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PubMed2001

Electroanalytical determination of tungsten and molybdenum in proteins.

Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.

Chemical Precipitation↗
PubMed1999

Suckling behaviour does not measure milk intake in horses, Equus caballus.

Studies of parental investment in mammals have frequently used suckling behaviour to estimate energy transfer from mother to offspring, and consequently to measure maternal input. Such studies assume that the more an offspring sucks, the more milk it will receive. This assumption has been questioned, and a review of the literature found little support for it. To test if suckling behaviour provided an accurate index of milk or energy intake we used a radioactive isotope technique to label the milk of thoroughbred mares and to measure milk transfer to foals. We found no significant linear relationship between usual measures of suckling behaviour and milk or energy intake. No behaviours associated with suckling nor with characteristics of mares and foals improved the relationship; only the number of butts associated with each suck episode even approached significance. If we had used suckling behaviour to test theories on differential maternal investment our conclusions would have been in error. For example, female foals tended to suck for longer than males did but there was no difference in the amount of milk transferred. Consequently, we show that measures of suckling behaviour do not adequately predict milk intake in the domestic horse and we suggest that conclusions about differential maternal investment in mammals based on suckling behaviour are likely to be in error. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1999

Targeting of green fluorescent protein expression to the cell surface.

We have previously reported on GPI-anchored fusion proteins that bind radioactive isotopes. We targeted their expression to the cell surface to obtain a marker protein detectable by nuclear and optical imaging (1, 2). Here we suggest a novel approach for targeting a model protein (GFP) to the exoplasmic surface of the plasma membrane. An expression vector (pcPEP-GFP) was constructed containing GFP cDNA fused with the fragment encoding the N-terminal cytoplasmic domain and signal peptide/membrane anchoring domain of the rabbit neutral endopeptidase (PEP-GFP). Flow cytometry showed green fluorescence in 45% of cells transfected with GFP and in 34% of cells transfected with PEP-GFP (24 h after transfection). Fluorescence microscopy of fixed cells stained with rhodaminated anti-GFP antibodies showed positive reaction only in the case of PEP-GFP-transfected cells indicating cell-surface expression. The PEP-GFP fusion protein was identified as a component of the light microsomal and Golgi fractions by immunoblotting.

Animals↗
PubMed2001

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11.

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.

Amino Acid Sequence↗
PubMed2002

Optical aspects of Na+ ions adsorption on sol-gel porous films used in optical fiber sensors.

Adsorption of Na+ ions from water solutions on the surface of a porous silica layer covering the optical fiber core was studied. It was shown that Na+ ions adsorbed on silica obtained by the sol-gel method influence optical signal transmitted by optical fiber. The dependence of optical signal intensity on pH was observed. The results of optical measurements are in good agreement with those obtained by the potentiometric titration and adsorption of Na+ cations with application of a radioactive isotope.

Journal Article↗
PubMed2002

Quantification of PCR amplification products of Brugia HhaI repeat DNA using a semiautomated Q-PCR system.

The sensitive, rapid and species-specific diagnosis of Brugia infections in humans or animal models is important in determining the level of parasitemia and the efficacy of chemotherapy or vaccinations. The HhaI family of highly repeated DNA sequences from Brugia have been useful in polymerase chain reaction (PCR)-based diagnosis of brugian filarial infections in blood samples and in mosquitoes. A PCR assay was developed using a biotinylated primer, a non-biotinylated primer and a species-specific chemiluminescent probe [tris[2,2'bipyridine] ruthenium (II) chelate, TBR] to detect PCR amplified Hhal family repeats. Individual blood samples from jirds infected with Brugia malayi or B. pahangi and with different levels of microfilaremia were tested in this assay. Known concentrations of Brugia DNA and DNA from the blood of uninfected control jirds were used as positive and negative controls, respectively. The PCR products generated by this method were analyzed using a semi-automated quantitative (Q)-PCR system. The levels of parasite DNA can be calculated from the luminosity units generated. Significant amounts of parasite DNA were detected in blood samples from infected jirds, and these values were correlated with the levels of microfilaremia. In contrast, reductions in circulating microfilaria following treatment with ivermectin correlated with low levels of measurable DNA. Using this system, we were also able to detect HhaI repeat DNA in the spleens of B. pahangi- infected jirds at 56 days post-infection when circulating microfilariae were not readily detectable. The results indicate that the species-specific Hhal Q-PCR detection and quantification method is rapid and sensitive, is useful in the detection of Brugia DNA in blood and other tissues and is suited for use in clinical settings because it does not require radioactive isotopes and gel-based protocols.

