Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.

Cells, Cultured↗

PubMed1982

Spontaneously metastasizing variants derived from MNU-induced rat mammary tumour.

N-methyl-N-nitrosourea (MNU) given i.v. to female rats of inbred strains induces mammary adenocarcinomas which are hormone-sensitive but do not spontaneously metastasize (Williams et al., 1981). Tissue culture and selection techniques have been used to derive metastasizing tumours from a mammary tumour induced with MNU in F344/N rats. Histologically, primary tumours and metastases in the lung and lymph nodes were similar. These systems may constitute useful models for the study of breast-cancer metastasis.

Adenocarcinoma↗

PubMed1982

Polymorphonuclear leucocyte hypersegmentation in heat stroke.

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Cell Nucleus↗

PubMed1982

Biological and immunological properties of Coxiella burnetii vaccines in C57BL/10ScN endotoxin-nonresponder mice.

Vaccines prepared from Formalin-killed whole cells of Coxiella burnetii (Ohio strain) or from chloroform-methanol residue (CMR) and extract (CME) of such cells were examined for biological and immunological properties in male C57BL/10ScN endotoxin nonresponder mice. Vaccines containing killed whole cells induced a high incidence of gross pathology, as evidenced by liver necrosis, significant splenomegaly, and significant hepatomegaly in mice. The degree and onset of these pathological changes were directly and inversely proportional, respectively, to the dosage of killed organisms administered. Conversely, no splenomegaly, hepatomegaly, or liver necrosis were observed in mice inoculated with CMR or CME. Moreover, killed whole cells were lethal for mice at dosages of 150 to 1,200 micrograms, whereas no deaths were seen in animals given 1,200 micrograms of CMR. In addition, antibodies against phase I and phase II antigens of C. burnetii were detected in the sera of mice inoculated with either whole cells or CMR. Enhanced blastogenic response of splenic lymphocytes was observed when animals were vaccinated with killed whole cells and CMR but not with CME. Moreover, 80 to 90% of mice inoculated with 300 micrograms of the CMR were protected against a lethal challenge of viable rickettsiae, whereas only 50% of the animals given 300 micrograms of killed whole cells were protected. Treatments with CME were essentially without value, since no antibodies were detectable and no significant protection was elicited. Collectively, these results show that, although killed whole cells induced immunity in C57BL/10ScN mice, they induced deleterious tissue reactions, whereas CMR, which also induced immunity, was essentially nondeleterious, based on the parameters employed. These observations suggest that the chloroform-methanol-extractable component(s) is implicated in the deleterious tissue reactions and that the phase I and II antigens may not be involved in the induction of the pathology observed in C57BL/10ScN mice.

Animals↗

PubMed1981

Further studies on the efficacy of fenbendazole against inhibited larvae of Ostertagia ostertagi.

Fenbendazole, at low dosage levels, was evaluated for its efficacy against inhibited early fourth stage larvae of Ostertagia ostertagi in naturally infected, yearling beef cattle in April. The mean number of inhibited larvae in 10 untreated control cattle was 51,500. At a dosage level of 1 mg per kg (pelleted fenbendazole) per day in feed over five days in 10 cattle, the percentage reduction in comparison to controls was: adults, 85.9 per cent; developing stages, 79.0 per cent; inhibited early fourth stage, 47.6 per cent; other abomasal genera (Haemonchus species and Trichostrongylus axei, 100 per cent). At a dosage level of 5 mg per kg (10 per cent liquid suspension) administered as an oral drench to 10 cattle, the percentage reduction in comparison to controls was: adults, 96.9 per cent; developing stages, 82.9 per cent; inhibited early fourth stage, 74.7 per cent; other genera, 98.6 per cent. Efficacy against parasite genera of the intestinal tract (primarily Cooperia species) was in excess of 94 per cent at both dosage levels. Some variability of efficacy was observed, in that large numbers of surviving inhibited O ostertagi larvae were recovered from two or three animals in treated groups.

Animals↗

PubMed1981

Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii.

