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PubMed1995

Quantitative chromatographic analysis of inositol phospholipids and related compounds.

The metabolism of phospholipids and the mobilization of second messengers such as inositol-1,4,5-trisphosphate, 1,2-diacylglycerol (DAG) and arachidonic acid (AA) from phospholipids is commonly studied by radiolabelling phospholipids with [3H]myo-inositol or [32P]ATP and measuring the incorporation of radioactivity in different phospholipids or their hydrolysis products. However, for the radiolabelling method to accurately reflect changes in the compound's mass, it is essential that the tissue is labelled to isotopic equilibrium which is difficult to achieve. To circumvent the disadvantages of the radiolabelling method, several analytical procedures have been developed for the mass analysis of phospholipids and inositolphosphates (IPs). Quantitation of the mass or the radiolabelling of phospholipids is a complex multi-step procedure that involves quantitative isolation of phospholipids, fractionation of individual phospholipids and either determination of radioactivity in each component or the measurement of their mass. Phospholipids, DAG and AA are extracted from tissue sample with organic solvents such as chloroform-methanol (2:1) containing HCl or formic acid. The extract is separated by TLC, cartridge-column chromatography or HPLC on a reversed-phase column. Phospholipids are quantitated by measuring inorganic phosphate, absorption at 200 nm or mass spectrometry. Inositol phosphates are extracted with perchloric acid or trichloroacetic acid and separated by ion-exchange cartridge-column or HPLC with an ion-exchange column. IPs are quantitated by measuring inorganic phosphate or by using enzymatic reaction, metal-dye coupling, NMR or mass spectrometry.

Arachidonic Acid↗

PubMed1977

Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase.

Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.

Adenylate Kinase↗

PubMed1990

Desorption kinetics and mobility of some radionuclides in sediments.

The desorption kinetics of five radionuclides, 85Sr, 137Cs, 60Co, 54Mn, and 65Zn, were studied for several coastal sediments, a few limnetic sediments, and one subsoil, using various aqueous solutions for extraction in batch systems. In a 2-wk desorption experiment, greater than 90% of most radionuclides sorbed were extracted by 6N-HCl, whereas in 1N-HCl approximately 30% of the 137Cs was retained within the sediments and a soil due to its fixation following intraparticle diffusion. For longer desorption periods (6 wk), reversible sorption with sediments could be seen for the 137Cs compared with the other four radionuclides in seawater. The EDTA-2Na solution extracted greater than 70% of 65Zn, 54Mn, and 60Co, and the ammonium oxalate extracted greater than 60% of 54Mn and 60Co. Readily exchangeable cations extracted by ammonium acetate were greater than 80% for 85Sr, approximately 50% for 137Cs, and 40% for 60Co and 54Mn. Many of the distribution coefficients for these radionuclides in coastal sediments obtained by desorption experiments were one order of magnitude or more larger than those obtained by the adsorption experiments. For 85Sr, larger-than-expected distribution coefficients were obtained in the desorption experiments, an indication of the irreversible formation of metal-oxyhydroxides during a slow reaction. Using batch and column diffusion experiments, it was recognized that 60Co and 54Mn are less strongly associated with coastal sediments under suboxic conditions, and their migration rates are greater in deeper layers of sediment than in the top layer, an indication that a single Kd model is inappropriate to forecast the behavior of radionuclides near sediment surface layers.

Adsorption↗

PubMed1988

Effect of selected insecticides on rat brain synaptosomal adenylate cyclase and phosphodiesterase.