Animals↗
PubMed1999

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.

Animals↗
PubMedDate not supplied

Determination of the pseudofacility by fluorophotometry in the human eye.

PURPOSE: Pseudofacility (decrease in aqueous humor flow in response to increase in the intraocular pressure (IOP)) has been evaluated in animals and in humans by a combination of different techniques that seriously alter the ocular physiology, such as the cannulation of the eyeballs, use of radioactive isotopes, perilimbic suction cups or tonography. Very different results have been obtained. Our objective is to relate the value of the aqueous humor flow in humans (as measured by fluorometry) with IOP. In order to obtain the widest range of IOP possible, we have performed this study in both normal as well as glaucoma patients. METHODS: 48 eyes were studied, corresponding to 31 subjects (mean age 58 +/- 14 years). Of these, 21 corresponded to normal subjects and 27 to glaucoma patients. The aqueous humor flow (F) was determined by fluorophotometry (Protocol of Yablonsky) and the IOP was measured thereafter. RESULTS: The mean value of F was 2.1 +/- 0.5 microliter/min for healthy eyes and 2.2 +/- 0.7 microliter/min for glaucoma-affected eyes. The values were not different (p > 0.1). The mean value of the IOP was 15.3 +/- 3.1 mmHg in the normal population and 22.1 +/- 4.3 mmHg in the glaucoma patients. A linear relationship between F and IOP was found (r = -0.41, p < 0.05, decrease in F = 0.081 microliter min-1 mmHg-1). CONCLUSIONS: The value of the pseudofacility in the humans can be estimated at 0.081 microliter min-1, mmHg-1, which constitutes 27% of the total outflow facility. This figure also makes it possible to estimate the role of the ultrafiltration in blood in the production of the aqueous humor since it is assumed that active secretion is pressure-independent. The results obtained are concordant with a secondary but no unimportant role of the plasmatic ultrafiltration with respect to the active secretion.

Aqueous Humor↗
PubMed1994

The effect of irradiation on blood flow through rabbit submandibular glands.

Microspheres labelled with radioactive isotopes were used to study blood flow through irradiated rabbit submandibular glands before and after injection of pilocarpine. The blood flow through the submandibular glands was measured by the microsphere technique with satisfactory accuracy. A single radiation dose of 13.1 Gy at 3 months of age reduced blood flow through the whole gland significantly 4 months later, and even more at 10 months after irradiation. However, the relative blood flow (per gram of gland tissue) was almost unaffected at both 4 and 10 months post-irradiation with 13.1 Gy because of the simultaneous reduction of gland parenchyma.

Animals↗
PubMed1978

Preparation and purification of estriol, estradiol, and progesterone labeled with radioactive iodine.

Steroids labeled with radioactive isotopes are useful tracers for radio-immunoassay and for in vivo nuclear medecine purposes. We here report and discuss methods for direct labeling of the steroids estriol, estradiol, and progesterone with radioiodine-125 or -131. The purification procedure and some chemical and physical properties of the purified labeled materials are referred to and discussed.

Chloramines↗
PubMed1985

In vivo distribution studies of radioactively labelled platinum complexes; cis-dichlorodiammine platinum(II), cis-trans-dichlorodihydroxy-bis-(isopropylamine) platinum(IV), cis-dichloro-bis-cyclopropylamine platinum(II), and cis-diammine 1,1-cyclobutanedicarboxylate platinum(II) in patients with malignant disease, using a gamma camera.

