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Biomedical subjects

J S Little

Publications and source records attributed to J S Little.

At least 37 records · Page 2Linked to original sources
PubMed1990

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Animals↗
PubMed1989

Partial characterization of an enzyme that hydrolyzes sarin, soman, tabun, and diisopropyl phosphorofluoridate (DFP).

The properties of a rat liver enzyme that hydrolyzes organophosphorus (OP) inhibitors of cholinesterases were studied. The rates of hydrolysis of OP inhibitors were determined by continuous titration of released hydrogen ions, using a pH stat method. Centrifugation of homogenates at 205,000 g for 30 min demonstrated that the activity was in the soluble fraction. Hydrolysis of sarin, soman, and diisopropyl phosphorofluoridate (DFP), but not of tabun, was stimulated by the addition of Mn2+ and Mg2+. Hydrolysis of sarin greater than soman greater than tabun greater than DFP. Unlike other OP hydrolases that preferentially hydrolyze the non-toxic isomers of soman, this enzyme hydrolyzed all four soman isomers at approximately the same rate. This result was obtained in vitro by gas chromatographic analysis of enzyme-catalyzed soman hydrolysis and confirmed in vivo by demonstrating reduced toxicity in mice of soman partially hydrolyzed by this enzyme. Km and Vmax were determined by fitting V vs [S] to a hyperbolic function using regression analysis. Km values ranged from 1.1 mM for soman to 8.9 mM for tabun. Vmax values ranged from 54 nmol/min/mg protein for DFP to 2694 for sarin. The enzyme was stable for at least 2 months at -90 degrees but was inactivated by heating at 100 degrees for 5 min. Elution profiles from gel filtration by high pressure liquid chromatography showed that the hydrolytic activity for the OP inhibitors eluted in a single peak, suggesting that a single enzyme was responsible for the observed hydrolysis. Further purification and characterization of this enzyme should prove useful for the development of methods for detection, detoxification, and decontamination of these cholinesterase inhibitors.

Animals↗
PubMed1986

Irrigating solutions used in arthroscopy and their effect on articular cartilage. An in vivo study.

The effect of arthroscopic irrigating solutions on articular cartilage was determined by the use of an animal model. Rabbit knee joints were irrigated continuously for two hours with either normal saline, Ringer's lactate, or sterile water. Subsequently, the rate of incorporation of 35SO4 by articular cartilage was used to measure the effect of the irrigants on chondrocyte metabolism. In addition, the irrigated groups were compared to an unirrigated control group. There was no significant difference in 35SO4 incorporation between the groups. This suggested that none of the irrigating solutions used in this study adversely affected articular cartilage function. On the basis of these findings, it appears that normal saline, Ringer's lactate, and sterile water can be safely used as irrigating solutions during most arthroscopic procedures.

Animals↗
PubMed1985

The effect of Streptococcus pneumoniae infection on the binding of triiodothyronine to nuclei isolated from rat liver.

Hepatic nuclei were isolated from control and Streptococcus (S.) pneumoniae-infected rats to determine the effect of S. pneumoniae infection on the binding capacity and affinity of hepatic nuclei for T3. Infection did not affect the purity or yield of the isolated nuclei. A significant decrease in serum total T3 was observed 40 h after inoculation with S. pneumoniae. Serum free T3 was significantly decreased by 20 h after inoculation, but returned to control levels by 40 h after inoculation. Scatchard analysis of nuclear T3 binding, determined under optimal conditions, confirmed the presence of high affinity, low capacity sites for T3 on nuclei isolated from both control and infected rats. During infection, the maximum binding capacity of the purified nuclei for T3 decreased significantly, but infection had no significant effect on the affinity of the receptor for T3. These results suggest that the decrease in serum T3 observed during infection is not contributed to by increased hepatic nuclear T3 receptor concentration and that the increased hepatic RNA, protein, and lipid synthesis observed during S. pneumoniae infection in the rat is not the result of increased binding or affinity of T3 to the nuclear receptor.

Animals↗
PubMed1985

Effect of thyroid hormone supplementation on survival after bacterial infection.