Previous reports from our laboratory and others clearly indicated that organochlorine insecticides such as chlordecone and DDT are potent inhibitors of ATPases involved in active ion transport. The present studies were initiated to study the effect of plictran, chlordecone, toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin on enzymes involved in cyclic AMP metabolism. Rat brain synaptosomes were prepared by Ficoll-sucrose gradient centrifugation method. Adenylate cyclase activity, which is involved in anabolism of cAMP, was determined using the radioactive method by measuring [32P]cAMP formed during hydrolysis of [32P]ATP. Phosphodiesterase activity, which is involved in the catabolism of cAMP, was estimated by measuring [3H]adenosine formed using [3H]cAMP as a substrate. Synaptosomal adenylate cyclase activity was inhibited significantly by plictran with an IC50 of 25 microM, and a maximum inhibition of 30% was observed with 50 microM chlordecone. Toxaphene, aldrin, dieldrin, endrin, isodrin, and telodrin did not affect the adenylate cyclase activity. Similarly, none of the insecticides studied inhibit the activity levels of synaptosomal phosphodiesterase. The significant inhibition of adenylate cyclase observed with plictran might be due to the tin component, since several heavy metals affect cAMP metabolism. The lack of inhibition of adenylate cyclase and phosphodiesterase with other compounds tested clearly supports our postulation that these organochlorine insecticides exert their neurotoxic action by the selective inhibition of ATPases in synaptosomes.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2004

Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer.

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the beta-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by beta-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.

Base Sequence↗

PubMed1992

Development of humoral immune responses against a macrocyclic chelating agent (DOTA) in cancer patients receiving radioimmunoconjugates for imaging and therapy.

The development of stable immunoconjugates by the advent of macrocyclic metal chelating agents (DOTA) has enabled us to study the ability of 111In-DOTA-labeled monoclonal antibodies to detect tumor lesions in a pilot radioimmunolocalization study, as well as to evaluate the kinetics, toxicity, and efficacy of i.p. administered 90Y-DOTA-labeled murine monoclonal antibody in a Phase I/II clinical trial of advanced ovarian cancer. The development of serum sickness-like reactions in three of six treated patients, in the absence of previous monoclonal antibody administration, led us to study the potential immunogenicity of the new chelate. Six patients with ovarian cancer received 25 mg of HMFG1 monoclonal antibody coupled with 90Y-DOTA (doses of radioactivity, 15 to 25 mCi), administered i.p. Eight patients with various malignant tumors received low doses (220 micrograms to 1 mg) of monoclonal antibodies, labeled with 111In-DOTA, i.v. for imaging studies. Using a solid-phase enzyme-linked immunosorbent assay method, the immunogenicity of DOTA was evaluated. Serial dilutions of patients' sera, before and after imaging or therapy with DOTA-coupled monoclonal antibodies, as well as sera from patients who did not receive DOTA-coupled antibody, were screened on enzyme-linked immunosorbent assay plates coated with human serum albumin (HSA), HSA-2-iminothiolane, and HSA-2-iminothiolane-benzyl-DOTA. All patients treated with i.p. monoclonal antibody developed anti-DOTA antibodies. Four of eight patients who received i.v. "imaging" doses of DOTA-coupled monoclonal antibody developed antibodies against DOTA. The levels of anti-DOTA response correlated with the amount of injected radioimmunoconjugate (r = 0.889, P less than 0.001). None of the patients who received DOTA-coupled antibody had detectable antibodies against the macrocycle before immunoconjugate administration. We then addressed further the restriction of the immune response against the macrocycle. We found that there was no or very low response against the aromatic ring attached to DOTA. Most, if not all, of the immune response is directed against the DOTA ring structure. Affinity purification of anti-DOTA antibody from serum enabled quantitation of these antibodies in the serum of patients. An inverse, statistically significant correlation was observed between the percentage of binding inhibition of a patient's serum to DOTA, by HSA-2-iminothiolane-DOTA (100 micrograms/ml) and the level of anti-DOTA immunoglobulin in the serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗

PubMed1993

Computer-aided methods for evaluating cancer risk in miners due to radiation exposure.

The paper presents some aspects of radiation hazard which occurs in a non-nuclear sector of industry, namely radiation hazard in non-uranium underground mines. The radiation hazard is caused in each type of underground mine by the naturally occurring noble radioactive gas-radon (222Rn) and radioactive products of its decay 218Po, 214Pb, 214Bi/214Po the so-called 'radon daughters' occurring in the mines' air. The paper presents the concept of how to provide a reliable system of assessment of miners' exposure by application of representative individual dosimetry, and also presents principles of computer-aided methods for interpretation of the results of miner's dosimetry useful for conversion of dosimetry data to the term of expected risk of cancer caused by exposure at miner's workplaces. The representative Individual Dosimetry system strengthened by computer-aided methods of analysis of results provided essential information on radiation cancer risk for miners employed in coal mines, metal-ore mines, chemical raw material mines in Poland. The coefficient of annual cancer risk induction is 1.5 x 10(-4) year-1 for coal mines, 1.40 x 10(-4) year-1 for metal ore mines and 1.5 x 10(-4) year-1 for chemical raw material mines. The radiation risk appears to be of the same magnitude as the conventional risk of life loss at work-related accidents. The average Lost Life Expectancy coefficient for both the radiation risk and conventional risk are 0.5 and 0.3 year per each miner, respectively.

