Receptor-mediated cellular uptake of cyclosporine.
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Cells, Cultured↗
Biomedical subjects
W F Diven
Publications and source records attributed to W F Diven.
Sequential changes of lamellar body hydrolases during ozone-induced alveolar injury and repair.
Lamellar body hydrolases in acutely damaged and regenerating type II cells were determined using an established rat model with well-defined stages of bronchiolo-alveolar injury and repair. Lamellar bodies were isolated from control and ozone-exposed (3.0 ppm for 8 hours) adult male rats by sucrose density gradient centrifugation and analyzed for their content of six different lysosomal hydrolases. Immediately after 3 ppm ozone exposure (zero-time) there was a significant decrease in specific enzyme activity (units/mg protein) of five lamellar body hydrolases and these activities remained depressed for at least 24 hours after exposure. In addition, total enzyme activity (units/lung) was reduced at zero-time for beta-hexosaminidase and at 24 hours postexposure for alpha-mannosidase and alpha-L-fucosidase. During the reparative and recovery stages (48 to 96 hours) the hydrolases demonstrated variable elevations in both specific activity and total activity (units/lung). Characteristically, beta-hexosaminidase and beta-galactosidase reached supranormal values at 96 hours, whereas alpha-mannosidase remained below normal levels through the recovery stage. Moreover, at 24 to 48 hours the lamellar body fraction demonstrated prominent enzyme depletion relative to the expanding pool of stored surfactant. It is concluded that acute ozone stress initiates the development of hydrolase deficiency within the lamellar bodies of injured and regenerating type II cells. This deficiency state is followed by asynchronous lamellar body hydrolase elevations that reflect distinct patterns of response rather than uniform return to normal condition. The lysosomal enzyme changes of lamellar bodies may be pathogenetically linked to the development of associated alterations in the storage and secretion of surfactant.
Animals↗
Regulation of bile acid synthesis. Isolation and characterization of microsomal phosphatases.
In preliminary experiments, we have shown that rat liver microsomes possess phosphatase activity which was inhibited in the presence of sodium fluoride. We have now separated six microsomal phosphatase fractions appearing to be isoenzymes. They all possess different kinetic constants and are not equally inhibited by tartrate and fluoride ions, inhibitors of phosphatase activity. One phosphatase fraction, in fact, is almost completely unaffected by fluoride ion. More pertinent to our interest, these isoenzymes exhibit differing abilities to modulate the activities of hydroxymethylglutaryl CoA reductase, acyl-CoA:cholesterol O-acetyltransferase, and cholesterol 7 alpha-hydroxylase. Interaction of four of the fractions with rat liver microsomes resulted in a decrease in cholesterol 7 alpha-hydroxylase activity; two were without effect.
Animals↗
Hydrolase activity in middle ear effusions. Effect of antibiotic therapy.
Hydrolytic enzymes have been shown to be present in middle ear effusions recovered from children with both persistent and acute otitis media. In the present study, we investigated the effect of ampicillin therapy on the expression of hydrolytic enzyme activity in acute middle ear effusions using the chinchilla animal model. The median values of enzyme activities were lower for the ampicillin-treated animals when compared with the nontreated control animals. For the ampicillin-treated animals, eight of 12 assayed activities were characterized by a time-dependent decay of enzymatic activity. For the untreated animals, the majority of assayed activities (seven of 12) showed an increase in activity with time. These results show that sterilization of the middle ear cleft and elimination of the hydrolytic enzyme activity may be benefits of antimicrobial therapy and prerequisite to the healing of the inflamed mucosa.
Acute Disease↗
Effects of deletions in mouse chromosome 7 on expression of genes encoding the urea-cycle enzymes and phosphoenolpyruvate carboxykinase (GTP) in liver, kidney, and intestine.
Chromosomal deletions at and around the albino locus on chromosome 7 of the mouse affect the enzyme activities and steady-state levels of mRNAs for five urea-cycle enzymes in liver. In newborn c3H homozygotes, activities of these enzymes were 43-62% of normal, while corresponding mRNA levels were 14-29% of normal. c14CoS deletion homozygotes expressed mRNA levels for these enzymes which were 32-48% of normal. However, transcription rates of these genes in hepatic nuclei of c3H/c3H mice were reduced only to 57-84% of normal. Since effects of the deletions had previously been noted in the kidney, mRNA levels for three enzymes expressed also in the kidney were examined. Mice homozygous for the c3H deletion, shown previously to have drastically reduced mRNA levels for phosphoenolpyruvate carboxykinase in the liver, expressed the same deficiency in the kidney, while mRNA levels for argininosuccinate synthetase and argininosuccinate lyase were reduced in the liver but remained unaffected in the kidney. However, mRNA levels for phosphoenolpyruvate carboxykinase, carbamyl phosphate synthetase I, and ornithine transcarbamylase were unaffected in the intestine of c3H homozygotes. The results suggest that a regulatory factor(s) encoded in the DNA encompassed by the deletion is involved in the normal developmental maturation of hepatocytes and certain cells in the kidney.