Coxiella burnetti, the etiologic agent of Q fever, is an oligate intracellular parasite of eukaryotes. Unlike the majority of successful bacterial parasites, which escape the bactericidal environment of the phagolysosome by various means, C. burnetii multiplies only in the phagolysosome. In view of the relatively harsh environment inhabited by C. burnetii, we have examined (i) the in vitro metabolism of glucose and glutamate by whole cells of C. burnetii under conditions designed to approximate the pH within the phagolysosome and (ii) the effect of manipulation of the phagolysosomal pH by lysosomotropic amines on the replication of C. burnetii in chicken embryo fibroblasts. The transport, catabolism, and incorporation of both glucose and glutamate were found to be highly stimulated by acidic conditions, whereas at pH 7.0 metabolism of these substrates was minimal. The transport processes were shown to be energy dependent and highly sensitive to inhibition by uncouplers of oxidative phosphorylation. Increasing the phagolysosomal pH of infected chicken embryo fibroblasts by use of the lysosomotropic agents chloroquine, methylamine, or ammonium chloride inhibited the multiplication of C. burnetii, thus demonstrating the in vivo requirement for the acidic conditions of the phagolysosome. This apparent dependence upon phagosome--lysosome fusion to generate pH conditions favorable to C. burnetii replication suggests a unique biochemical mechanism of parasite activation. A pathogenic mechanism based on regulation of microbial metabolism by H+-dependent stimulation of cell function is proposed.

Animals↗

PubMed1981

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.

Antigens, Bacterial↗

PubMed1981

Immunological and biological characterization of Coxiella burnetii, phases I and II, separated from host components.

Coxiella burnetii, phase I and II, cells cultivated in the yolk sac of chicken embryos were separated from host cell components by two cycles of isopycnic Renografin gradient centrifugation. Initial steps in the purification of viable C. burnetii involved differential centrifugation and sedimentation through an aqueous solution of 30% sucrose and 7.6% Renografin. After the first, but not the second, cycle of Renografin gradient centrifugation, the cells were passed through microfilter glass filters which facilitated the removal of host components. The integrity of morphologically different cell variants was maintained during purification procedures by suspending highly purified C. burnetii in phosphate-buffered saline-sucrose solutions. C. burnetii, phases I and II, obtained by these methods appeared to be free from host cell components by serological methods while retaining morphological integrity and infectivity for yolk sacs and experimental animals. Average yields of C. burnetii were 2.83, 1.5, and 0.84 mg (dry weight) per yolk sac of the Ohio strain (phase I), 9 Mile strain (phase I), and 9 Mile strain (phase II), respectively. Recovery of phase I cells averaged about 70%, whereas the recovery of phage II cells was approximately 40%. The temporal sequence of phase I and II antibody response was demonstrated in infected and vaccinated animals. Also, no antibody response in mice and guinea pigs to yolk sac antigens was detectable after two injections of vaccine or viable cells. Importantly, this is the first report of the separation of viable phase II cells of C. burnetii free of host components.

Animals↗

PubMed1981

Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiations.

Coxiella burnetii is a gram-variable obligate intracellular bacterium which carries out its development cycle in the phagolysosome of eucaryotic cells. Ultrastructural analysis of C. burnetii, in situ and after Renografin purification, by transmission electron microscopy of lead-stained thin sections has revealed extreme pleomorphism as demonstrated by two morphological cell types, a large cell variant (LCV) and a small cell variant (SCV). Potassium permanganate staining of purified rickettsiae revealed a number of differences in the internal structures of the cell variants. (i) The outer membrane of the sCV and LCV were comparable; however, the underlying dense layer of the SCV was much wider and more prominent than that of the LCV. The periplasmic space of the SCV was not readily visualized, whereas the periplasmic space of the LCV was apparent and resembled that of other gram-negative bacteria. (ii) Complex internal membranous intrusions which appeared to originate from the cytoplasmic membrane were observed in the SCV. The LCV did not harbor an extensive membranous system. (iii) Some LCVs contained a dense body in the periplasmic space. This endogenous structure appeared to arise in one pole of the LCV as an electrondense "cap" formation with the progressive development of a dense body approximately 130 to 170 nm in diameter which was eventually surrounded by a coat of at least four layers. Our observations suggest that the morphogenesis of C. burnetii is comparable, although not identical, to cellular differentiation of endospore formation. A developmental cycle consisting of vegetative and sporogenic differentiation is proposed.

