Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase alpha. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10(8) nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs. The specific DNA polymerase alpha activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10(8) cells, it decreases in mature cells to 57 units/10(8) cells, and in cells from senescent animals to 36 units/10(8) cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.     

Gas chromatographic determination of chloramphenicol residues in shrimp: interlaboratory study

An interlaboratory study of a gas chromatographic method for determining chloramphenicol (CAP) residues in shrimp was conducted. An internal standard (Istd), the meta isomer of CAP, was added to the shrimp, and the treated shrimp were homogenized with ethyl acetate. The ethyl acetate extract was defatted with hexane, and the CAP was partitioned into ethyl acetate from an aqueous salt solution. The ethyl acetate was evaporated, and the dried residue was treated with Sylon, a trimethylsilyl derivatizing agent, to yield the trimethylsilyl derivative of CAP. A portion of the solution containing the derivative was injected into a gas chromatograph equipped with an electron capture detector. Levels of fortified and incurred CAP were calculated from the peak area ratio of standard CAP to Istd. Recoveries of CAP from tissue directly fortified at 5 ppb were 102% (within-laboratory relative standard deviation [RSDr] = 5.6%), 104% (RSDr = 5.5%), and 108% (RSDr = 6.3%) from Laboratories 1, 2, and 3, respectively. Incurred-CAP residues at 5 and 10 ppb levels were also determined, with the following results: Laboratory 1: composite A, 4.56 ppb (RSDr = 14.0%); composite B, 8.38 ppb (RSDr = 11.6%); Laboratory 2: composite A, 4.17 ppb (RSDr = 12.5%); composite B, 8.90 ppb (RSDr = 5.60%); Laboratory 3: composite A, 4.66 ppb (RSDr = 14.9%); composite B, 11.0 ppb (RSDr = 11.8%).     

Cell fates in leech embryos with duplicated lineages

We have examined the fates of the progeny of supernumerary embryonic stem cells (O/P teloblasts) generated by microinjecting polyadenylic acid into newborn O/P teloblasts in embryos of the leech, Helobdella triserialis. In normal development, each O/P teloblast generates a rostrocaudal column of daughter cells (primary blast cells) that contribute distinct segmentally iterated O or P sets of epidermal and neural progeny to the mature leech. Previous results suggest that primary blast cells derived from ipsilateral pairs of O/P teloblasts are equipotent and equivalent at birth; that they and their progeny assume distinct O or P fates according to hierarchical and position-dependent interactions; and that the P fate is the primary, or default, fate and the O fate is the secondary fate. In the work presented here, one O/P teloblast was experimentally induced to undergo a supernumerary equal division, and the developmental fates of the progeny of the three (two "duplicate" and one "nonduplicate") ipsilateral O/P teloblasts were determined at stages 8 and 10. We find that some supernumerary O/P teloblasts produce supernumerary P progeny, whereas others generate supernumerary O progeny. When three O/P-derived bandlets are present, bandlets derived from the duplicate O/P teloblasts give rise to progeny of the same (O or P) fate. When the nonduplicate bandlet is absent, the duplicate bandlets assume distinct O and P fates. These results suggest that ipsilateral sister O/P teloblasts, while equipotent, might not be equivalent.     

Thromboxane stimulation of mesangial cell fibronectin synthesis is signalled by protein kinase C and modulated by cGMP

Thromboxane (TX) has been implicated in the pathogenesis of glomerulosclerosis in several models of glomerular injury. In the present study, we examined the role of the protein kinase C (PKC) signalling system in expression of the action of the TXA2/PGH2 analogue U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat mesangial cells (MC), and the influence of cGMP on this MC response. U-46619 activated PKC and enhanced Fn synthesis in MC in a time and concentration dependent fashion. Both responses to U-46619 were blocked by GF 109203X, a selective inhibitor of PKC activity, as well as by calphostin C and staurosporine, PKC inhibitors structurally distinct from GFX. Down-regulation of PKC by prior sustained exposure of MC to 0.5 microM phorbol myristate acetate similarly blocked increases in Fn synthesis induced by U-46619. The TXA2/PGH2 receptor antagonist Sq-29548 also prevented activation of PKC and stimulation of Fn synthesis by U-46619, consistent with transduction of these responses via specific high affinity TXA2/PGH2 receptors on MC. Addition of exogenous 8-Br-cGMP or stimulation of endogenous cGMP generation with atrial natriuretic peptide (ANP) suppressed both U-46619 activation of PKC and stimulation of Fn synthesis. cGMP did not alter TXA2/PGH2 receptor number of affinity in MC, but significantly suppressed phorbol ester activation of PKC. Thus, cGMP inhibition of U-46619 actions is expressed at steps distal to TX receptor binding and may involve effects at and proximal to activation of PKC. Interactions between the PKC and cGMP cellular signalling systems may be important determinants of MC matrix protein production in response to TX.     