During nonthyroidal illness in man and a number of experimental animals, serum total thyroid hormone levels are decreased. To determine whether the reduction in thyroid hormones that occurs during nonthyroidal illness is beneficial or harmful to the host, an experimental animal model was developed. Male Sprague-Dawley rats were infected with Streptococcus pneumoniae type I, a known human pathogen. By 40 h after inoculation, serum total and free T4 and total T3 decreased significantly. Serum free T3 also decreased slightly but not significantly. When infected animals were injected daily with T4, there was a decrease in the time to death and significant increase in the total number of deaths compared to controls receiving vehicle alone. Control experiments showed that T4 did not affect the growth of S. pneumoniae on blood agar plates, and the injected T4 did not cause hyperthyroidism. These results suggest that the decrease in serum thyroid hormone that occurs during nonthyroidal illness is beneficial and may serve a role in maintaining homeostasis by conserving metabolic energy during infection as an important adaptation that prolongs survival.

Animals↗
PubMed1984

Biochemical and cytochemical localization of inosine-5'-diphosphatase in rat liver microsomal fractions.

A procedure has been developed for the biochemical and cytochemical localization of inosine-5'-diphosphatase (IDPase) in rat liver microsomal fractions. The concentration of Pb2+ ions used in the cytochemical incubation medium inhibited IDPase by 32%. IDP formed during the incubation was not hydrolysed further to inosine, and no significant non-enzymic hydrolysis of substrate occurred. When microsomal fractions were incubated cytochemically for IDPase and separated from unchanged substrate under conditions such that the microsomal membrane was not permeable to EDTA, approx. 80% of the reaction product was solubilized by EDTA. In the electron microscope, lead precipitate was observed on the cytoplasmic side (outside) of a major fraction of the vesicles and on the cisternal side (inside) of others. After extraction with EDTA, the lead precipitate was observed only on the cisternal side. Since it was shown that phosphate is trapped on the same side of the membrane as it is released, it is concluded that IDPase can release phosphate on either the cisternal or the cytoplasmic side of the microsomal membrane.

Acid Anhydride Hydrolases↗
PubMed1984

The effect of Streptococcus pneumoniae infection on the binding of thyroxine to purified rat liver plasma membranes.

Hepatic plasma membranes were isolated from control and Streptococcus pneumoniae-infected rats in order to determine the effect of S. pneumoniae infection on the binding capacity and affinity of hepatic plasma membranes for T4. Infection did not affect the purity or the yield of isolated membranes. A significant decrease in both total and free serum T4 was observed during infection. Scatchard analysis of membrane binding, determined under optimal conditions, confirmed the presence of high affinity, low capacity sites, as well as low affinity sites for T4 on membranes isolated from both control and infected rats. T4 maximum binding capacity (MBC) of the high affinity sites decreased significantly as the infection became more severe. However, the affinity of these receptors did not change. Neither MBC nor affinity of the low affinity sites was altered by infection. The observed decrease in the MBC of the high affinity binding sites for T4 on hepatic plasma membranes cannot account for the decrease in serum T4 or the hepatic metabolic alterations also known to occur during S. pneumoniae infection.

Animals↗
PubMed1983

An assay of RNA synthesis in hepatic nuclei from control and Streptococcus pneumoniae-infected rats.

Hepatic nuclei were isolated, purified, and partially characterized from control and Streptococcus pneumoniae-infected rats. Biochemical and morphologic examination showed little contamination by other cell organelles. An in vitro system for the incorporation of 2-[14C]uridine-5'-triphosphate into ribonucleic acid (RNA) was developed and characterized. Although the stimulatory effects of cytosol on incorporation of labeled precursors into RNA have been previously reported, nuclei isolated from the livers of S. pneumoniae-infected rats were stimulated to a significantly greater extent than were nuclei isolated from the livers of control rats. In the presence of cytosol prepared from either control or infected rats, the increased incorporation of labeled precursor into RNA by nuclei isolated from infected rats was observed over broad pH and temperature ranges. The increased activity of infected nuclei was eliminated when albumin was substituted for cytosol, and could not be accounted for by differences in endogenous precursor pool size. These results are consistent with other infection-induced alterations in hepatic RNA and protein synthesis.

Animals↗
PubMed1983

Intracellular fate of phase I Coxiella burnetii in guniea pig peritoneal macrophages.