Cell Membrane↗

PubMed1981

Stability of the adenosine 5'-triphosphate pool in Coxiella burnetii: influence of pH and substrate.

The ability of Coxiella burnetii to couple oxidation of metabolic substrates to adenosine 5'-triphosphate (ATP) synthesis in axenic reaction buffers was examined. Pyruvate, succinate, and glutamate were catabolized and incorporated at the highest rates of 11 substrates tested. Glutamate oxidation, however, resulted in the greatest stability of the ATP pool and highest intracellular ATP levels over a 48-h period. At pH 4.5, the optimum for metabolism by C. burnetii, glutamate oxidation resulted in maintenance of the ATP pool at a concentration of approximately 0.7 nmol of ATP per mg of dry weight over a 96-h period. In the absence of substrate, ATP declined by 96 h to less than 0.01 nmol/mg of dry weight. When cells were maintained at pH 7.0 in the presence or absence of glutamate, ATP pools were considerably more stable, presumably due to the minimal metabolic activity displayed by C. burnetii at pH 7. The stability of the ATP pool reflected viability as there was greater than an 8-log decrease in viable C. burnetii after incubation for 7 days at pH 4.5 in the absence of glutamate. Viability was retained in the presence of glutamate at pH 4.5 or 7.0 in the absence of any added substrate. The stability of the ATP pool was due to endogenous synthesis of ATP coupled to substrate oxidation as shown by depression of ATP levels in the presence of inhibitors of electron transport or oxidative phosphorylation. In addition, the adenylate energy charge increased from an initial value of 0.57 to 0.73 during glutamate oxidation with a concomitant rise in the total adenylate pool size. C. burnetii therefore appears able to regulate endogenous ATP levels in response to substrate availability and pH, thus effecting a conservation of metabolic energy in neutral or alkaline environments. Such a mechanism has been proposed to play a role in the resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome in which this organism replicates.

Adenosine Monophosphate↗

PubMed1981

Parathyroid ablation in dystrophic hamsters. Effects on Ca content and histology of heart, diaphragm, and rectus femoris.

Cumulative evidence indicates that there is an increased accumulation of calcium in dystrophic muscle and that this may have a pathophysiological role in the progression of the dystrophic process. The accumulation may be related to a defect of the plasma membrane. Because parathyroid hormone (PTH) stimulates calcium influx into the cytosol, the chronic effects of surgical ablation of the parathyroid glands on muscle Ca, Mg, protein synthesis, and histology, as well as plasma creatine phosphokinase (CPK), Ca, and Mg, were studied in normal and dystrophic (BIO 14.6) hamsters. Thyroparathyroidectomized (TPTX) hamsters receiving replacement doses of l-thyroxine were killed at age 90 d, 55 d after TPTX. In intact dystrophic hamsters, the Ca content in the heart was 20 times higher than in normal animals and was reduced by half in TPTX dystrophic hamsters. Similar results were observed in diaphragm and rectus femoris. No abnormalities in Mg content were observed in intact or TPTX dystrophic hamsters. Ether-extractable fat of the heart and diaphragm was reduced in dystrophic hamsters and was not modified by TPTX. Protein synthesis was enhanced in the diaphragm of dystrophic hamsters but was not changed by TPTX. The concentration of CPK in plasma was elevated in dystrophic hamsters and fell significantly after TPTX. In the latter animals, microscopic examination of the heart showed lesser signs of dystrophy, particularly in the degree of fibrosis. To determine the degree of dystrophy at the age when TPTX was performed, identical analyses were made in 35-d-old hamsters. Definitive histological signs of dystrophy were observed, and although the Ca content in heart, diaphragm, and rectus femoris was elevated, the values were lower than in 90-d-old intact and TPTX dystrophic hamsters. This indicates that chronic TPTX in dystrophic hamsters reduces, but does not arrest, the dystrophic process. In normal hamsters, a 50% reduction in plasma Ca concentration was observed 6 h after TPTX; 55 d after TPTX, however, plasma Ca was within normal limits in both normal and dystrophic hamsters. No parathyroid tissue was observed in serial sections of the trachea and adjacent tissues in TPTX animals. This suggests that in chronically TPTX hamsters fed a standard laboratory diet, plasma Ca can be maintained by mechanisms independent of parathyroid function. THE DATA INDICATE THAT IN DYSTROPHIC HAMSTERS TPTX CAUSES A MARKED REDUCTION IN: (a) muscle Ca accumulation, (b) levels of plasma CPK and, (c) intensity of histological changes in the heart. These changes were independent of the levels of plasma Ca and were not observed in normal hamsters. We conclude that PTH accentuates the dystrophic process, probably by enhancing the already increased Ca flux into muscle (apparently caused by defective sarcolemma). We postulate that normal secretion of PTH may have a deleterious effect in congenital or acquired conditions associated with altered plasma membranes.

