reductase genes (RCBR5 and RCBR6). However, there was a difference of one amino acid residue between the sequences of peptides from the purified enzyme and the corresponding region in the amino acid sequences deduced from the two cDNAs. The purified carbonyl reductase was confirmed to exhibit no reactivity towards menadione, even though the transient expression of the two cDNA for rabbit liver carbonyl reductase has been reported to cause a marked increase of menadione reductase activity in COS7 cells. The enzyme purified from rabbit liver was inactivated by thiol-specific reagents, 5,5'-dithiobis(2-nitrobenzoic acid) and sodium tetrathionate, suggesting that menadione probably interacts with the functional cysteine residue(s), and cannot serve as a substrate of the purified enzyme. Based on these results, it is concluded that the carbonyl reductase purified from rabbit liver is not the product of cloned carbonyl reductase gene (RCBR5 or RCBR6).

Alcohol Oxidoreductases↗

[Analysis of the vascular reactivity of isolated and blood-perfused rabbit thoracic aortas with support rabbits].

We have developed a new method for perfusing isolated and blood-perfused rabbit thoracic aortae by using conscious support rabbits. The vasoconstrictory and vasodilatory response to vasoactive substances were recorded as the decrease or the increase in intra-chamber pressure of the glass container in which the rabbit thoracic aorta was mounted. Examined were the vascular responses to dl-norepinephrine (NE) and l-isoproterenol (Iso) at different pressure loads to the vessel. NE produced a dose-dependent vasoconstriction which was effectively suppressed by i.v. phentolamine (3 mg/kg). In contrast, Iso produced beta-adrenoceptor-mediated vasodilations in a dose-dependent manner (dl-propranolol, 0.5 mg/kg, i.v.). The present experiments were performed at different pressure loads (pressure changes both in the vessel-mounted chamber and a Starling pneumatic resistance set connected in the way of a series circuit). Our newly developed system for perfusion of isolated vascular vessels with conscious support rabbits has some advantages over the existing methods for analysing vascular reactivity in terms of being perfused with arterial blood, having a simulated peripheral resistance apparatus in the circuit and showing a distinct vasodilatory response to i.a. Iso.

Animals↗

Studies on an inhibitor of erythropoiesis. II. Inhibitory effects of serum from uremic rabbits on heme synthesis in rabbit bone marrow cultures (38483).

The effects of sera from anephric rabbits on the rate of heme synthesis in erythroid cells were studied in vitro in rabbit bone marrow cultures. Sera from nephrectomized rabbits significantly (p smaller than 0.05) inhibited 59-Fe incorporation into heme in normal rabbit bone marrow cultures incubated with erythropoietin (ESF) when compared with the response in control culutres with normal serum and ESF. This inhibitory activity increased exponentially with increasing concentrations of the serum in cultures suggesting that a higher concentration of the inhibitor is present in uremia and may be the result of the failure to excrete the inhibitor. This suggests that the retention of this inhibitor may play an important role in the mechanism of the anemia of uremia. The inhibitor of heme synthesis is a low molecular weight substance.

Animals↗

Studies on catabolism of 125I-labelled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: methodologic aspects.

The catabolism of rabbit fibrinogen labelled with radioactive iodine was studied over a period of 7-9 days in normal rabbits and in rabbits with indwelling intravenous catheters. The labelled fibrinogen was highly clottable and homogeneous on immunoelectrophoresis and disc electrophoresis. The protein-bound 125I-activity amounted to at least 99%. Normal rabbits with a weight between 1.6 and 2.2 kg exhibited a mean plasma volume of 41.65 ml/kg. A mean half-life time of 1.69 days and a mean fractional catabolic rate of 59.8% of the plasma fibrinogen pool per day was computed by tracing labelled fibrinogen. The absolute catabolic rate revealed a mean value of 58.7 mg/kg body weight/day. Comparable results were obtained for all three labelled fibrinogen batches. A siliconized polyvinyl tubing with an outer diameter of 2.0 mm inserted into a jugular vein up to a length of 10-12 cm and indwelling for more than 10 days did not significantly influence the parameters studied for controlling the fibrinogen catabolism except for the absolute catabolic rate of 112.8 mg/kg/day which differed significantly from that of the control group. Thus, the indwelling intravenous catheter had no or only a minor effect on the activation of intravascular coagulation.

Animals↗