Cultivated guinea pig peritoneal macrophages were infected with radio-labeled phase I Coxiella burnetii in order to assess the intracellular distribution of ingested rickettsiae. Localization of organisms was determined by fractionation of macrophage homogenates by equilibrium density centrifugation on sucrose gradients. Macrophages isolated from either nonimmune or immune guinea pigs and infected with C burnetii opsonized with immune serum yielded equilibrium density distribution for rickettsiae similar to lysosomal enzymes, suggesting sequestration within macrophage lysosomes. To confirm these observations nonimmune or immune guinea pigs were injected with Triton WR-1339 prior to macrophage harvest to decrease the density of macrophage lysosomes. Triton-laden macrophages infected with opsonized rickettsiae resulted in equilibrium density distribution for lysosomal enzymes and organisms in less dense regions of the gradient. In contrast, when either nonimmune or immune macrophages were infected in the presence of normal guinea pig serum, the distribution of labeled rickettsiae in the gradient did not correspond with lysosomes. We conclude that in the absence of immune serum, ingested C burnetii are not sequestered within macrophage lysosomes. Phagolysomal fusion and subsequent degradation of rickettsiae within the lysosomes of the macrophages appear to occur only when C burnetii are opsonized with immune serum.

Animals↗
PubMed1981

Effect of Streptococcus pneumoniae infection in rats on hepatic water content, electrolyte concentration, and chemical composition.

Total hepatic water content, dry weight, protein, lipid, carbohydrate, RNA, DNA, and electrolyte concentrations were determined in control and Streptococcus pneumoniae-infected rats. During infection, there was a significant (P = 0.001) increase in total liver weight. This increase was the result of increased total liver water content and increased total liver dry weight. Intracellular water content, diameter of hepatocytes, and all measured constituents of hepatic dry weight, excluding DNA, increased significantly (P varied from 0.05-0.001) during infection. Concentrations of liver Na+ and Cl- increased significantly (P varied from 0.05-0.005), whereas the concentration of liver K+ decreased significantly (P = 0.01). Seemingly, there is an enlargement of hepatocytes due to increased intracellular water and increased dry weight during pneumococcal infection in the rat. The mechanism of these liver changes may be due, in part, to a shift of Na+ and K+ across liver cell plasma membranes.

Animals↗
PubMed1980

In vitro studies of interaction of rickettsia and macrophages: effect of ultraviolet light on Coxiella burnetii inactivation and macrophage enzymes.

The inactivation of Coxiella burnetii in suspension or in cultures of guinea pig peritoneal macrophages by ultraviolet (UV) light was studied. The effect of UV treatment on the activity of macrophage organelle marker enzymes and their subsequent equilibration in linear sucrose gradients was also determined. It was shown that UV treatment of 600 mu W/cm2 for 15 s at a distance of 10 cm inactivated C. burnetti, either in suspension (10(8) organisms per ml) or within guinea pig peritoneal macrophages. Similar UV treatment had little effect on the activity or equilibration of macrophage organelle marker enzymes in linear sucrose gradients. However, longer exposure caused considerable inactivation of these enzymes.

Acetylglucosaminidase↗
PubMed1980

Elevated glycosyltransferase activities in infected or traumatized hosts: nonspecific response to inflammation.

Streptococcus pneumoniae infection leads to multifold increases in sialyltransferase, galactosyltransferase, alpha 2-fucosyltransferase, and alpha 3-fucosyltransferase activity of rat liver. Such changes may reflect an increased demand for glycosylation of acute-phase proteins synthesized and secreted by the liver during inflammatory processes. Serum sialyltransferase became elevated in bacteria-infected or burned rats and sandfly fever-infected humans, but did not correlate with acute-phase serum protein changes. These data suggest that nonparenchymal liver cells, such as macrophages, may contribute substantially to elevated sialyltransferase activity in the circulation during infection and, as such, represent a general host response to infection and tissue trauma.

Acetylglucosamine↗
PubMed1978

Synthesis, transport, and secretion of plasma proteins by the livers of control and Streptococcus pneumoniae-infected rats.