Adipose Tissue↗

PubMed1981

N-nitrosomethylurea-induced rat mammary tumours as models of human breast cancer.

A histopathological study is reported of the autopsy finding in two strains of rats treated with N-methyl-N-nitrosourea (MNU). The induced mammary tumours were histologically indistinguishable from those produced by dimethylbenz(a)anthracene (DMBA). A number of other primary tumours were found and there was no evidence of metastases.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1981

N-methyl-N-nitrosourea-induced rat mammary tumors. Hormone responsiveness but lack of spontaneous metastasis.

N-Methyl-N-nitrosourea (MNU) given iv to rats 50-55 days old induced mammary tumors in 70% of F344/N and 91% W/ICRF inbred females with mean latency periods of 149 and 93 days, respectively. Reduction of the MNU dose did not affect tumor incidence in W/ICRF rats. Of the mammary tumors, 98% were classified histologically as adenocarcinomas, which grew progressively. Primary tumors of nonmammary origin were detected at low incidence. Upon histologic examination, no evidence was found for metastases of either the mammary or other primary tumors. No evidence for tumor-induced hypercalcemia was found. Oophorectomy at the time of MNU administration prevented tumor development; oophorectomy when at least 1 tumor/animal was palpable caused growth delay or regression. All MNU-induced and 7,12-dimethylbenz[a]anthracene-induced mammary tumors tested contained cytoplasmic estrogen receptor (ER) at similar concentrations and were indistinguishable histologically. MNU-induced tumors in F344 rats were transplantable and retained ER through three transplantations.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1981

Efficacy of ivermectin against inhibited larvae of Ostertagia ostertagi.

The efficacy of ivermectin against inhibited early 4th-stage larvae of ostertagia ostertagi and other nematodes of the abomasum and intestinal tract was determined in naturally infected yearling beef cattle. The time when large numbers of inhibited larvae were acquired was determined by monthly slaughter of monitor cattle, beginning in January. In April, 12 animals were removed from pasture and maintained free of further helminth exposure until slaughter (21 days). At 9 days after the cattle were removed from pasture, ivermectin was administered to the principals by subcutaneous injection (200 micrograms/kg); the other 6 animals were given subcutaneous injections of the ivermectin vehicle. both groups were klled and necropsied at 12 days after treatment. Mean numbers of O ostertagi in the 6 controls were: adults, 41,906; developing 4th stage, 73,813; and early 4th stage, 334,965. The mean proportion of early 4th-stage larvae was 73.7%. In the 6 principals (treated with ivermectin), the following reductions were observed: O ostertagi adults, 100%; developing 4th stage, 99.8%; and early 4th stage, 99,9%. Small numbers of dead and degenerated O ostertagi of all developmental stages were recovered from abomasal washings before fixation; few viable worms were recovered.

Abomasum↗