Animals↗
Predicting outcome after resuscitation from clinical death.
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Animals↗
CSF alpha 2-macroglobulin and C-reactive protein as aids to rapid diagnosis of acute bacterial meningitis.
alpha 2-Macroglobulin (AMG) and C-reactive protein (CRP) levels in cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis have been analyzed by a rate nephelometric method to determine if these acute phase proteins can aid in differentiation of bacterial from aseptic meningitis. The mean CSF concentrations of AMG and CRP were 15 and 3.5 times greater, respectively, in the bacterial compared to the aseptic meningitis group. Also, the range of AMG levels showed minimal overlap between the two groups. The elevated levels of the proteins persisted after CSF cultures became negative. Quantitation of specific acute phase proteins in CSF may assist the differentiation of bacterial from aseptic meningitis.
Adolescent↗
Efficacy of two third-generation cephalosporins in prophylaxis for head and neck surgery.
Optimal antimicrobial prophylaxis during head and neck surgery is as yet unclear. The semisynthetic third-generation cephalosporin antibiotics would appear to have potential usefulness because of their broad antibacterial spectrum, favorable pharmacokinetic properties, and record of safety. Eighty patients were prospectively randomized into this placebo-controlled, double-blind study to receive either placebo, cefoperazone sodium, or cefotaxime sodium preoperatively and for 24 hours postoperatively. Infection rates were 78% for the placebo group and 10% for the group receiving the cephalosporin antibiotics. Infections were polymicrobial. Anaerobic bacteria constituted 42% of the pathogens isolated. Wound and serum antibiotic concentrations exceeded the minimal inhibitory concentration of the bacterial flora. This study establishes the necessity of antimicrobial prophylaxis for contaminated oncologic procedures of the head and neck. Our experience indicates that cefoperazone and cefotaxime are particularly useful.
Bacteria↗
Properties of cholesterol 7 alpha-hydroxylase in rat liver microsomal acetone powder.
Rat liver microsomes were extracted with acetone, and a microsomal powder preparation was obtained. The cholesterol 7 alpha-hydroxylase activity of acetone powder was linear with time, the amount of protein, and the amount of cholesterol in human or rat serum. Unesterified lipoprotein cholesterol was also an effective substrate, and the Km values increased progressively from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) to very-low-density lipoprotein (VLDL), suggesting that HDL-free-cholesterol was the better substrate.
Acetone↗
Addition of rifampin to ticarcillin-tobramycin combination for the treatment of Pseudomonas aeruginosa infections: assessment in a neutropenic mouse model.
The efficacy of ticarcillin (100 mg/kg), tobramycin (1 mg/kg), and rifampin (43 and 7.2 mg/kg) individually and in combination was assessed in neutropenic mice infected with an LD90 of one of four Pseudomonas aeruginosa isolates. The study end point was survival at 120 hours after infection. Treatment with the triple combination, ticarcillin plus tobramycin plus rifampin (43 mg/kg), was significantly superior to the double combination of ticarcillin plus tobramycin (p less than 0.01). Although treatment with rifampin (43 mg/kg) alone yielded results similar to treatment with the triple combination in mice infected with three of the four isolates, rifampin-resistant mutants (minimal inhibitory concentration greater than 1000 micrograms/ml) of P. aeruginosa were frequently isolated from surviving mice (26% of mice sampled). In contrast, in mice treated with the triple combination, rarely were rifampin-resistant mutants isolated (3% of mice sampled). Rifampin alone was active against P. aeruginosa isolates only when peak serum concentrations of rifampin exceeded the rifampin minimal bactericidal concentration of the infecting isolate. The addition of rifampin to a "standard" therapy of antipseudomonal penicillin plus aminoglycoside may be useful in the treatment of serious P. aeruginosa infection.
Animals↗
Effect of detergents on in vitro 7 alpha-hydroxycholesterol formation by rat liver microsomes.
Formation of 7 alpha-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7 alpha-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7 alpha-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7 alpha-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (Ks greater than Kp). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.