The activation of prothrombin by the prothrombinase complex. The contribution of the substrate-membrane interaction to catalysis

The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholine and phosphatidylserine (PCPS). The concentration of PCPS was varied to alter the concentration of free phospholipid available for substrate binding without influencing the concentration of membrane-assembled prothrombinase. In fluorescence stopped-flow measurements, increasing concentrations of PCPS resulted in an increase in the rate of product formation. Assessment of bond cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following rapid chemical quench using 125I-prothrombin revealed that the activation reaction proceeded through the ordered cleavage at Arg323-Ile324 followed by cleavage at Art274-Thr275 at all concentrations of PCPS. Increasing the PCPS concentration resulted in a large increase in the Arg323-Ile324 cleavage reaction with a much smaller effect on the subsequent cleavage at Arg274-Thr275, thereby leading to an increase in the extent of accumulation of the intermediate, meizothrombin. Fluorescence stopped-flow and rapid chemical quench measurements were also conducted using prethrombin 2 plus fragment 1.2 or meizothrombin as substrates to assess the influence of PCPS on the individual cleavage reactions. The rate of cleavage at Arg323-Ile324 by prothrombinase was increased approximately 60-fold with increasing PCPS, whereas the cleavage at Arg274-Thr275 was increased by a factor of approximately 5. These differential effects of PCPS on the two cleavage reactions adequately explain changes in the extent of accumulation of meizothrombin during prothrombin activation. Proteolytic removal of the membrane binding fragment 1 domain of the substrates, meizothrombin and prethrombin 2-fragment 1.2, had no effect on the cleavage at Arg274-Thr275 at saturating PCPS but completely eliminated the membrane-dependent rate enhancement for cleavage at Arg323-Ile324. Thus, membrane binding by the substrate is essential for the first cleavage reaction at Arg323-Ile324, which leads to the conversion of prothrombin to meizothrombin. In contrast, the substrate-membrane interaction mediated by the fragment 1 domain has no detectable effect on the second cleavage reaction at Arg274-Thr275 which is required for the conversion of meizothrombin to thrombin.     

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.     

Dye leakage of four root end filling materials: effects of blood contamination

OBJECTIVES: A measurement of cell DNA content would be highly useful in determining the malignant nature of thyroid tumours in cases without distinctive features such as metastases, capsule invasion or emboli. Abnormal cell ploidy can be recognized with flow cytometry, but it is not known whether such results have diagnostic value. We therefore compared--in a double blind prospective study--the results of flow cytometry and pathologic diagnosis in fresh tumoural and non-tumoural thyroid cells. METHODS: Fifty unselected cold thyroid nodules were obtained from 50 consecutive patients (40 women, 10 men; age 18-80 years; mean 46) who underwent surgery within a 6 month period. Surrounding non-tumoural tissue was also obtained in 46 of them. Cell ploidy and the percentage of cells in each cell phase was determined with flow cytometry for both tumoural and nontumoural tissues. Two pathologists, unaware of the flow cytometric results, independently established the histologic diagnosis according to the WHO classification. RESULTS: The pathologic diagnosis was carcinoma in 7 cases (papillary carcinoma 6, vesicular carcinoma 1) and benign adenomas in 43 (29 macrovesicular, 11 microvesicular, 3 oncocytal). All the non-tumoural tissue samples were diploid. All 7 carcinomas were diploid and 10 of the 43 benign adenomas were aneuploid (4 near-diploid, 3 hyperploid, 1 near-tetraploid, 2 multiploid). The mean proliferation index was increased in 5 diploid tumours. CONCLUSION: These findings confirm that cell ploidy measured by flow cytometry is of no diagnostic value in the thyroid gland. It was also revealed that aneuploidy in adenomas may be related to tissue rearrangements of undetermined prognostic significance.     