Electronic Data Processing↗

PubMed2004

153Sm and 166Ho complexes with tetraaza macrocycles containing pyridine and methylcarboxylate or methylphosphonate pendant arms.

A set of tetraaza macrocycles containing pyridine and methylcarboxylate (ac(3)py14) or methylphosphonate (MeP(2)py14 and P(3)py14) pendant arms were prepared and their stability constants with La(3+), Sm(3+), Gd(3+) and Ho(3+) determined by potentiometry at 25 degrees C and 0.10 M ionic strength in NMe(4)NO(3). The metal:ligand ratio for (153)Sm and (166)Ho and for ac(3)py14, MeP(2)py14 and P(3)py14, as well as the pH of the reaction mixtures, were optimized to achieve a chelation efficiency higher than 98%. These radiocomplexes are hydrophilic and have a significant plasmatic protein binding. In vitro stability was studied in physiological solutions and in human serum. All complexes are stable in saline and PBS, but 20% of radiochemical impurities were detected after 24 h of incubation in serum. Biodistribution studies in mice indicated a slow rate of clearance from blood and muscle, a high and rapid liver uptake and a very slow rate of total radioactivity excretion. Some bone uptake was observed for complexes with MeP(2)py14 and P(3)py14, which was enhanced with time and the number of methylphosphonate groups. This biological profile supports the in vitro instability found in serum and is consistent with the thermodynamic stability constants found for these complexes.

Blood Proteins↗

PubMed2003

Radiolabeling of avidin with very high specific activity for internal radiation therapy of intraperitoneally disseminated tumors.

PURPOSE: For the effective internal radiation therapy of i.p. disseminated tumors, we developed avidin (Av)-dendrimer-chelate complex, which can be labeled with indium-111, emitting Auger and conversion electrons, with very high specific activity, and we studied its internalization, biodistribution, and therapeutic effect in nude mice with i.p. tumors. EXPERIMENTAL DESIGN: Generation 4 dendrimer (G4) was biotinylated and conjugated with 52 1B4M chelates. (111)In-G4-bt was mixed with Av to form (111)In-G4-Av complex. (111)In-G4-Av was incubated with ovarian cancer cells (SHIN-3), and the rate of internalization of the radiolabel into SHIN-3 cells was followed. (111)In-G4-Av was i.p. injected into nude mice that had i.p. disseminated SHIN-3 tumors, and the biodistribution was determined. Nude mice bearing i.p. disseminated tumors received i.p. injection of (111)In-G4-Av (9.25 or 18.5 MBq x 2, with a 1-week interval) and were followed for the formation of malignant ascites. RESULTS: Av could be labeled with (111)In with specific activity as high as 37 GBq/mg. More than 75% of the radioactivity was internalized 24 h after binding to cancer cells. (111)In-G4-Av accumulated rapidly and highly in the i.p. tumors (128.20% injected dose/gram of tissue at 2 h, 114.91% injected dose/gram of tissue at 24 h for unsaturated compound) with high tumor:background ratios. Treatment with a high dose of (111)In-G4-bt-Av was tolerable and showed dose-dependent therapeutic effect. CONCLUSIONS: G4-Av complex, which could be labeled with (111)In with very high specific activity and showed efficient internalization into cancer cells and high accumulation to i.p. tumors, appears to be suitable for the internal radiation therapy of i.p. disseminated tumors using metallic radionuclides emitting Auger and conversion electrons.

Animals↗

PubMed2000

Mobilization and retardation of uranium DOC species at three mine piles in Schlema/Alberoda, Saxony, Germany.