The in vivo distribution in man of the four platinum derivatives cis-dichlorodiammine platinum(II) (DDP), cis-trans-dichlorodihydroxy-bis-(isopropylamine) platinum (IV) (CHIP), cis-dichloro-bis-cyclopropylamine platinum(II) (CP), and cis-diammine 1, 1-cyclobutanedicarboxylate platinum(II) (CBDCA) has been observed. The availability of these compounds labelled with the radioactive isotope of platinum, platinum-191, has made serial in vivo imaging of their distribution possible. Injection of 17-35 MBq (5-28 mg) of the labelled compound IV was followed by imaging, using a gamma camera, with particular reference to the kidneys, liver, and tumour site. Hepatic and renal clearances were observed in all nine patients, but no unequivocal evidence of tumour uptake was found. The left kidney uptake was estimated at times up to 7 days after injection. Mathematical analysis of some of the uptake curves failed to show any significant difference between the clearance times observed. However, the two patients who received CBDCA did show a higher initial renal uptake, falling within the 1st day to levels comparable with those of the other compounds, and the three patients who received DDP showed consistently liver uptake.

Adult↗
PubMed1996

Oxidation of D(-)3-hydroxybutyrate administered to rats with extensive burns.

Although the suppressive effect of 3-hydroxybutyrate (3-OHB) on post-traumatic protein catabolism in traumatized patients is well documented, the oxidation of exogenously administered 3-OHB during catabolic stress has not been investigated. The present study was designed to evaluate, using radioactive isotopes, total body oxidation in rats with and without burn stress to which 3-OHB had been exogenously administered, in comparison with total body oxidation in such rats that had received glucose. The rats were divided into four groups, based on whether or not a 30% full-thickness burn was inflicted, and the type of infusate they received after the burn, namely, 3-OHB or glucose. The total exhaled CO2 was collected for 6h after the infusion was commenced, and 14CO2 was assayed in a liquid scintillation spectrometer. Oxidation of the infusate was calculated from the percentage of exhaled 14CO2 derived from the infused substrates. The plasma concentration of 3-OHB was significantly increased after the infusion in both the burned and non-burned rats. The total exhaled 14CO2 from the rats infused with glucose decreased from 48.2 +/- 2.4% to 40.8 +/- 3.7% (means +/- SD, P < 0.001) after thermal injury. However, the total exhaled 14CO2 from the rats infused with 3-OHB appeared sooner, and there was no difference in the total expired 14CO2 derived from 3-OHB between the burned and non-burned rats, at 68.1 +/- 2.7% vs 66.4 +/- 3.4%, respectively. These findings suggest that even under conditions of burn stress, 3-OHB can be oxidized normally if the plasma concentration of 3-OHB is elevated by exogenous administration.

3-Hydroxybutyric Acid↗
PubMed1980

The influence of trauma, Dextran 1000, and Dextran 40 on the lodgement of circulating tumor cells.

Earlier studies have shown that trauma and infusion of Dextran 1000 increased the formation of pulmonary metastases after i.v. tumor cell injection in rats. Infusion of Dextran 40 enhanced the pulmonary metastasis formation in traumatized but not in non-traumatized animals. In this study tumor cells labelled with a radioactive isotope were used to register the lodgement circulating tumor cells after trauma and infusion of Dextran 1000 and Dextran 40. It was found that the pulmonary lodgement of tumor cells was increased after trauma and infusion of Dextran 1000. Dextran 40 did not influence the lodgement in a significant way either in normal or in traumatized animals. Thus, the increased formation of metastases after trauma and infusion of Dextran 1000 seems to depend upon an increased tumor cell lodgement.

Animals↗
PubMed1981

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13.

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, 13N as NO3-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of 13N internally was ammonium and amino acids; the amino acid labeling pattern indicated that 13NO3- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO3- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar Km values, 7 muM. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.

Ammonium Sulfate↗
PubMed1986

Coupling between sodium and succinate transport across renal brush border membrane vesicles.