Animals↗
Partial purification and characterization of particulate acid phosphatase of Leishmania donovani promastigotes.
1. More than 90% of the total acid phosphatase activity in a sonicate of L. donovani promastigotes is contained in a particulate fraction (200,000 X g 30 min). The enzyme can be quantitatively extracted and solubilized with the aid of Triton X-100 (0.2 g/100 ml) and purified over 200-fold with 54% yield by chromatography on DEAE-Sephadex, QAE-Sephadex, Sepharose 4B and concanavalin-A Sepharose. 2. The phosphatase is a true acid hydrolase (pH optimum, 5.0-5.5) and has a rather broad substrate specificity; it will catalyze the hydrolysis of 4-methylumbelliferylphosphate, thymolphthalein diphosphate, pyridoxal phosphate, fructose 1,6-diphosphate, glucose 6-phosphate, glucose 1-phosphate, ADP and AMP. 3. It is a large (170,000 daltons in the presence of Triton X-100), stable and acidic enzyme (pI = 4.1) that has the electrophoretic mobility of a type zero or type 1 isoenzyme in acid (pH 4.3) polyacrylamide gels. 4. The enzyme is inhibited by sodium fluoride, 2-mercaptoethanol and mumolar amounts of a number of polyanionic molybdenum and heavy metal complexes that include the following: [C(NH2)3]4[(C3H7O3PO3)2Mo5O15] X 3H2O, [C(NH2)3]2[(C6H5)2AsMo4O15H] X H2O, (NH4)4[SiMo12O40] X H2O and (NH4)6[P2Mo18O62] X 9H2O. 5. L. donovani promastigotes contain very low levels of 10 other acid pH optimum hydrolytic enzymes, with the exception of modest levels of alpha-fucosidase.
Acid Phosphatase↗
Metabolism of lysosomal enzymes in the protein-deficient weanling rat.
We have shown that the protein-deficient weanling rat fed a 3% casein diet, within 2 to 4 wk, exhibits marked changes in serum lysosomal hydrolases similar to those observed in children suffering from protein-calorie malnutrition: serum hexosaminidase, alpha-mannosidase, and beta-glucuronidase activities increase 3-fold, 2-fold, and 50%, respectively, whereas the acid phosphatase levels decrease by 50%. Rehabilitation of the protein-deficient animals with a diet containing 25% protein (i.e., casein) results in a rapid restoration of the plasma lysosomal hydrolase profiles to normal in less than 1 wk. The specific activities of various tissue lysosomal enzymes change significantly in the protein-deficient animals; however, no overall consistent pattern of change is apparent. In general, the greatest number of changes in lysosomal enzymes occurs in the kidney, whereas the brain exhibits the smallest differences between experimental and control animals in this regard. Perfusion experiments have shown that the rate of release of lysosomal enzymes from livers of rats fed the protein-deficient diet is profoundly altered when compared to that of control animals. Studies of the variation of enzyme secretion with time have demonstrated that the rate of secretion of hexosaminidase by the liver remains low and then rises markedly (3-fold) after the animals have been consuming the 3% casein diet for 16 days. In contrast, the secretion of both acid phosphatase and beta-glucuronidase is markedly depressed in the early phase of protein malnutrition (i.e., 7 to 16 days), and then increases greatly by the 3rd wk. These results demonstrate that changes occur in the rate of secretion of lysosomal enzymes by the liver during the course of experimental protein malnutrition.
Acid Phosphatase↗
Determination of cefoperazone concentration in serum and muscle tissue with a versatile high-pressure liquid chromatographic method.
A rapid, specific, and reproducible high-pressure liquid chromatographic method was developed for the determination of cefoperazone concentration in serum and tissue. The assay uses a simple methanol extraction, with cefoxitin as the internal standard. The limits of detection are 1 to 150 micrograms/ml; the maximum coefficient of variation is 7.4%. Using the same chromatography column, muBondapak phenyl, and mobile-phase 0.005 M tetrabutylammonium buffer-acetonitrile (80:20), the method can be easily adapted for the analysis of cefoxitin and moxalactam.
Cefoperazone↗
Altered alpha-mannosidase isoenzymes in the liver in hepatic cirrhosis.