The modeling of geo-chemical processes needs the detailed and comprehensive knowledge of all chemical interactions existing in the flow path of waste dumps, subsoil and aquifer. This includes the adsorption, displacement and transport of heavy metal species of fulvic and humic acids, which represent the main amount of DOC in the liquid/solid system of the flow path. Comparative measurements of DOC concentrations in the input and output flow at the three waste dumps in the district Schlema/Alberoda indicated that DOC is produced and/or supplied within the waste dumps. The speciation of heavy metal compounds of fulvic and humic acids was achieved by means of sequential chromatographic analysis SCA and isoelectric focusing IEF. Between 5% and 20% of uranium exist in the flow path in the form of stable fulvic and humic acid species. Their distribution coefficients are strongly correlated with the pH values of the geo-chemical system.

Benzopyrans↗

PubMed1993

Pharmacokinetics of an anti-carcinoembryonic antigen monoclonal antibody conjugated to a bifunctional transition metal carborane complex (venus flytrap cluster) in tumor-bearing mice.

An anticarcinoembryonic antigen (CEA) monoclonal antibody, T84.66, has been conjugated to a metallocarborane complex (Venus flytrap cluster, VFC) containing 57Co. This radioimmunoconjugate, 57Co-VFC-T84.66, retained > 90% immunoreactivity, was stable in serum (7 days) and demonstrated good localization in LS174T tumor xenografts. Pharmacokinetics of 57Co-VFC-T84.66 in tumor-bearing mice were compared to T84.66 Mab conjugated with either DTPA or its benzylisothiocyanate derivative (BzDTPA) labeled with 111In. Whole-body half-life for VFC-T84.66 was less (t1/2 = 62 hr) than that for either DTPA-T84.66 (t1/2 = 157 hr) or BzDTPA-T84.66 (t1/2 = 167 hr). Blood clearance was similar for all three radioimmunoconjugates (t1/2 = 22 hr). Hepatic uptake of the radiolabel was rapid and remained constant for 7 days for both DTPA radioimmunoconjugates (DTPA radioimmunoconjugate = 13.7 +/- 1.5 %ID/g; BzDTPA radioimmunoconjugate = 10.4 +/- 1.7%ID/g). For VFC, however, liver radioactivity decreased from 19.1 +/- 0.6%ID/g at 1 hr to 0.9 +/- 0.1 %ID/g 7 days postinjection, suggesting a possible role for VFC radioimmunoconjugate in the imaging and therapy of liver metastases.

Animals↗

PubMed2004

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1975

Metal ion contents of gel-filtered platelets from patients with storage pool disease.

Platelets from nine patients with storage pool disease (SPD) and from ten control subjects were isolated by gel filtration into a suspension medium permitting the direct determination of platelet Mg-2+, Ca-2+, and K+ levels. The total intracellular levels of ATP and ADP, as well as the incorporation patterns of 14-C-adenine into the metabolic nucleotide pool, were also determined in these platelet suspensions. The gel-filtered platelets (GFP) from SPD patients exhibited slightly lowered levels of ATP and substantially reduced amounts of ADP, in agreement with previous studies using PRP suspensions. Diminished aggregation responses to ADP, epinephrine, and to collagen in particular, similar to those observed previously in PRP, were obtained in GFP from SPD patients. However, GFP from the patients exhibited more variable aggregation responses to addition of ADP and epinephrine than did GFP from the control subjects. Increases in the extent of radioactive hypoxanthine formation, observed previously in normal platelets as a result of isolation into the suspension medium used in these studies, were significantly reduced in the GFP from SPD patients. The levels of platelet Mg-2+ and K+ determined in GFP from the patients were not significantly different from the levels of these ions in GFP from control subjects. However, substantial reductions in platelet Ca-2+ were found in the SPD platelets. A strong correlation was obtained between this reduction in platelet Ca-2+ and the reduction in ADP in these platelets. No such correlation was apparent between the ATP and Ca-2+ deficiencies. These results suggest that a major portion of platelet Ca-2+ may be located in the dense granules and support previous hypotheses that granular ADP and/or Ca-2+ may play a role in the release reaction. The finding of normal levels of platelet Mg-2+ and K+ in SPD platelets, however, suggests that these latter ions are not located in the dense granules.