The coupling between Na+ and succinate transport through the renal brush border Na+-dicarboxylate cotransporter was examined in a vesicle preparation. Initial rates of Na+ and succinate uptake were measured simultaneously using radioactive isotopes under zero-trans (Na+ and succinate-free intravesicular solutions) and voltage-clamped conditions (p.d. = 0 mV). The coupling coefficient is defined as the ratio of the succinate-dependent Na+ flux to the Na+-dependent succinate flux. The cis Na+ and succinate concentrations were varied from 0.05-5 mM succinate and 25-150 mM Na+. The coupling coefficient was approximately 3 and was independent of the succinate and Na+ concentrations tested. These results suggest that only the free carrier and the fully loaded carrier (carrier + 3 Na+ + succinate2-) are able to cross the brush border membrane; i.e., there is little evidence of slippage in the coupling between Na+ and succinate fluxes through the membrane via this carrier. A coupling coefficient of 3 is consistent with the electrogenic nature of the Na-dicarboxylate cotransporter.

Animals↗
PubMed1996

Use of fluorochromes in the determination of chemotaxis and haptotaxis of granulocytes by micropore filter assays.

Most conventional assays for the in vitro measurement of polymorphonuclear leukocyte (PMN) migration are modifications of the Boyden chamber technique, which requires quantification of migrated cells on micropore filters. This quantification is accompanied by several disadvantages, comprising errors in direct cell counting, low sensitivity of turbidimetric methods, the loss of marker enzymes by activation of PMNs, or the use of radioactive isotopes. We set up an improved fluorometric method to measure chemotactic and haptotactic migration of PMNs using polycarbonate filter-bearing Transwell culture plate inserts. This improved fluorometric method allows the evaluation of effects of fluorescent-dye labeling on chemotaxis and haptotaxis - defined as cell migration due to cell surface- or matrix-bound gradients of chemoattractants - by collecting data in an automated system. Results were compared with data obtained from direct microscopic cell counting in Transwell experiments as well as in conventional 48-multiwell Boyden chamber assays. Calcein-AM and BCECF-AM proved to be fluorochromes with minimal effect on both types of PMN migration. We conclude that the fluorochromes are powerful tools for the analysis of PMN migration and allow modifications of chemotaxis/haptotaxis assays for automated quantification.

Chemotaxis↗
PubMedDate not supplied

Volume-dependent echocardiographic parameters are not useful for estimating baseline blood volume but are useful for detecting acute changes in vascular filling state.

To assess the hypothesis that echocardiographic variables vary as a function of baseline blood volume and change in response to changes in vascular filling state, we evaluated the relationship between the atrial and ventricular dimensions and left ventricular function, as measured by combined Doppler and M-mode echocardiography on the one hand, and blood volume as measured by a standard radioactive isotope technique on the other. Furthermore, we determined the effect of an increment in vascular filling state induced by plasma volume expansion. Test subjects were ten male and six female volunteers. Blood volume/kg was the only factor not different between male and female subjects and therefore suitable for our study. None of the echocardiographic variables correlated with blood volume/kg. Volume loading increased stroke volume, left atrial diameter, right atrial area, left ventricular end-diastolic dimension, mean velocity of circumferential fiber shortening and left ventricular ejection time. It is concluded that echocardiographic variables are unsuitable for estimating blood volume. Serial measurements of atrial dimensions and left ventricular dimensions are useful for detecting rapid changes in vascular filling state.

Adult↗
PubMed1987

Advances in the use of nucleic acid probes in diagnosis of viral diseases of man. Brief review.

A variety of methods are now available for the preparation and labelling of viral nucleic acids for use as probes in diagnostic virology. Some of these are assessed including the use of synthetic oligonucleotides in place of molecularly cloned nucleic acids, and alternatives to labelling with radioactive isotopes such as biotin, enzymes and fluorochromes. Dot blot, sandwich, indirect sandwich and in situ hybridization are covered, and examples given of the current use of nucleic acid probes in detection of human viral infections. The potential and limitations of nucleic acid hybridization are discussed in the light of these new methods.

DNA, Viral↗