The synthesis, intracellular transport, and secretion of plasma proteins by the liver was studied in both control and Streptococcus pneumoniae-infected rats. Rats were injected with [3H]leucine; at various time intervals, the components of the intracellular secretory system were isolated. The isolation and partial characterization of rough microsomes, smooth microsomes, and Golgi from both control and infected animals are described. After infection, the specific activity of the label in the homogenate and in all isolated cell fractions was significantly increased. In both control and infected animals, the kinetics of labeling suggested that the secretory pathway of the newly synthesized protein was from the rough endoplasmic reticulum to the smooth endoplasmic reticulum to the Golgi and finally to the circulation. Even though infected animals synthesized, transported, and secreted significantly more plasma protein, infection did not significantly alter the secretion time (time between injection of isotope and appearance of labeled protein in the circulation). It is concluded that, after S. pneumoniae infection, the liver still maintains its capability to perform the basic functions of protein synthesis, intracellular transport, and secretion and that newly synthesized plasma protein follows the same intracellular pathway in both control and S. pneumoniae-infected rats.

Animals↗
PubMed1977

Effects of pneumococcal infection on rat liver microsomal enzymes and lipogenesis by isolated hepatocytes.

Modification in the enzymatic complement and lipogenic functions of rat liver endoplasmic reticulum (ER) were shown to occur during pneumococcal sepsis. Glucose-6-phosphatase, 5'nucleotidase, esterase, and NADPH cytochrome C reductase decreased in activity by as much as 50% with respect to controls. Hydroxymethylglutaryl-CoA and NADH cytochrome C reductases were increased 6-and 2-fold, respectively. Alkaline phosphatase and inosine-5'-diphosphatase did not differ with respect to fasted controls. The lipogenic capacity of the ER was shown to be enhanced. In vitro [14C]acetate incorporation into cholesterol and other lipids by hepatocytes isolated from infected rats was increased 2-to 10-fold. It is concluded that the flow of acetyl-CoA in liver cell of Streptococcus pneumoniae-infected rats is toward lipogenesis rather than ketogenesis.

Animals↗
PubMed1977

Isolation and partial characterization of plasma membranes from the livers of control and Streptococcus pneumoniae-infected rats.

Plasma membranes were isolated from the livers of control and Streptococcus pneumoniae-infected rats. This work, therefore, represents the first isolation of plasma membranes from infected actron microscopy and by the use of enzyme markers for microsomes (glucose-6-phosphatase), mitochondria (glutamate and malate dehydrogenases), and lysosomes (acid phosphatase). Plasma membranes from infected cells banded at the same sucrose density as plasma membranes from uninfected cells. Moreover, equivalent amounts of plasma membranes could be isolated from control and infected rat livers. There were, however, significant alterations in the enzyme complement of the plasma membrane after infection. 5'-Nucleotidase activity was significantly decreased, whereas alkaline phosphatase activity was significantly increased. Kinetic analysis demonstrated that only the Vmax and not the Km of these two enzymes was changed, suggesting that the altered affinity of the enzymes for substrate was not the mechanism responsible for the observed alterations. No change in the mitochondrial enzyme markers was observed after infection, but the specific activity of microsomal glucose-6-phosphatase decreased significantly. Possible explanations for the observed alterations are discussed.

Acid Phosphatase↗
PubMed1975

Evidence for the translocation of 5'-nucleotidase across hepatic membranes in vivo.

Hepatic 5'-nucleotidase (EC 3.1.3.5; 5'-ribonucleotide phosphohydrolase) activity has been studied in cisternal elements of the Golgi complex and in secretion vacuoles, both isolated after ethanol administration to rats in vivo. The enzyme in secretion vacuoles was latent, so that a 5-fold increase in activity was observed when incubations were carried out in the presence of detergent; evidence is presented that the latency is caused by the impermeability of the membrane to substrate. Essentially no latency was observed in Golgi cisternae. Confirming the results of Farquhar et al. [(1974) J. Cell Biol. 60, 8-25], reaction product from 5'-nucleotidase was localized by cytochemical procedures on the inside of secretion vacuoles and on the cytoplasmic side of Golgi cisternae. After solubilization in detergent, the enzyme from both fractions reacted almost identically with both antibody to the purified enzyme and concanavalin A. In contrast, when intact fractions were incubated with an excess of antibody or concanavalin A, only 22-23% of the enzyme was inhibited in secretion vacuoles whereas 51-84% was inhibited in Golgi cisternae. Sonication of secretion vacuoles in the presence of antibody or concanavalin A increased the inhibition 2- to 3-fold. It is suggested that during the formation of secretion vacuoles from the Golgi cisternae, 5'-nucleotidase is translocated from the cytoplasmic side of the membrane to the inside.

Animals↗