Plasmid profile analysis of a salmonellosis outbreak and identification of a restriction and modification system.

After an outbreak of salmonellosis in humans caused by Salmonella typhimurium bacteriophage type 135, 62 isolates from human, animal, and water sources were retained for further analysis. Most of the isolates (92%) could be placed in one of five plasmid pattern groups, with a majority containing a common 60-kilobase plasmid and a smaller 3.8-kilobase-pair plasmid. This small plasmid, pIMVS1, was labeled with [32P]phosphate and used as a probe in subsequent colony and Southern hybridization studies. We concluded that pIMVS1 from isolates obtained from humans was genetically different from plasmids of a similar size found in isolates from chickens. Studies to characterize pIMVS1 were undertaken to determine if it codes for known virulence factors. It did not appear to be associated with the formation of attachment pili or major outer membrane proteins. By using transposon mutagenesis techniques, Tn3(Apr) was inserted into pIMVS1, and the existence of a restriction and modification system was deduced.     

Comparison of the properties of a soluble form of glucocerebrosidase from human urine with those of the membrane-associated tissue enzyme

Human urine contains a soluble form of glucocerebrosidase, an enzyme associated with the lysosomal membrane in cells and tissues. Urinary glucocerebrosidase is identical to the enzyme extracted from tissues with respect to the following parameters: Km for natural and artificial substrates, inhibition by conduritol B-epoxide, and stimulation by taurocholate. The enzyme is greater than 90% precipitable by polyclonal anti-(placental glucocerebrosidase) antiserum. Upon isoelectric focussing of urinary glucocerebrosidase multiple peaks of activity were observed. Partial deglycosylation (removal of sialic acid, N-acetylglucosamine and galactose) of the urinary enzyme increased the isoelectric point to a value identical to that of the main form found after partial deglycosylation of the placental enzyme. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by immunoblotting, the immunopurified urinary enzyme shows the same molecular mass forms as the enzyme immunopurified from brain and kidney. In placenta the apparent molecular mass is somewhat higher but upon removal of sialic acid, N-acetylglucosamine and galactose the urinary and the placental enzyme show identical molecular masses of 57 kDa. We conclude that the enzymes extracted from urine and tissue are identical and that differences in apparent molecular mass and isoelectric point are probably due to heterogeneity in the oligosaccharide moieties of the molecules.     

Effect of long-term Intralipid administration in mice

Intralipid was administered intravenously to mice at a level of 2 g kg-1 day-1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 micron multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 micron liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 micron MLV of phosphatidylcholine:cholesterol (2:1). Intralipid was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.     

Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation

In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H₂, CO₂, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe.     

Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450

In order to elucidate the isozyme specificity of complex formation between cytochrome P-450 and the isosafrole metabolite the effect of complex dissociation on different steroid hydroxylation pathways was studied in hepatic microsomal fractions. Isosafrole induction was found to increase the 16 beta- and 7 alpha-hydroxylation of androst-4-ene-3,17-dione approximately 2.8- and 1.7-fold, respectively, whereas the 16 alpha-hydroxylation pathway was decreased to about one-quarter of control activity; 6 beta-hydroxylation was unchanged from control activity. More striking changes were apparent following dissociation of the isosafrole metabolite from its complex with ferricytochrome P-450 by the steroid substrate. Thus an approximate fourfold elevation of 16 beta-hydroxylase activity was observed after displacement and 6 beta-hydroxylation increased about twofold; 7 alpha-hydroxylase activity was decreased to 0.75-fold of undisplaced activity and 16 alpha-hydroxylase activity was unchanged. These data provide convincing evidence that at least two forms of phenobarbital-inducible cytochrome P-450 (cytochromes P-450PB-B and P-450PB/PCN-E) are present to some extent in a catalytically inactive complexed state in isosafrole-induced rat hepatic microsomes. Furthermore, there is now evidence to suggest that the constitutive isozymes cytochrome P-450UT-A and cytochrome P-450UT-F are not complexed to any degree in hepatic microsomes from isosafrole-induced rats.     

Eimeria tenella: parasite-specific incorporation of 3H-uracil as a quantitative measure of intracellular development

An assay has been developed using parasite-specific incorporation of 3H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, 3H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional 3H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of 3H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.     

Polyploid cells in blastocysts and early fetuses from Australian Merino sheep

Cytogenetic examination was made of 103 13-14-day-old blastocysts and 116 24-32-day-old fetuses from untreated and androstenedione-7-HSA-immunized Merino ewes. There were no differences in the chromosome composition of blastocysts or fetuses from treated or untreated ewes and so the data were combined. At Days 13-14 a 1N/2N mosaic and a 2N - 1/2N/4N mosaic embryo were observed. In addition, 52 of the blastocysts were 2N/4N mosaics, with 8 of these also containing 8N cells, and one blastocyst was a 2N/8N mosaic. No aneuploid fetuses were observed, but 80 of the 116 fetuses contained polyploid cells, including 4N, 6N and 8N cells. The polyploid cells observed in the blastocysts and fetuses should not be considered as abnormal cells as they appear to be a normal part of the developmental processes leading to trophoblast formation and fetal differentiation.     

Fertilization and early embryonic development in androstenedione-immunized Merino ewes

Ewes were immunized against androstenedione (Fecundin) and assigned to be mated 14 days (179 ewes Group C) or 25 days (174 ewes Group B) after a booster immunization with Fecundin. The anti-androstenedione titres at these times were 6790 and 3240 respectively (P less than 0.01). The remaining 169 ewes were untreated controls (Group A). Ewes were mated to entire rams (12 rams to 180 ewes) at their second oestrus after synchronization of oestrus. Immunization against androstenedione caused a shortening of the time from sponge removal to mating (Day 0) and a decrease in the percentage of ewes mated by the rams. Also, ovulation rate was increased after immunization (P less than 0.01), being 1.42, 2.16 and 1.93 for Groups A, C and B respectively. Egg recovery rates on Day 2 were lower in immunized ewes and there was some indication that fertilization rates were lowered. On Day 13 after mating a higher proportion of blastocysts was recovered from ewes in Group A than from those in Groups B and C. Immunization resulted in lower fertilization rates and smaller blastocysts with lower mitotic indexes (P less than 0.01). At Days 24-32 of pregnancy fetal weight was lower in the immunized ewes. At all sampling stages, the proportion of ewes pregnant (fertility) was lowered in immunized ewes. The results of the present study show that significant reproductive wastage occurs in androstenedione-immunized Merino ewes, with lower rates of embryo recovery and delayed embryonic development being found in comparison to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.     

Modulation of collagen production in cultured fibroblasts by a low-frequency, pulsed magnetic field

Primary cultures of chicken tendon fibroblasts have been exposed for various periods to a low-frequency, pulsed magnetic field, and the effects on protein and collagen synthesis have been examined by radioisotopic incorporation. Total protein synthesis was increased in confluent cells treated with a pulsed magnetic field for the last 24 h of culture as well as in cells treated for a total of 6 days. However, in 6 day-treated cultures, collagen accumulation was specifically enhanced as compared to total protein, whereas after short-term exposure, collagen production was increased only to the same extent as total protein. Levels of cyclic AMP were significantly decreased after 6-day pulsed magnetic field treatment, probably as a consequence of diminished adenylate cyclase activity. Exposure to pulsed magnetic field had no effect on cell proliferation or collagen phenotype. These results indicate that a pulsed magnetic field can specifically increase production of collagen, the major differentiated function of fibroblasts, possibly by altering cyclic-AMP metabolism.