Acetylator phenotype, aminobiphenyl-hemoglobin adduct levels, and bladder cancer risk in white, black, and Asian men in Los Angeles, California

BACKGROUND: There is a large body of epidemiologic and experimental data that have identified a number of arylamines as human bladder carcinogens. Metabolic activation is required to biotransform these arylamines into their carcinogenic forms, and N-hydroxylation, which is catalyzed by the hepatic cytochrome P4501A2 isoenzyme, is generally viewed as the first critical step. On the other hand, the N-acetylation reaction, catalyzed by the hepatic N-acetyltransferase enzyme, represents a detoxification pathway for such compounds. The N-acetyltransferase enzyme is coded by a single gene displaying two phenotypes, slow and rapid acetylators. In the United States, cigarette smoking is a major cause of bladder cancer in men, and carcinogenic arylamines present in cigarette smoke are believed to be responsible for inducing bladder cancer in smokers. PURPOSE: Our purpose was to test the differences in three ethnic/racial groups for the prevalence of acetylator phenotypes and to ascertain whether slow acetylators actually have higher levels of activated arylamines in comparison with rapid acetylators. METHODS: One hundred thirty-three male residents of Los Angeles County who were either white, black, or Asian (Chinese or Japanese) and over the age of 35 years were assessed for their acetylator phenotype and levels of 3- and 4-aminobiphenyl (ABP) hemoglobin adducts. Subjects were either lifetime nonsmokers (n = 72) or current cigarette smokers of varying intensity (n = 61). RESULTS: The proportion of slow acetylators was highest among whites (54%), intermediate among blacks (34%), and lowest among Asians (14%). Similarly, geometric mean levels of both 3- and 4-ABP-hemoglobin adducts were highest in whites (1.80 and 49.2 pg/g hemoglobin [Hb], respectively), intermediate in blacks (1.54 and 38.5 pg/g Hb), and lowest in Asians (0.73 and 36.0 pg/g Hb). As expected, cigarette smokers had significantly higher mean levels of both 3- and 4-ABP-hemoglobin adducts relative to nonsmokers, and the levels increased with the number of cigarettes smoked per day (P < .0005 for both adducts). Slow acetylators consistently exhibited higher mean levels of ABP-hemoglobin adducts relative to rapid acetylators, independent of race and level of smoking. CONCLUSION: The present cross-sectional survey supports acetylation phenotype as an important determinant of bladder cancer risk and a possible major factor in the varying bladder cancer risk among whites, blacks, and Asians.     

Identification of drugs in pharmaceutical dosage forms by X-ray powder diffractometry

A simple X-ray powder diffractometric (XRD) method was developed for the identification of the active ingredient in a variety of dosage forms. The method was successfully used to unambiguously identify the active ingredient(s) in tablet, capsule, suppository and ointment formulations. The unique feature of the method is that it provides information about the solid-state of the drug. Thus, a capsule formulation containing anhydrous ampicillin was readily distinguished from that containing ampicillin trihydrate. The USP stipulates the use of the beta-polymorphic form of anhydrous carbamazepine in carbamazepine tablets. Contamination by the alpha-polymorph (down to a level of 1.4% w/w of the formulation) could be detected. In some of the multicomponent formulations, there was a pronounced overlap of the powder patterns of ingredients which made identification difficult. This problem was solved by using a pattern subtraction technique, which permitted selective subtraction of the XRD pattern of the constituents of the formulation from the overall XRD pattern. Such an approach enabled identification of the drug even when it constituted only 5% w/w of the formulation. The method also permitted simultaneous identification of the multiple active ingredients in trimethoprim-sulfamethoxazole and acetaminophen-aspirin-caffeine formulations.