The immunomodulatory effects of urine from patients with superficial bladder cancer receiving intravesical evans BCG therapy

Summary Bladder cancer cells were stimulated with urine obtained from patients with superficial bladder cancer who had received treatment using intravesical bacillus Calmette-Guérin (BCG). The urine from the first 12 h following each of six BCG instillations was collected and examined for its biological effect. We evaluated effects that had previously been attributed to cytokines detected in the urine of such patients. The modulation of MHC class II antigen and intercellular adhesion molecule-1 (ICAM-1) expression were studied. Using neutralizing polyclonal antibodies to interferon and tumour factor the relative contribution of these molecules to the effects investigated were determined. When cells were stimulated for up to 48 h with first-instillation urine, little effect was seen in any of the parameters investigated. Urine from the sixth instillation, however, proved to be a potent immunomodulatory agent, inducing MHC class II molecule and ICAM-1 expression. Urine from instillations two to five mediated increasing immunomodulatory effects. When sixth-instillation urine samples were treated with neutralizing antibodies to interferon prior to their addition to the bladder cancer cells, a marked and significant decrease in their potency was observed. Only in urine from one patient did any immunomodulatory capability remain after antibody treatment. Neutralizing antibodies to tumour necrosis factor , however, failed to reduce the ability of any patient's urine to induce ICAM-1 expression. When both antibodies were used simultaneously no further decrease in potency was observed. These studies demonstrate for the first time the potential immunomodulatory and cytotoxic effects of urine produced by patients receiving intravesical BCG. Furthermore, in all samples tested, the major immunomodulatory component was shown to be interferon . Although tumour necrosis factor is produced as a result of BCG therapy, this cytokine did not appear to contribute to the parameters investigated. namely the induction of HLA class II antigens, and cell-surface ICAM-1.     

Stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography-mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97-99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1-3% for samples prepared concurrently and 2-11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.     

The design,synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

Summary The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore--lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors ofEscherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of theE. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate-and catechol-carbacephalosporin conjugates were most effective inhibitors ofE. coli X580.     

Steroid analysis and xenosteroid potentials in two small streams in southwest Germany

The presence of estrogenic substances in thewater of the small streams Körsch (Kö)and Krähenbach (Kr), Southwest Germany, wasdetermined by chemical and biological analysis.Because a large proportion of the Kö waternear its mouth consists of sewage treatmentplant (STPs) effluents, the impact of STPs onlevels of estrogens in surface water is anenvironmental issue of concern. In July 1996,water samples were taken from Kr and Kö(four sites) and tested in the E-Screen assaywith human MCF-7 breast cancer cells. AllKö samples induced estrogen-dependent cellproliferation resulting in 17-estradiolequivalent concentrations (EEQ) between 3.3 and9.7 ng/L while the Kr water showed no effect.In 1998/99 eight samples from Kö (near itsmouth) and nine samples from Kr were collectedand tested in the E-Screen after solid phaseextraction. Some estrogenicity was detectablein three Kr samples but Kö samples had amedian EEQ of 3.1 ng/L (range: 1.2–42 ng/L).GC/MS analysis revealed differences in thelevels of 17-estradiol and estronebetween the two streams. 17-estradiolwas detectable in five Kö samples only (0.7–1.8 ng/L). Estrone was found in the Köfrom 2.5 to 38 ng/L (median: 7.6 ng/L) and inthe Kr between 0.8 and 22 ng/L (median: 1.7 ng/L). Analysis for nine phenolic xenoestrogensrevealed rather low levels for five compoundswhich occurred more frequently and in higherconcentrations in the Kö. After asemi-field exposure of adult male rainbow troutfor 4 weeks in Kö water, plasmavitellogenin levels were significantly highercompared to those levels detected in the sameanimals before exposure.     

Studies on steroids : CLXV. Determination of isomeric catechol estrogens in pregnancy urine by high-performance liquid chromatography with electrochemical detection

A method for the determination of 2- and 4-hydroxylated estrone and estradiol in pregnancy urine by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is described. The urine catechol estrogens were deconjugated, purified by adsorption on alumina, and subjected to HPLC-ECD. Two pairs of isomeric catechol estrogens were distinctly separated on a μBondapak C₁₆ column with acetonitrile-0.5% ammonium dihydrogen phosphate (pH 3.0). The amounts of these four compounds were satisfactorily determined with a quantitation limit of 1 ng using 4-hydroxy-16-oxoestradiol 17-acetate as an internal standard. The validity of the present method for the determination of urine catechol estrogens was verified by the recovery test.     

Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

Variations in the natural abundance of 15N in ryegrass/white clover shoot material as influenced by cattle grazing

Biological N2 fixation in clover is an important source of N in low external-N input farming systems. Using the natural 15N-abundance method, variations in N2 fixation were investigated in grazed and mowed plots of a ryegrass/white clover field. Ryegrass 15N varied considerably, from 0.2 to 5.6 under mowed conditions and from –3.3 to 11.6 under grazed conditions. Variations in 15N white clover were lower than in ryegrass, especially in the mowed plots (SE = 0.05, n = 20). The variations in the percentage of nitrogen derived from the atmosphere (%Ndfa) in white clover were highest in the grazed plots where it ranged from 12 to 96% (mean = 64%) compared with the mowed plots where it ranged from 64 to 92% (mean = 79%). Thus, the N2 fixation per unit white clover DM in the grazed ley was lower and more variable than under mowing conditions.Urine from dairy cows equivalent to 0, 200, 400 and 800 kg N ha-1 was applied to a ryegrass/white clover plot 6, 4 or 2 weeks before harvest. Without urine application 15N of ryegrass was positive. By increasing urine application (15N = –1) two weeks before sampling, the 15N of ryegrass decreased strongly to about –7 (P < 0.001). However, this effect was only observed when urine was applied two weeks before sampling. When applying 800 kg N four and six weeks before sampling, 15N in ryegrass was not significantly different from the treatment without urine application. White clover 15N was unaffected by whatever changes occurred in 15N of the plant-available soil N pool (reflected in 15N of ryegrass). This indicates that within the time span of this experiment, N2 fixation per unit DM was not affected by urine. Therefore, newly deposited urine may not be the main contributing factor to the variation in %Ndfa found in the grazed fields. This experiment suggested that the natural abundance method can be applied for estimating %Ndfa without disturbance in natural animal-grazed systems.     

Enzymatic measurement of phosphatidic acid in cultured cells

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.     

Distribution of Fucoidan Hydrolases and Some Glycosidases among Marine Invertebrates

Thirty-three species of marine invertebrates from the Sea of Japan were analyzed for contents of fucoidan hydrolases and some glycosidases. Fucoidan hydrolase activity was assessed by examining the effect of animal tissue extracts on fucoidans from the two brown seaweeds Laminaria cichorioides and Fucus evanescens, which have different structural characteristics. The activity of glycosidases (-glucosidase, -galactosidase, -fucosidase, and -mannosidase) was determined using p-nitrophenyl derivatives of sugars as substrates. It was found that glycosidases and fucoidan hydrolases of different specificities are fairly widely distributed among marine invertebrates. Mollusks and some species of echinoderms and arthropods showed the highest enzymatic activity. This research will enable us to choose organisms for the separation and study of fucoidan hydrolases and glycosidases, which may be useful in determining the structure of fucoidans.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

Bacterial metabolism of substituted phenols

4-(Methylmercapto)-phenol (MMP) and 4-(methylsulfinyl)-phenol (MSP) are oxidized by the soil isolate Nocardia spec. DSM 43251, which is closely related to Nocardia calcarea. The rate of degradation depends on the capability of a substrate to support growth and is strongly enhanced in the presence of a second carbon source under the conditions of cooxidation. MMP and MSP are cometabolized by hydroxylation of the benzene ring with the formation of the substituted catechol following by ring cleavage between carbon atoms 2 and 3 (metafission) to give 2-hydroxy-5-methylmercapto-or 2-hydroxy-5-methylsulfinylmuconic semialdehyde. Oxidation of MMP to MSP represents a bypath of MMP-oxidation. The intermediates were identified on the basis of their physical properties. The enzymes responsible for the metabolism of MMP and MSP are induced by growth with MMP or MSP, but not with glucose. MMP- and MSP-induced cells catalyze the oxidation of a variety of substituted phenols. This indicates a rather low substrate specificity of the enzymes induced by MMP and MSP.List of Abbreviations MMP 4-(methylmercapto)-phenol - MSP 4-(methylsulfinyl)-phenol - 3M-4MMP 3-(methyl)-4-(methylmercapto)-phenol - MMC 4(methylmercapto)-catechol - MSC 4-(methylsulfonyl)-catechol - MSV 4-(methylsulfinyl)-veratrol - MSOP 4-(methylsulfonyl)-phenol - MM-HMSA 2-hydroxy-5-methylmercaptomuconic semialdehyde - MS-HMSA 2-hydroxy-5-methylsulfinylmuconic semialdehyde - HMSA 2-hydroxymuconic semialdehyde - d₆-DMSO deuterated dimethylsulfoxide - CDCI₃ deuterochloroform - tlc thin layer chromatography     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Cereal β-amylase: Immunochemical study on two enzyme-deficient inbred lines of rye