Adenine↗

PubMed1975

Evidence for an essential histidine in carboxypeptidase Y. Reaction with the chloromethyl ketone derivative of benzyloxycarbonyl-L-phenylalanine.

The possible role of histidine residues in the catalytic function of carboxypeptidase Y from bakers' yeast has been investigated using site-specific reagents. Among the reagents tested, benzyloxy-L-phenylalanylchloromethane (Z-PheCH2Cl) was the most powerful inhibitor of the enzyme. It irreversibly inactivated both the peptidase and esterase activities with an apparent second order rate constant of 3.8 M-minus 1 S-minus 1; the D isomer caused essentially no effect on either activity. Inhibition by L-Z-PheCH2Cl, the reaction retarded by certain competitive inhibitors of the enzyme. Using radioactive L-Z-PheCH2Cl, the reaction with the enzyme was shown to be essentially stoichiometric. Diisopropylphosphorofluoridate (iPr2PF)-inactivated enzyme failed to react with Z-PheCH2Cl, and conversely, the Z-PheCH2Cl-inhibited enzyme failed to react with radioactive iPr2PF. Amino acid analyses of the Z-PheCH2Cl-inactivated enzyme revealed the loss of essentially 1 residue, with a concomitant yield of a 0.62 residue of N-t-carboxymethylhistidine. Since carboxypeptidase Y has a reactive serine at its active center, we concluded from these results that the mechanism involves a charge-relay system in the hydrolysis of peptide and ester substrates, as in chymotrypsin. An -SH group of carboxypeptidase Y was not affected during the reaction with L-Z-PheCH2Cl. The generic name "serine carboxypeptidase" has been proposed for carboxypeptidase Y and for the iPr2PF-sensitive carboxypeptidases from plants, molds, and animal tissues, in order to distinguish them from "metal carboxypeptidase" to which carboxypeptidase A (EC 3.4.12.2) and B (EC 3.4.12.3) belong.

Amino Acids↗

PubMed1980

Calcium and pancreatic beta-cell function. IX. Demonstration of lanthanide-induced inhibition of insulin secretion independent of modifications in transmembrane Ca2+ fluxes.

beta-Cell-rich pancreatic islets were microdissected from noninbred ob/ob-mice and used to examine the mode of action of trivalent lanthanide ions on insulin secretion. La3+, Sm3+, and Tm3+ were equally effective inhibitors of basal and glucose-stimulated insulin release. As indicated by perifusion experiments with Tm3+, the inhibitory action was prompt, sustained, and readily reversible. Despite the similarities among the lanthanides in inhibiting insulin secretion, these cations differed considerably in their ability to impair transmembrane 45Ca fluxes. Using 10 different members of the lanthanide series, it was possible to demonstrate that their effectiveness to inhibit 45Ca uptake increased with ionic radius. La3+ markedly inhibited intracellular uptake and superficial binding of 45Ca at both 3 and 20 mM glucose. However, Tm3+ failed to affect intracellular 45Ca uptake and only reduced superficial binding of 45Ca at 3 mM glucose. In efflux experiments, Tm3+ did not affect basal or glucose-stimulated 45Ca washout from islets perifused with a medium containing 1.28 mM Ca2+. In a Ca2+-deficient medium, Tm3+ caused a slight transient increase, followed by reduction of 45Ca washout. However, when glucose was omitted, there was a prompt increase in the washout of radioactivity in the presence of Tm3+. Accordingly, the potent inhibitory action of Tm3+ on insulin secretion is not matched by changes in transmembrane Ca2+ fluxes. Since the lanthanides do not penetrate intracellularly, we propose the existence of cationic binding sites in the beta-cell plasma membrane with direct inhibitory effects on insulin secretion.

Animals↗

PubMedDate not supplied

Oligonucleotide labelled lipase as a new sensitive hybridization probe and its use in bio-assays and biosensors.