Homogenates of liver from cases of hepatic cirrhosis due to alpha 1-antitrypsin deficiency (PiZZ) alcoholism were analyzed for their content of various lysosomal enzymes. Also determined were the specific activities of lactate dehydrogenase, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase, and creatine phosphokinase in the extracts of liver from cases of both kinds of hepatic cirrhosis: all of these activities were within the range of control values. Similarly, the specific activities of the following lysosomal hydrolases were unremarkable: acid phosphatase, beta-mannosidase, beta-fucosidase, beta-glucuronidase and beta-glucosidase. Hexosaminidase specific activity was increased twofold in livers from the cases of cirrhosis due to alpha 1-antitrypsin deficiency. The specific activity of alpha-mannosidase (measured at pH 4.5) in homogenates of livers from PiZZ individuals with cirrhosis and those with alcoholic cirrhosis was increased two- to four-fold. Chromatography of the high-speed supernatant fraction from homogenates of livers of cirrhotic and noncirrhotic individuals on columns of DEAE-cellulose resolved alpha-mannosidase activity into two components: under the conditions employed, acid pH optimum (pH 4.5) alpha-mannosidase did not bind to the resin, whereas intermediate pH optimum (pH 5.5) alpha-mannosidase could be eluted with 0.1 mol/l NaCl. Liver from one case of (PiZZ) alpha 1-antitrypsin deficiency and emphysema, without demonstrable cirrhosis, was found to contain normal levels of both acid alpha-mannosidase and intermediate alpha-mannosidase. However, cases of cirrhosis due to alpha 1-antitrypsin deficiency contained twice as much acid alpha-mannosidase and only one third to one fourth as much intermediate alpha-mannosidase as controls. The deficiency in hepatic intermediate alpha-mannosidase was also observed in 5 of 5 cases of alcoholic cirrhosis.
Humans↗
CSF lysosomal hydrolase activity as an aid in the diagnosis of bacterial meningitis.
The activity of the lysosomal enzymes acid phosphatase, beta-glucuronidase, alpha-mannosidase and hexosaminidase were determined in CSF obtained from patients with proven bacterial meningitis and from patients with various other diagnoses. The mean value for CSF beta-glucuronidase from bacterial meningitis was elevated 73-fold when compared to the aggregate mean of all control groups. Acid phosphatase and alpha-mannosidase means were 26-fold and 33-fold elevated respectively while hexosaminidase was threefold elevated. Measurement of CSF acid phosphatase and beta-glucuronidase should prove a rapid useful test in establishing the diagnosis of bacterial meningitis. Chromatography of CSF samples on DEAE Sephadex allowed the resolution of hexosaminidase and beta-glucuronidase into individual isozymes. The ratio of hexosaminidase A to hexosaminidase B was generally higher in CSF from patients with bacterial meningitis but was very variable. The isozyme distribution for beta-glucuronidase was identical to that found in serum and no differences in pattern were found between patients and control subjects.
Acid Phosphatase↗
An improved fluorometric leukocyte beta-glucosidase assay for Gaucher's disease.
Three fluorometric leukocyte beta -glucosidase assays were compared for their ability to diagnose Gaucher's disease and identify carriers of the disorder: the acid beta-glucosidase assay of Beutler and Kuhl [2], a pH 5.5-sodium taurocholate-dependent assay and a new procedure which employs conduritol B epoxide, an active-site specific inhibitor of glucocerebrosidase. All three assays unambiguously identified patients with Gaucher's disease. With regard to identifying carriers the bile salt dependent assay of Peters et al. and the conduritol B epoxide-dependent procedure gave the greatest discrimination between the mean beta-glucosidase values for the control and heterozygote samples when evaluated using Student's t test. The most reliable assay for the identification of the carrier state was the conduritol B epoxide-dependent procedure which can be expected to provide the fewest false negative results when classifying heterozygotes (5%). However, the fact that none of these methods will completely separate control and heterozygote samples indicates that their use in screening programs will result in a significant number of incorrect assignments.
Adolescent↗
Measurement of serum and tissue concentration of moxalactam using high pressure liquid chromatography.
We describe a sensitive high pressure liquid chromatographic (HPLC) procedure for the analysis of the new beta-lactam antibiotic moxalactam. Conditions are described for either measurement of total drug concentration or the concentration of the individual isomers. The proteins in a 1.0 ml plasma sample are denatured with isopropyl alcohol which is then extracted into a chloroform reagent, leaving the drug in the aqueous phase. An aliquot is then injected into a mu-bondapak phenyl column. A similar extraction procedure was employed for tissue homogenates. Linear regression analysis and comparison of the HPLC assay with the microbiological assay gave a correlation coefficient of 0.97. Analysis of tissue samples indicated that significant concentrations of moxalactam were obtained at the site of infection.
Biological Assay↗