Two inbred lines of rye (Secale cereale L), the kernels of which displayed a very low level of -amylase activity (1–3% of the levels generally found in rye), were investigated in comparison with a third normal line. An anti-wheat -amylase immune serum which cross-reacted with the rye enzyme was used in this study.The anti-wheat -amylase immune serum absorbed the -amylase activity in the three lines which were investigated. Comparably small amounts of enzymatic antigen corresponded to the small levels of activity detected in the enzyme-deficient lines. The three inbred lines were equally able to germinate. One of the enzyme-deficient lines was further investigated and neither the level of activity nor the amount of enzymatic antigen were notably changed upon germination.The results indicate that the reduced activity is due neither to the presence of an inhibitor nor to the production of inactive enzymes. Germination can proceed normally without late production of -amylase.     

Chitovibrin: a chitin-binding lectin fromVibrio parahemolyticus

A novel 134 kDa, calcium-independent chitin-binding lectin, chitovibrin, is secreted by the marine bacteriumVibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers >dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0–4m NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bindV. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.Abbreviations (GlcNAc)₂ N,N-diacetylchitobiose - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PTS phosphotransferase system     

Normal coordinate analysis of 2′-deoxythymidine and 2′-deoxyadenosine

The proposed valence force field allows us to reproduce the vibration modes of 2-deoxythymidine and 2-deoxyadenosine. The present calculations are based on the Wilson GF-method and a non-redundant set of symmetrical coordinates. The calculated wavenumbers have been compared to the available Raman and infrared peak positions observed in solid, amorphous or aqueous samples. Moreover, the results obtained with the present force field allow us to assign some of the characteristic vibration modes for the thymidine and adenosine residues involved in DNA double-helical chains.     

A genetic component in human lysosomal enzyme excretion

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of -glucuronidase, -galactosidase, -hexosaminidase, and -galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that -glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.This study was supported by grants from the National Institutes of Health (GM-19521) and the Council for Tobacco Research—U.S.A., Inc. (1080). The work was done while the authors were in the Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Characterization of γ-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries

Summary Endothelial cells were isolated with high viability (>93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of -glutamyl transpeptidase. The -glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70±10h. In the absence of collagen, -glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the -glutamyl transpeptidase to 30% of the initial value.     

Tailoring molecular sensing for peptide displaying engineered enzymes

Regulable enzymes displaying foreign peptides are valuable instruments for molecular targeting and fast analyte detection in homogeneous assays. Both the specificity and the intensity of the signal generated by the sensor are critical parameters that can be manipulated by trial-and-error protein engineering in the vicinity of the active site. An alternative approach is presented to enhance signal-background ratio in -galactosidase-based molecular sensors by an optimisation of the sensing conditions. The screening of the enzymatic response in a set of engineered enzymes has revealed an antibody-dependent increase in their specific activity up to 500% for the enzyme, HD72CA, that is reached with 0.25 pmol enzyme per reaction in presence of 1.75 mM substrate. This value, much higher than 200% enzyme activation achieved only by protein engineering, represents a step further in enhancing the enzyme's responsiveness. On the other hand, engineered -galactosidases are also highly dynamic without preliminary antibody incubation, rendering activation factors around 300% after global reaction times shorter than 15 min. Therefore, this enzymatic system has been revealed as extremely robust and suitable for efficient and fast molecular detection in the diagnosis of infectious diseases.