Radiolabelled polynucleotide probes have been employed extensively for the detection of complementary nucleic acids by specific hybridization. Within the last few years, various methods have been developed using enzyme-labelled probes to avoid unstable and hazardous isotopes. These assays, based on photometry, fluorescence, and chemiluminescence, have helped to overcome the use of radioactive probes. To increase the performance of a non-radioactive DNA detection system, the labelling enzyme should remain stable under hybridization conditions which allow the formation of a 15-25 bp long DNA-DNA duplex (Tm = 50-70 degrees C). Therefore, the use of unstable phosphatase and peroxidase conjugates must be avoided due to the composition of the hybridization mixture and the high temperature. By screening various hydrolytic enzymes to fit the special demands, fungal lipases turned out to be the most practical. They offer high sensitivity due to an extremely high turnover number, stability at room temperature for several years, thermostability under working conditions and an easy design of various chromogenic, fluorescent and electrochemical active substrates. Several types of silanized, oxidized and unmodified metal sensors and also standard microtitre plates modified with amino groups were used for the immobilization of oligonucleotides. A sandwich hybridization using the lipase-labelled oligonucleotide probe and a terminal immobilized capture DNA on a microtitre plate or sensor surface combined with a rapid hybridization in solution simplifies and improves the performance of the DNA detection system.

Alkaline Phosphatase↗

PubMed1997

Internalisation of short-chain isoprenyl diphosphates by chromaffin cells from bovine adrenal medulla.

The uptake of FPP and GGPP was studied using primary cultures of chromaffin cells as an experimental model. The translocation of the isoprenyl diphosphates was shown to be both dose- and time-dependent. After incubation and a series of thorough washing cycles the residual radioactivity could only be released by either detergent or alkali treatment endorsing true internalisation rather than sorption onto the external cell surface. Preliminary experiments suggest that the isoprenyl diphosphates are internalised-at least for the greater part-without a preceeding dephosphorylation step. The translocation process does not seem to follow regular Michaelis-Menten kinetics. Upon varying pH (pH-range 6-8) or temperature (0 degree-40 degrees C) of the incubation medium only moderate changes in internalisation rate were observed. The metabolic poisons (CN-, F-, chloroquine) administered were without any significant effect, suggesting the transport phenomenon not to be energy dependent nor to proceed via receptor-mediated endocytosis. Upon inclusion of metallic ions in the test medium a pronounced increase in uptake was registered for Zn(2+)-ions, while most other cations did not cause any significant alterations. From the amino acid modifying agents screened sulphydryl reagents (NEM, PCMB, IA) resulted into a moderate unexpected elevation of uptake. An interesting feature was the activatory effect on the uptake of the diphosphorylated derivatives elicited by the corresponding free alcohols farnesol and geranylgeraniol; shorter and longer chain alcohols were without effect. In order to assess the specificity of the transport system a number of (pyro)phosphorylated compounds were included in the incubation medium indicating dolichylmonophosphate and isopentenylpyrophosphate to promote the uptake.

Adrenal Medulla↗

PubMed1991

Cadmium and zinc kinetics in fish: studies on water-borne 109Cd and 65Zn turnover and intracellular distribution in minnows, Phoxinus phoxinus.

This study investigates the short- and long-term turnover of zinc and cadmium in fish. Juvenile minnows, Phoxinus phoxinus, were exposed to water-borne 109Cd and 65Zn for 24 hr. Tracer uptake, redistribution, elimination, and intracellular tracer distribution were studied for 60 days in tracer-free water. The influx of 65Zn from the water to the internal organs was more rapid than the influx of 109Cd, and the 65Zn turnover in the liver and kidney was considerably faster than the turnover of 109Cd. Moreover, in the gills and kidney, intracellular 65Zn was more mobile between intracellular compartments than 109Cd. The liver and kidney accumulated the major part of the 109Cd originating from the gills, whereas the head and skin, and muscle and bone-fractions retained the majority of the 65Zn. This indicates that the metals have different pathways in the fish body, after entering the body from the water. The cadmium levels in the gills, liver and kidney increased drastically because of the cadmium exposure, but the zinc levels in the organs were not markedly affected by a fifteen-fold increase in external zinc concentration, indicating a homeostatic control of zinc in the minnow.

Animals↗